List of Abbreviations and Nomenclature

11.1 Introduction

Sulfur content of residual fuel varies widely according to various local restrictions. The major part of the sulfur contained in fuel oil is in the form of thiophene and its derivatives and are found mainly in the resins (70%) and the remainder is equally distributed in the oils and asphaltenes. It is difficult to dispose high sulfur fuel oil HSFO with an S-content > 3 wt.%. Its primary outlet is the bunkers market, in cement manufacture, or as a liquid fuel for combustion uses in plants with flue gas desulfurization (FGD) or gasification units. The limits of sulfur content of fuel oil for use in most combustion plants without FGD was set to be 1 in 2005, which was then tightened to reach 0.3–0.5%. Although the economic and environmental regulations drove the switch to natural gas, fuel oil sales make up less than 15% of the world petroleum market. This presents a challenge to many refiners, considering the potentially large investments for upgrading the bottom of the barrel.

Heavy gas oil (HGO) is an important fraction of petroleum, as it is an intermediate fraction obtained from vacuum distillation used in the production of diesel and some lubricants. It is a byproduct of oil atmospheric and vacuum distillation that contains a high amount of sulfur-containing organic molecules. Since HGO is used in diesel composition, the application of a BDS process can result in products with low pollutant concentrations and would prevent the poisoning of catalysts (Otsuki et al., 2000; Kilbane and Le Borgne, 2004; Gupta et al., 2016).

There are three major types of transportation fuels, gasoline, diesel, and jet fuels, and there is a growing demand for transportation fuels which are produced from naphtha (BP < 175 °C) and middle distillates (boiling point < 370 °C) (Exxon Mobil 2016). The common types of sulfur compounds in liquid fuels are listed in Table 11.1.

Diesel fuels and domestic heating oils have boiling ranges of bout 400 °-700 °F. The desirable qualities required for distillate fuels include controlled flash and pour points clean burning, no deposit formation in storage tanks, and a proper diesel fuel cetane rating for good starting and combustion. In fractions used to produce diesel oil, most of the sulfur is found in BT, DBT, and their alkylated derivatives (Monticello and Finnerty, 1985). Deep reduction of diesel sulfur (from 500 to < 15ppm sulfur) is dictated largely by 4-methyldibenzothiophene (4-MDBT) and 4,6-dimethydiben-zothiophene (4,6-DMDBT), where the sulfur atoms sterically hindered by substitutions in positions 4 and 6 are the most difficult to remove by HDS; 3,6-DMDBT has been shown to be particularly recalcitrant to HDS (Kabe et al., 1997). The deep HDS problem of diesel streams is exacerbated by inhibiting effects of co-existing problems of polyaromatics and nitrogen compounds in the feed, as well as H₂S in the product (Song and Ma, 2003).

According to the Environmental Protection Agency, the standard sulfur level in gasoline fuel is 15 mg/L (Song, 2003). To meet the stringent emission standard postulated by regulatory organics, the petroleum refining industry should reach towards ultra-low sulfur fuels. Thus, ultra-deep removal of sulfur from transportation fuels are mandatory. Nevertheless, to achieve ULS fuels or the recent “no sulfur” specification, the conventional hydrodesulfurization (HDS) method (Chapter 2) needs higher temperature and pressure and expensive catalysts to remove the recalcitrant high molecular weight polyaromatic sulfur heterocyclic compounds (PASHs), such as dibenzothiophene (DBT) and substituted DBTs. Some other technologies, such as oxidative desulfurization (ODS) (Chapter 2), extractive desulfurization (EDS) (Chapter 2), and adsorptive desulfurization (ADS) (Chapter 2) have been proposed.

However, ADS has some drawbacks that need to be solved. If the selectivity of the adsorbent is low, it is easy to be regenerated, but it will lead to heat loss due to the competitive adsorption. Upon the increase of selectivity, the regeneration of spent adsorbent becomes more difficult. Thus, adsorbents should be well designed to achieve suitable selectivity. Solvent extraction and calcination in the air are the two widely used methods to regenerate desulfurization adsorbents. However, they have also some drawbacks that should be also solved. For the solvent extraction method, it is difficult to separate sulfur compounds from organic solvents and reuse these solvents, while for the calcinations method, sulfur compounds and aromatics are burned out causing fuel heat loss.

For reaching ultra-low sulfur fuels, deep HDS processes need large new capital investments, larger reactor volumes, longer processing times, new expensive catalysts, higher temperatures and pressures, and substantial hydrogen and energy inputs. Such extreme conditions to desulfurize high molecular weight recalcitrant polyaromatic sulfur heterocyclic compounds (PASHs) would lead to the deposition of carbonaceous coke on the catalysts. Sometimes, exposure of crude oil fractions to severe conditions, including temperatures above about 360 °C, decreases the fuel value of the treated product. The large amounts of produced H₂S poisons the catalysts and shortens their useful life. Moreover, deep HDS is affected by components in the reaction mixture such as organic hetero-compounds and polyaromatic hydrocarbons. Furthermore, deep HDS has higher operating costs (Mohebali and Ball, 2008, 2016; El-Gendy and Speight, 2016).

The 4S-pathway that removes sulfur without altering the octane value of fuels has been reported (Malik, 1978), but it did not take much concern because of the high light petroleum reserves, flexibility of environmental rules, and profitability of conventional processes at that time. By the 1990s, due to the depletion of light crude oil reserves, increasing worldwide fuel demand, more restrict environmental emission regulations, and the exploration of new reservoirs of sulfur rich oils (e.g. heavy crude oil, tar sands, and oil shale), the petroleum industry shared efforts with academia to research and develop the applicability of biodesulfurization (BDS) throughout specific oxidative BDS. Recently, biodesulfurization (BDS) catches the wave of the applied desulfurization technologies throughout the selective desulfurization of DBT and its derivative without affecting their hydrocarbon skeleton, keeping the heat value of the treated oil. Contrary to HDS, BDS offers mild processing conditions and reduces the need for hydrogen. Both these features would lead to high energy savings in the refinery. Further, significant reductions in greenhouse gas emissions have also been predicted if BDS is used (Linguist and Pacheco, 1999). However, although BDS has the potential benefits of lower operational cost and production of valuable by-products, it suffers from the slow rate of the desulfurization process. Therefore, there is still a need to increase the rate of BDS. Thus, BDS is recommended to be used as a complementary method to the conventional oil refining technologies.

Kilbane (1989) proposed a hypothetical oxidative desulfurization pathway that, if it ever existed in nature, could specifically remove sulfur from DBT. The pathway was named as the ‘4S-pathway’, which implied consecutive oxidation of DBT sulfur to sulfoxide (DBTO), sulfone (DBTO₂), sulfinate (HPBSi) and/or sulfonate (HPBSo), and, finally, to the oil soluble product 2-HBP or 2,2’-BHBP, respectively, which finds its way back into the petroleum fractions while retaining the fuel value (Figure 11.1). The use of this pathway has been proposed for the desulfurization of petroleum in production fields as well as refineries (Kim et al., 1996; Soleimani et al., 2007).

There is a particular interest for microorganisms capable of carbon-sulfur (C-S) bond-targeted DBT degradation since they do not alter the calorific value of the fuel. There are 4 genes which are involved in complete C-S bond-targeted DBT degradation (McFarland, 1999; Abbad Andaloussi et al., 2003ab; Gray et al., 2003): dszC, which encodes a DBT-monooxygenase, dszA, which encodes a DBT-sulfone monooxygenase, dszB, which encodes a 2-hydroxybiphenyl sulfinate desulfinase, and dszD, which encodes an NADH-flavin mononucleotide oxidoreductase. The proprietary of a BDS process relies on these four genes.

Although Rhodococcus erythropolis IGTS8 is the widely used bacterium in BDS research all over the world and is able to completely desulfurize DBT and some substituted DBTs in model oil system, it is reported to not significantly reduce the sulfur content in crude oil (Kaufman et al., 1999). Generally, work on BDS has focused on model compounds in aqueous systems and, sometimes, in biphasic systems using model solvents, such as tetradecane or hexadecane, little has been reported on the BDS of real refinery feeds. This bears little resemblance to the conditions the biocatalyst would encounter in industrial application and limits the ability to assess the commercial potential of BDS. In fact, the BDS rates of diesel oil were much smaller than those obtained for pure DBT (Rhee et al., 1998). The experimental results indicated that the BDS efficiency would decrease slightly in a diesel oil system compared to a single C_X-DBT system. Folsom et al. (1999) and Okada et al. (2003) reported that BDS of diesel oil was smaller than that obtained in model oil and attributed that to the existence of other organic sulfur compounds (OSCs), for example 4-MDBT, that might be less desulfurizable than DBT. The accumulation of toxic metabolites in the medium may cause a decrease in BDS activity (Chang et al., 2001). According to Abbad-Andaloussib et al. (2003b), this decrease in BDS might be due either to the inhibitory or toxic effects by certain sulfur compounds on the cells. Zhongxuan et al. (2011) and Zhang et al. (2011) attributed this phenomenon to an apparent competitive inhibition of substrates. Moreover, according to Reichmuth et al. (2000) the sulfur requirement for microbial growth is low compared to the level of sulfur in oil feeds, so microorganisms would cease desulfurization before the bulk of the sulfur is removed from the oil feed. Constanti et al. (1996), reported that a Gram-negative Agrobacterium sp. MC501 could oxidize alkanes at the terminal or subterminal methyl group and then continue through the β-oxidation pathway and consume more energy and time to obtain sulfur from sulfur compounds for growth which led to a decrease in their BDS efficiency in a real oil feed.

However, considerable research on the BDS of model OSCs, diesel, gasoline, and crude oil by different microorganisms has been done. BDS of diesel has been demonstrated using both growing and resting cells (Mohebali and Ball, 2008). On the contrary, the BDS of real oil feeds has been reported to be higher than model oils with model compounds such as DBT (Chang et al., 2000a, Dinamarca et al., 2010, 2014) since real oil feeds, such as LGO and HGO, contain various easily desulfurizable compounds, such as thiols and sulfides, besides recalcitrant compounds such as DBT (Chang et al., 2000a). Setti et al. (1992) reported that the presence of n-alkane is seen to favor the removal of sulfur aromatic compounds, which act as a co-substrate, ensuring the growth of the culture, which also permits the solubilization and the emulsifying of the sulfur aromatic compounds. According to Setti et al. (1995), microorganism adsorption to the oil-phase is the most likely mechanism for explaining n-alkane biodegradation. Substrate uptake presumably occurs through diffusion or active transport at the point of contact. It is well known that most of the aerobic microorganisms adhere to the n-alkanes (below n-C₁₆) which are in a liquid form at room temperature, where the n-alkane form a film around the aromatic sulfur compound and, as this film is easily attacked by aerobic microorganisms, the bioavailability of sulfur compounds increases. Studies with a strain of Candida in two-phase systems (oil-water mixture) suggested that the rate of biodegradation might be also related to the interfacial area because a large part of the biomass, which characteristically is hydrophobic, adheres to the non-aqueous phase layer (NAPL)-water interface as a biofilm (Ascón-Cabrera and Lebault, 1995), where the smaller the interfacial tension, the larger the uptake of the dissolved compounds in NAPL and the higher the biodegradation of aliphatic hydrocarbons present. According to Setti et al. (1997), although aerobic microorganisms can remove high amounts of organic sulfur, the sulfur percentage in the residual heavy oil may increase as a consequence of simultaneous aliphatic compound biodegradation.

The difference in desulfurization efficiency within different microorganisms might be related to differences in cell surface hydrophobicity, mass transport through the cell walls and membranes, as well as metabolic regulations inside the cell (Bustos-Jaimes et al., 2003). Thus, the BDS efficiency in real oil feeds depends mainly on the applied microorganisms and its broad versatility on different OSCs and there is no general trend for the efficiency of microorganisms in model or real feed oils.

BDS of petroleum results in total sulfur removals between 30 and 70% for mid-distillates (Grossman et al., 1999; Pacheco et al., 1999), 24 to 92% for hydrotreated diesel (Rhee et al., 1998; Maghsoudi et al., 2001; Ma et al., 2006), 65–70% for partially HDS-treated mid-distillates (Folsom et al., 1999), ≈ 90% for extensively HDS-treated mid-distillates (Grossman et al., 2001), 20–60% for light gas oils (Chang et al., 1998; Ishii et al., 2005), 75–90% for cracked stocks (Pacheco et al., 1999), and 25–60% for crude oils (Mcfarland, 1999; Premuzic and Lin, 1999b). Although the obtained removals are significant, this level of desulfurization is insufficient to meet the required sulfur level for all oil derivatives (Grossman et al., 1999; Alves, 2007).

The majority of the work on BDS was performed for middle distillate fractions. The US DOE announced that research also started on diesel BDS (Le Borgne and Quintero, 2003). These results may serve as a background for the desulfurization of other streams. The treatment of oils using free cells has encountered some limitations, such as high cost of the biocatalyst and low volumetric ratio between the organic phase and the aqueous one. Also, separation of oil product from oil-water-biocatalyst emulsion is troublesome and the free cells are difficult for recycling (Choi et al., 2003; Konishi et al., 2005). In view of industrial application of BDS, cell immobilization is considered to be one of the most promising approaches (Mukhopadhyaya et al., 2006; Huang et al., 2012).

This chapter summarizes the worldwide achievements in biodesulfurization of crude oil and its distillates, at what point it’s reached, and to what point it aims to reach.

11.2 Biodesulfurization of Crude Oil

Oil refineries usually separate crude oil into several fractions and then desulfurize them separately.

Refineries can make substantial cost savings if most of the sulfur is removed from the crude oil before it is fractionated. Also, it has been suggested that due to the high content of water in crude oil, the BDS of crude oil is more practical compared to that of diesel oil and gasoline (Zhou and Zhang, 2004). Nowadays, the petroleum industry is increasingly dependent on heavy crude oil to meet the domestic demand for gasoline and distillate fuels. Heavy oils with high viscosity are expensive to recover, transport, and process and have a lower market value than less viscous oils since asphaltene fraction is a major component of heavy oils. So, to use these as a fuel, they must be upgraded by reducing the average molecular weight of their constituents and remove heteroatoms. For bio-modification of asphaltenes, the reactions with organosulfur moieties could be very significant because sulfur is the third most abundant element in asphaltenes behind carbon and hydrogen and has an important role in the molecular structure of asphaltenes (Sarret et al., 1999). Because the diversity and complexity of asphaltene molecular structures can be attacked, heme proteins were the biocatalysts chosen for investigations on the enzymatic modification of asphaltenes. In a survey of several heme proteins, including horseradish, lignin, manganese chloroperoxidase, and cytochrome c, they were found to be able to modify the greatest number of organosulfur compounds, including sulfur heterocycles and sulfides, and to have superior specific activity (Vazquez-Duhalt et al., 1993).

The high viscosity of many crude oils is a factor contributing to the underutilization of these valuable natural resources. Viscosity greatly complicates and may even defeat the extraction of many types of crude oil from the earth. It remains a concern following extraction, as high viscosity significantly hampers the pumping, transportation, refining, and handling of crude oil. Because of this, the petroleum industry has long recognized the need for a safe, economical, and effective methods for reducing the viscosity of valuable fossil fuel resources.

Under certain circumstances, standard-refining processes such as hydrotreating or hydrodesulfurization (HDS) can favorably affect the viscosity of crude oil during refining. Some reduction in viscosity is also achieved through the breakdown of complex hydrocarbons (e.g. aromatic hydrocarbons) into simpler hydrocarbons of low molecular weight.

A generally accepted and controllable method of modulating crude oil viscosity during refining involves diluting viscous crude oil with low viscosity crude oil refining fractions, usually light-end distillates. Light-end distillates that are used as viscosity lowering diluents are referred to as cutter fractions. Thus, “cutting” it with such a light-end distillate lowers the viscosity of heavy crude oil or bitumen. This technique is useful at some stages of the petroleum refining process, but is not economical for large-scale use or to assist with the extraction of viscous crude oil from the earth.

Microorganisms have been used for the relatively controlled destruction of certain compounds in petroleum with the result that viscosity of the treated product is stabilized. For example, Hitzmann (1962) describes a method for stabilizing the viscosity of jet fuels when stored, as in military installations over seawater. Biocatalytic viscosity reduction uses bacteria to partially transform less valuable crude oil components to surface-active compounds (alcohols and carboxylic acids) that reduce crude oil viscosity is under investigation (Stringfellow, 2001).

A method for reducing the viscosity of viscous petroleum liquids, such as heavy crude oil and bitumen, has been disclosed. The method is appropriate for use with viscous petroleum liquids that contain sulfur-bearing heterocycles, the physicochemical properties of which contribute significantly to the viscosity of the liquid. The method comprises of contacting the viscous petroleum liquid with a biocatalyst that converts sulfur-bearing heterocycles into molecules that make the physicochemical properties conducive to viscosity. The biocatalyst works in a sulfur-specific manner such that the sulfur-bearing heterocycle is altered at the sulfur heteroatom thereof. Via biocatalysis, carbon-sulfur bonds are cleaved and/or polar substituents, such as hydroxyl groups, are joined to the sulfur heteroatom, the hydrocarbon framework of the sulfur-bearing heterocycle, or both. Preferred biocatalysts for viscosity reduction include preparations of Rhodochrous erythropolis ATCC 53968 microorganisms and enzymes obtained there from (Monticello et al., 1996; Monticello, 2000). A need remains for a viscosity reducing treatment that can be used to facilitate the handling of viscous petroleum liquids at any desired stage of the extraction and/or refining processes. A suitable viscosity reducing treatment would not require specialized equipment or safety procedures and would not degrade the caloric (fuel) value of the treated petroleum.

In 1982, the Atlantic Richfield Company patented a biodesulfurization process for crude oils using a strain of Bacillus Sulfasportare ATCC 39909 (Atlantic Richfield Company, 1986). Referring to the biotreatment of vacuum residual oil, sulfur removal ranged from 17–23 wt.% (originally 4.21 wt.% S) and 45 wt.% for Maya crude oil. The removal of organic sulfur from oil was not combined by a loss of carbon content.

In 1987 it was reported that an aerobic process has been developed by the Unocal Corporation. The process was based on hydroxylation of the sulfur heterocycles to water-soluble products by a Pseudomonas strain, which also produced a surfactant, helping oil/water contact. The disadvantages of the process included low efficiency and loss of fuel value (Monticello and Finnerty, 1985).

Treatment of high-sulfur crude oil with the soil isolate Pseudomonas alcaligenes (DBT-2) (Finnerty and Robinson, 1986), which efficiently oxidizes DBT to water-soluble products, resulted in the loss of greater than 70% of organic sulfur from the pentane-soluble fraction of crude oil. Major deficiencies exist in this bioprocess technology. First, DBT-2 does not remove organic sulfur from the heavy fractions (asphaltenes and resins) of the crude oil which contain major concentrations of organic sulfur. Secondly, the biocatalyst causes a significant loss of BTU content in the treated oil. Thirdly, a byproduct is produced of sulfur-containing, water-soluble product for which there is no apparent end use.

Removal of organic sulfur from the crude oil or from its different fractions by employing microorganisms such as Thiobacillus ferroxidans, Thiobacillus thioxidans, Thiobacillus thioparus, and Thiophysovolutans has been attempted, but it is not suitable because catabolic desulfurization of organic molecules mostly involves the utilization of hydrocarbon portions of these molecules as a carbon source, resulting in loss of high calorie petroleum components (Hartdegen et al., 1984; Indian Institute of Petroleum, 1996).

It was reported by Setti et al. (1997) that in an experiment of heavy oil desulfurization in which R. erythropolis IGTS8 was used, non-destructive desulfurization was followed by the complete degradation of aliphatic fraction which consisted 60% of the oil.

All Egyptian crude oils are characterized by being dominated with mixtures of substituted BTs and DBTs. Specifically, Suez Gulf oils show higher abundance of BTs relative to DBTs, as compared to Western Desert crude oils (Hegazi et al., 2003). Salama et al. (2004) reported the BDS of Egyptian crude oil (Balaeem Barry) by Rhodococcus rhodochrous ATCC 53968 (which is later identified as R. erythropolis IGTS8). The optimized parameters affecting the BDS process were found to be: 24 h, 30 °C, pH 7.2, and 1% oil/water, 10 g/L glucose and 3 g/L ammonium chloride that recorded 45.4% total sulfur compound (TSC) removal. The biodesulfurized crude oil indicated a pronounced drop in sulfur content in asphaltenes, while no difference was monitored for saturates, aromatics, and resins. Microbial desulfurization of Balaeem crude oil was associated with a decrease in paraffins and isoparaffins with C-numbers, C13-C18, with a slight degradation of the biomarker phytane, but it was associated with an increase in those having 21–30 carbon atoms. In another study performed on Balaeem crude oil by Egyptian isolate, Bacillus subtilis, the same optimum conditions for maximum BDS-efficiency were found to be the same, but at pH7, recording 38% TSC-removal (Ibrahim et al., 2004). This was accompanied by a pronounced decrease in polynuclear aromatic, phytane, polynuclear naphthene compounds, and asphaltenes, but B. subtilis raised paraffins with higher carbon number (C20–30) and isoparaffins (C21 -30). Both ATCC 53968 and isolated B. subtilis recorded 36.5% and 22.6% reduction in asphaltene content during the BDS process. IGTS8 was also reported to remove up to 45% of DBT through seven days of incubation from Ras Badran asphaltene fraction. Biodesulfurization of aromatic fractions showed that IGTS8 removed up to 63% of sulfur content from that fraction (Moustafa et al., 2006).

Gunam et al. (2006) reported biodesulfurization of the majority of the sulfur (59% w/w) from Liaoning crude oil within 72 hours using Sphingomonas subarctica T7B.

Yu et al. (2006a) reported the BDS of two crude oils by 16 g DCW/L of resting cells of Rhodococccus erythropolis XP in shaken flasks with 1:20 (O/W) and found that the total sulfur content (TSC) of Fushun crude oil was decreased from 0.321 to 0.122 wt.% and that of Sudanese crude oil was decreased from 0.124 to 0.0656 wt.% within 72 h.

The BDS of MFO 380, with adding surfactants, was investigated to explore the potential improvement of BDS efficiency of heavy oil through adding surfactants (Li and Jiang, 2013). According to the hydrophobicity and the solubility of bunker oil, three kinds of surfactants, Tween 20, Triton X-100, and PEG 4000, were used to test the influence of surfactants on the BDS of MFO380. The ratio of surfactant to water was 1:50 (w/w). The ratio of oil phase to aqueous phase was 1:50 (w/w). The biocatalyst was obtained by enrichment with oil sludge as the seed and using DBT (0.5 mM) as the sole sulfur source. The oil sludge was mixed with BSM at a volume ratio of 1:5 at 200 rpm/min and room temperature for 4 days to digest the remaining oil and detach the microbial seed. The results show that the sulfur in MFO 380 has not been utilized during the biotreatment process when received soil or oil sludge was used directly. The possible reason for this is that the specific microorganism for BDS did not exist in the received seed. It is recommended that specific microbial strains should be concentrated by enrichment of a mixed culture system by model compounds first, as this will help in establishing the effectiveness of BDS of heavy oil. On the other hand, the low solubility of MFO 380 in the aqueous phase might be another major limitation to the poor BDS of heavy oil. When MFO 380 was added into the aqueous phase, large particles or irregular balls were formed. A large amount of heavy oil was tightly pasted on the wall of the flasks. Thus, sulfur compounds in heavy oil cannot contact and react with biocatalysts efficiently. Reducing the viscosity of bunker oil and increasing the contact of bunker oil with biocatalysts are the critical steps in the BDS process. The results of bunker oil BDS showed that the removal efficiency of sulfur in MFO380 was only 2.88% after 7 days of incubation, but this could be significantly improved by adding surfactants Triton X-100 or Tween 20. This effect could be attributed to the greatly reduced viscosity of heavy oil and increased mass transfer of sulfur compounds from heavy oil into water. Adding Triton X-100 achieved the highest removal efficiency of sulfur, up to 51.7 % after 7 days of incubation. The optimal amount of Triton X-100 was 0.5 g/50 mL medium.

Chauhan et al. (2015) reported the batch BDS of crude oil (1:5 O/W) using Gordonia sp. IITR 100, where the GC-MS revealed the production of 2-HBP, 1-methyl hydroxy-biphenyl, and BNT-hydroxide, which were possibly formed from DBT, 1-methyl DBT, and BNT.

Studies on the BDS of heavy crude oil are rather limited. The maximum reduction in sulfur content achieved with different microorganisms using heavy crude oil as the sulfur source are in the range of 47–68% (Setti et al., 1992; EL-Gendy et al., 2006; Torkamani et al., 2008 a,b; Agarwal and Sharma, 2010; Bhatia and Sharma, 2010, 2012; Adlakha et al., 2016).

The BDS of heavy crude oils is considered to be an upgrading process, as it not only decreases the S-content, but also decreases the viscosity of heavy oils. A decrease in viscosity is highly desirable as it can make the heavy crude oil more amenable for the refining process. Viscosity in heavy crude oil is largely due to asphaltenes which contain aromatic heterocyclic rings linked with aliphatic C-S bridges. Thus, a bacterium which will target aliphatic CS bonds will reduce the size of the asphaltene suprastructure and, thereby, viscosity (Kirkwood et al., 2005).

Mohamed et al. (2015) reported a 10% reduction in the S-content of heavy crude oil (with initial S-content of 3%) in a batch BDS of 10% O/W, at 30 °C and 250 rpm, within 7 days’ incubation period, using resting cells of Rhodococcus sp. strain SA11 collected in the exponential phase of DBT-cultures. Moreover, total S and S-speciation analyses of the hexane soluble fraction of the heavy crude oil (i.e. the de-asphaltened oil fraction or the maltenes) revealed 18% total S reduction including a wide range of thiophenic compounds, such as DBT, BT, and their alkylated derivatives.

Adlakha et al. (2016) reported a viscosity reduction of 31.28% with 76% BDS of heavy oils in a batch BDS of 1/3 O/W ratio using growing cells of Gordonia sp. IITR100, at 30 °C and 250 rpm, within 7 days’ incubation period. Adlakha et al. (2016) reported approximately 61% BDS of heavy crude oil with an initial S-content of 1.083 wt.% after two successive BDS-rounds, each of 7 days, using Gordonia sp. IITR100.

Li and Jiang (2013) and Jiang et al. (2014) studied biodesulfurization of model thiophenic compounds and heavy oil by mixed cultures enriched from oil sludge, where the BDS of bunker oil by microbial consortium enriched from oil sludge recorded 2.8 % sulfur removal without de-asphalting within 7 d. After de-asphalting, the BDS efficiency was significantly improved (26.2–36.5 %), which is mainly attributed to fully mixing the oil and water, due to the decreased viscosity of bunker oil. Most recently, Martínez et al. (2016) reported enhanced desulfurization of oil sulfur compounds by using engineered synthetic bacterial consortia.

The desulfurization capability of B. subtilis Wb600 on light crude oil was examined by Nezammahalleh (2015). This bacterial species grows on a sulfur-free basal salt medium in the presence of light crude oil with a total sulfur content of 1.5%. B. subtilis Wb600 utilizing the sulfur-containing compounds of the oil as a source of sulfur without degrading the hydrocarbon skeleton. The microorganism produces biosurfactants which facilitates the transfer of the organic sulfur compounds into the aqueous phase. The quantity of biosurfactants produced in the microbial culture is about two times higher than the ones produced in the control culture with an inorganic sulfur source. This bacterial species decreases the total sulfur content of the light crude oil to about 40% during 35 h with an oil/water phase ratio of 0.2.

11.3 Biodesulfurization of Different Oil Distillates

Biodesulfurization has been studied extensively by researchers at Energy BioSystems Corporation (EBC, named later Enchira) for reducing sulfur content in various hydrocarbon fractions.

Energy Bio Systems Corporation was reported to use a five-barrel/day pilot plant to study the BDS of diesel fuel (Rhodes, 1995) by R. erythropolis IGTS8, which was previously known as R. rhodochrous IGTS8, before its final identification by 16S rRNA and physiological studies (Monticello, 1994).

Monticello and Kilbane (1994) investigated the formation of a reversible micro-emulsion between the biocatalytic agent and the petroleum liquid in an attempt to facilitate the recovery of the treated product. Firstly, the experiments were performed on model compounds such as DBT and alkylated DBT. The aqueous catalytic agent Rhodococcus rhodochrous selectively cleaves organic C-S bonds in the presence of the emulsifying agent Triton. Then, Monticello and Kilbane (1994) presented one example showing the enhanced performance of biocatalytic desulfurization of a residual fuel oil in an emulsion. Six runs were conducted using an emulsifying agent in some of the runs to form an appropriate emulsion between the residual fuel oil and the aqueous phase. BDS was found to be enhanced when an appropriate emulsion between the organic (substrate) and aqueous (biocatalyst) phases is formed. It was concluded that the intimateness of contact between the oil and water phases is the rate-limiting factor of the BDS process.

Applications of growing cells of Nocardia sp. CYKS2 in desulfurization of a model oil (10 mM DBT in n-hexadecane), revealed a decrease in S to approximately 1.8 mM within 80 h with a desulfurization rate of 0.279 mg sulfur/L dispersion/h at 1/10 O/W. This was 2.8 times as high as that for a DBT/ethanol system, while in the case of the BDS of diesel oil, the sulfur content decreased from 0.3 to 0.24 and 0.2 wt% within 48 h at 1/10 and 1/20 O/W with desulfurization rates of 0.909 and 0.992 mg sulfur/L dispersion/h, respectively. This was equivalent to 10.0 and 20.8 mg sulfur/L oil/h, respectively. Such results for two different phase ratios implies that the rate of sulfur removal from oil can be increased by decreasing the phase ratio. However, such an advantage is counterbalanced due to an increased reaction dispersion volume requiring almost the same reactor size as for the treatment of a given amount of oil in a given time (Chang et al., 1998).

Paenibacillus sp. strains A11–1 and A11–2 grew well in the presence of LGO (20% O/W) with a recorded decrease in sulfur content from 800 ppm to 720 ppm (Konishi et al., 1997).

Gordona sp. CYKS1, via the S-specific pathway, yielded a 70 and 50% reduction in the S-content of a middle-distillate unit feed (MDUF) and LGO, respectively, in a batch BDS of 190% O/W (Rhee et al., 1998), where the S-content decreased from 0.15% to 0.06% and from 0.3% to 0.25 % after 12 h, respectively. The specific desulfurization rates of MDUF and LGO were 5.3 and 4.7 µM S/g DCW/h, respectively.

The total sulfur in light gas oil which has been hydrodesulfurized decreased from 800 ppm to 310 ppm by resting cells of R. erythropolis KA2–5-1. The ratio of light gas oil was 50% in the reaction mixture (Ohshiro and Izumi, 1999).

Studies were carried out at both a shake flask level, as well as in bioreactors, and most of the studies were performed using Pseudomonas and Rhodococcus. Srivastava (2012) reported that biodesulfurizing bacteria can reduce the S-content of diesel oil from 535 to 75 ppm within only 24 h. McFarland et al. (1998) used resting cells of Rhodococcus sp. for the BDS of diesel in a continuous stirred tank reactor and observed 50–70% reduction in sulfur.

Rhodococcus sp. strain ECRD-1 was evaluated for its ability to desulfurize a 233–343 °C middle-distillate (diesel range) fraction of Oregon basin (OB) crude oil. Up to 30% of the total sulfur in the straight-run middle distillate cut was removed and oxidized another 35% into oil-soluble products. It has been found that ECRD-1 is able to degrade alkanes, but is unable to attack aromatic hydrocarbons (Grossman et al., 1999).

Chang et al. (2000b) reported the decrease of the S-content of nonhydrotreated-middle distillate unit feed from 1500 mg/L to 610 mg/L, with a desulfurization rate of 9.4 mmol/kg DCW/h using resting cells of Gordona strain CYKS1. It was worthy to mention that Gordona sp. CYK1 expressed a higher desulfurization rate in real oil feed than the model oil (12 mmol DBT in n-hexadecane), recording 0.34 and 0.12 mg sulfur/g cell, respectively.

Jiang et al. (2002) found that surfactants could improve the desulfurization rate, where, Pseudomonas delafieldii strain R-8 removed 72% of the organic sulfur from low sulfur diesel oil (S<300 mg/L) within 72 h at 250 r/min with the addition of Tween-80.

Mingfang et al. (2003) reported that the BDS of straight-run diesel oil was treated by resting cells of Nocardia globerula R-9 in a batch process of 1:8 (O/W). The sulfur content of the diesel oil was reduced from 1807 to 741 mg/L after 3 days of reaction. The mean desulfurization rate was found to be 5.1 mmol S/kg DCW/h.

The desulfurization gene cluster from R. erythropolis strain KA2–5-1 was transferred into 22 Rhodococcal and Mycobacterial strains. The resting cells of recombinant strain MR65 from Mycobacterium sp. NCIMB could desulfurize 68 ppm of sulfur in light gas oil (LGO) containing 126 ppm sulfur. This strain expressed about 1.5 times higher LGO desulfurization activity as much as of R. erythropolis strain KA2–5-1. The application of a recombinant was effective in enhancing LGO BDS (Noda et al., 2003a,b). In another study performed by Watanabe et al. (2003b), the dsz gene cluster from R. erythropolis KA2–5-1 was transferred into R. erythropolis MC1109, which was unable to desulfurize LGO. Resting cells of the resultant recombinant strain, named MC0203, decreased the sulfur concentration of LGO from 120 mg/L to 70 mg/L within 2 h. The LGO desulfurization activity of this strain was about twice that of strain KA2–5-1.

For higher achievement of desulfurization value, sequential BDS of real oil feeds has been reported. Ten cultivation rounds each of 3 days’ incubation were done using Mycobacterium phlei WU-0103 to achieve 52% reduction in total sulfur in light gas oil (Ishii et al., 2005).

R. erythropolis XP was adopted to desulfurize fluid catalytic cracking (FCC) and straight-run gasoline (SRG) and the first publication on the BDS of gasoline by free whole cells was conducted. Approximately 29% of the total sulfur of Jilian FCC gasoline (from 495 to 338 ppm) and 32% of that of Qilu FCC gasoline (from 1,200 to 850 ppm) were removed, respectively. About 85% of the sulfur content of SRG (from 50.2 to 7.5 ppm) was also removed (Yu et al., 2006b).

Considering that benzothiophenes predominate in gasoline and the toxicity of gasoline to bacterial cells, the BDS of gasoline is a significant achievement. The alginate immobilization of M. goodii X7B is reported to minimize the toxicity of gasoline to the biocatalyst. Li et al. (2005a) studied the ability of Mycobacterium goodie X7B to desulfurize gasoline in an immobilized-cell system. Cells were immobilized by entrapment with calcium alginate. Cells were harvested in the mid-exponential phase of growth by centrifugation, washed twice with a sodium chloride solution (0.85%), and re-suspended in the same solution containing 0.3% Tween80 and 2% sodium alginate at a concentration of 12.4 mg of dry cells/mL. The mixture was then dropped into a 5% calcium chloride solution containing 0.3% Tween 80 to obtain beads of immobilized cells (about 1.0 mm in diameter). Ten milliliters of gasoline were added for desulfurization and the volumetric phase ratio of the aqueous phase to oil was 9. Dushanzi straight-run gasoline 227 (DSRG227) and DSRG275 were used in the BDS process. The numbers 227 and 275 refer to the concentrations of sulfur in the oil in parts per million. When DSRG227 was treated with immobilized cells of strain X7B for 24 h, the total sulfur content significantly decreased from 227 to 71 ppm at 40 °C, corresponding to a reduction of 69%. In addition, when immobilized cells were incubated at 40 °C with DSRG275, the sulfur content decreased from 275 to 54 ppm in two consecutive reactions corresponding to a reduction of 81%. With this excellent efficiency, strain X7B is considered a good potential candidate for industrial applications for the BDS of gasoline.

A new type of airlift reactor with immobilized Gordonia nitida CYKS1 cells on a fibrous support was designed by Lee et al. (2005) and used for the biocatalytic desulfurization (BDS) of diesel oil. Its performance was evaluated at different phase ratios of the oil to aqueous medium (or oil phase fractions) and different sucrose concentrations. A 7-L jar ferment reactor was modified to fabricate a new type of airlift immobilized-cell reactor as shown in Figure 11.2. A glass draft tube (16 and 9 cm in height and diameter, respectively) was placed inside the reactor (32 and 17 cm in height and inner diameter, respectively). Eight strings (5 g for each) of nylon fiber bunches were installed as the cell carrier. When the growth reached an exponential growth phase, the culture was inoculated into the airlift reactor containing a minimal salt medium (MSM) with 0.3 mM DBT and 10g/L sucrose. Cells were successfully immobilized in the nylon fiber bunches by incubating them in MSM with 0.3 mM DBT and 10 g/L sucrose for 3 days without supplying diesel oil. After immobilizing the cells, the medium was drained completely. The reactor was then filled with a mixture of MSM containing 2.9 g/L sucrose and diesel oil. Total biomass immobilized in nylon fibers measured at end of run was 21.6 g and the immobilized cell density was 0.54 g DCW/ g support. When the reaction mixture contained 10% diesel oil (v/v), 61–67% of sulfur was removed and the sulfur content decreased from 202–250 to 76–90 mg/ L within 72 h. The sulfur content did not decrease any further because the remaining sulfur compounds were recalcitrant to BDS. During desulfurization, strain CYKS1 consumed hydrocarbons in the diesel oil, mainly n-alkanes with 10–26 carbons, as a carbon source even though an easily available carbon source, sucrose, was supplied. The results obtained from the airlift reactor operation provided important information on the future development of BDS processes for deep desulfurization of diesel oils and other fuel oils.

Thus, briefly, Lee et al. (2005) reported 61–67% S-removal from diesel oil accompanied by a loss of C10–C26 carbons using immobilized Gordonia nitida CYKS1 cells in an airlift reactor. Similarly, when using Pseudomonas delafieldii R-8 in a 5 L reactor 47% sulfur removal from diesel was achieved (Guobin et al., 2006).

Yang et al. (2007) reported 12% BDS of diesel with a sulfur content of 12,600 ppm, where most of the sulfur compounds (>80%) in the diesel were thiophene, benzothiophene, DBT, and its derivatives in a two-layer continuous bioreactor (1/4 O/W) using growing cells of Rhodococcus globerulus DAQ3. The total sulfur reduction from 12,600 to 11,100 ppm in 250 mL of non-hydro-treated diesel was performed in a two-layer continuous bioreactor (1:4 O/W), where the organic phase and the cell-containing aqueous phase were kept as two layers and the cells were grown under steady-state conditions in the aqueous phase by carefully controlling the continuous operation. The OSCs diffused from the organic layer into the aqueous phase where BDS occurs in the cells and the produced water-soluble inhibitory compounds from cell metabolism or cell lysis were washed out from the aqueous phase of the bioreactor, consequently, avoiding the accumulation of possible inhibitors.

The total S-content of a middle distillate unit feed (MDUF) decreased from 0.15% (w/w) to 0.06% and that of a light gas oil (LGO) decreased from 0.3% (w/w) to 0.15% by resting cells of Gordonia sp. CYKS1 after 12 h incubation (Li et al., 2009).

Bhatia and Sharma (2010) examined the capability of Pantoea agglomerans D23W3 to desulfurize different oil feeds: light crude oil (0.38% S), heavy crude oil (2.61% S), diesel oil (0.15% S), hydrodesulfurized diesel (0.07% S), and aviation turbine fuel (ATF, 0.41% S) in a batch BDS of 1:9 (O/W) at 30 °C, 200 rpm, and 120 h. The recorded BDS percentage ranged between 26.38 and 71.42%.

Dinamarca et al. (2010) studied the efficiency of adsorption of Pseudomonas stutzeri on silica (Si), alumina (Al), sepiolite (Sep), and titania (Ti) and their influence on the BDS of gas oil (4700 mg/L of sulfur). The most active biocatalysts were P. stutzeri/Si and P. stutzeri/Sep, recording a desulfurization capacity of 2.80 x 10^–13 g sulfur/cell/h and 2.57 x 10^–13 g sulfur/cell/h, respectively, while P. stutzeri/Ti showed the lowest activity, recording 1.03 x 10^–13 g sulfur/cell/h.

Nassar (2010) reported that Bacillus sphaericus HN1 is considered to have a sufficiently broad substrate specificity to degrade major organic sulfur compounds found in diesel oils. The effect of microbial treatment on the sulfur content and on the hydrocarbon skeleton of the total resolvable components (TRP) of diesel oil at different ratios of oil to water (O/W) (1/9, 1/4, 2/3, 1/1, and 3/2) was studied using GC-FPD and GC-FID analysis, respectively. When the oil phase ratio was increased from 1/9 to 1/4 (O/W) phase ratio, the sulfur content was decreased from 9,594 mg/L to 3,393 mg/L (%BDS 64.63%) and to 1,743 mg/L (%BDS 81.83%), respectively. The efficiency of total sulfur removal was decreased to 78%, 20.94%, and 14.93% at a higher o/w phase ratio of 2/3, 1/1, and 3/2, respectively. The biodegradation capacity of DBT from diesel oil was 62.75%, 78.71%, 74.86%, 29.46%, and 0.53% in 1/9, 1/4, 2/3, 1/1, and 3/2 (O/W) ratio cultures, respectively, and the biodegradation of BT was 60.96%, 41.34%, 35.19%, 32.49%, and 24.75% in 1/9, 1/4, 2/3, 1/1, and 3/2 (O/W) ratio cultures, respectively. HN1 showed an excellent ability to remove high alkylated DBTs (4-MDBT and 4,6-DMDBT) from diesel oil. The biodegradation capacity of 4-MDBT was 66.22%, 78.66%, 75.89%, 14.87%, and 11.07% in 1/9, 1/4, 2/3, 1/1, and 3/2 (O/W) ratio cultures, respectively, and the biodegradation of 4,6-DMDBT was 63.07%, 80.85%, 77.50%, 32.11%, and 32.47% in 1/9, 1/4, 2/3, 1/1, and 3/2 (O/W) ratio cultures, respectively. In this study, the percent of degradation of total petroleum hydrocarbons (TPH) was 63.05%, 86.87%, 57%, 53.90%, and 39.24% in 1/9, 1/4, 2/3, 1/1, and 3/2 (O/W) ratio cultures, respectively, indicating that the maximum TPH degradation occurred in 1/4 (O/W) ratio cultures.

Irani et al. (2011) used the growing cells of Gordonia alkanivorans RIPI90A for the BDS of diesel oil in an internal airlift bioreactor. The effect of initial sulfur concentration on growth and sulfur reduction during the growth phase were investigated. All the sulfur compounds in diesel oil (30 mg/L) were lumped into a pseudo-compound and then diluted at different dilution ratios using hexadecane to give final sulfur concentrations. Hydrodynamic characterization and BDS assays were evaluated in an internal airlift bioreactor. A reactor vessel was 0.12 m in diameter and its overall height was 0.7 m. The draft-tube was 0.07 m in internal diameter and 0.35 m tall and was located 0.06 m above the bottom of the tank. The vessel was sparged in the concentric zone through a 0.0006 m diameter sparger. The working volume and the overall volume of the reactor were 5 and 8 L, respectively. A dissolved oxygen electrode and a pH-meter were placed in the bioreactor. Air was supplied to the reactor through a filter and a rotameter. Moreover, the effect of superficial gas velocity (U_g) and working volume (v) on volumetric gas liquid mass transfer coefficient was studied in an airlift bioreactor for the BDS of diesel. Under optimum conditions in the airlift bioreactor, the superficial gas velocity (U_g) and working volume (v) was set at 2.5 L/min and 6.6 l, respectively, and an oil/water phase ratio of 30% and initial sulfur concentration of 28 mg/L, about 50% removal of sulfur from diesel occurred within 30 h incubation using Gordonia alkanivorans RIPI 90A (Irani et al., 2011).

Tang et al. (2013) sonicated a 1 g bunker oil/45 mL BSA solution mixture at different amplitude ratios and times. After 7 d of incubation using a mixed culture from an oil-contaminated soil, approximately 18.4% sulfur reduction in ultrasound pretreated bunker oil compared to 13.8% sulfur reduction in the mechanically stirred and surfactant-supplemented positive control experiment. This experiment proved that the concept of ultrasound pretreatment leads to greater sulfur removal efficiencies in a shorter period of time.

Dinamarca et al. (2104a) evaluated the BDS of gas oil in a bioreactor packed with a catalytic bed of silica containing immobilized Rhodococcus rhodochrous. For immobilization, the cells were circulated through the packed bed in a downward direction at 10 mL/min for 72 h. Bacterial numbers were adjusted by measuring the OD₆₀₀ and normalized by the mass of support (g) in the catalytic bed. The cell numbers adsorbed were in the range of 74–95 OD₆₀₀. To remove the non-adsorbed bacteria on the support, saline solution was circulated through the catalytic bed. 0.623 M DBT in n-hexadecane was used as the only sulfur source and gas oil containing 4.7 g sulfur/L. A glass bioreactor of 125 mL total volume (2 cm diameter, 40 cm length) was constructed. The bioreactor was continuously sparged with air at 70 mL/min in an upward direction. A feed flow rate of sulfured substrates was provided by a peristaltic pump. The temperature inside the bioreactor was maintained at 20 °C by a thermostatically-controlled water bath. The bioreactor was filled with silica particles to evaluate three packed bed heights (5, 10, and 15 cm) for each of two particles size (0.71–1.18 mm or 3.35–5.6 mm). DBT or gas oil was fed continuously into the packed bed bioreactor in a downward direction at 0.45 or 0.9 mL/min. The desulfurized substrate was collected after a 2 h trickling operation. Samples were taken over 1 h at intervals of 20 min to confirm steady-state operation. After each BDS run, the bed was washed with a saline solution and then a fresh bacterial suspension was circulated through the packed bed. Results indicated that the BDS of DBT was dependent on the length of the bed. The conversion at a flow rate of 0.45 mL/min and bed length of 15 cm was 57 %, whereas lengths of 10 and 5 cm gave 41 and 16 %, respectively, at the same flow rate. With a constant flow, it is clear that a greater length of the bed results in a higher retention time, allowing a sufficient contact time for expression of the absorbed bacterial metabolism. Similar to the BDS of DBT, sulfur removal from gas oil was greater using a larger bed length. However, BDS conversion values were lower for gas oil than for DBT in all the studied systems. It is possible that the presence of organic molecules in gas oil affects the bacterial metabolism and, therefore, the BDS process. Desulfurization of DBT was improved when particles of silica in the range of 3.35–5.6 mm were used to absorb cells. Because BDS of DBT with Rhodococcus is a process that depends on the aeration variable (Del Olmo et al. 2005), the results suggest that higher particle size improves aeration of the bed by offering a greater inter-particle volume that can be occupied by both liquid and air. The effect of a large particle size dominates over the higher specific area for cell immobilization offered by a smaller particle size and shows the importance of the meso- and macro-porosity of the catalytic bed in the configuration of packed bed bioreactors for BDS. Immobilized R. rhodochrous cells maintained BDS activity after three cycles, retaining at least 84% of the initial desulfurization conversion.

In another study, Dinamarca et al. (2014b) studied the effect of adding biological surfactants to immobilized biocatalysts formed by adsorption of R. rhodochrous on Si, Al, and Sep and their influence on the BDS activity of gas oil. For the BDS reaction of immobilized cell systems with surfactants, the bacterial cells (4.0 × 10⁹ –1.8 × 10¹⁰) were adsorbed on 1 g of each support (Al, Sep and Si) and were placed in 25-mL flasks containing 10 mL of sulfur-free Medium A and the surfactants (Tween 80 or biosurfactant). One milliliter of DBT or gas oil was added for desulfurization. The reaction was carried out in a 10 mL medium with bacterial cell numbers ranging from 1.62 × 10¹⁰ to 2.14 × 10¹⁰ with a concentration of biosurfactant and Tween 80 in a range of 0.1–0.6% w/v and 1 mL of gas oil (4700 mg/L of sulfur). The reaction was carried out at 30 °C in a rotary shaker at 200 rpm for 24 h. A greater effect can be noted on the BDS activity in non-immobilized cells with surfactants than in the immobilized cells, essentially in the Rhodochrous/Si and Rhodochrous/Al systems. In the case of Rhodochrous/Sep systems, the greater interaction between bacterial cells and the support optimized the effect of immobilization. The addition of biosurfactant to the immobilized systems increased the desulfurization of the gas oil in the three supports. However, this increase was at its maximum with the Rhodochrous/Sep system. The optimal interaction between bacterial cells and Sep and the solubilization of gas oil by the presence of the biosurfactant in the reaction medium explained that behavior. Adding Tween 80 to the immobilized systems increased the BDS of gas oil in the three supports. However, the increase, in the case of the Rhodochrous/Sep system, was less than when biosurfactant was added. This effect demonstrated that, in the case of gas oil samples, the formation of micelles is more critical than increasing particle mobility for further solubilization.

Arabian et al. (2014) reported the BDS of kerosene with an initial S-content of 2333 ppmw by Bacillus cereus HN, at an oil phase fraction of 0.2, using an inoculum size of 3.6×10⁷ (cell/mL), 180 rpm at 40 °C. The total S-content decreased to 1557 ppm (≈ 33% BDS) within 72 h.

Maass et al. (2014) evaluated the effectiveness of the strain R. erythropolis ATCC 4277 for the desulfurization of a synthetic model oil for diesel (3 mM DBT in n-dodecane) in a batch reactor with different O/W phase ratios of 20, 80, and 100% (v/v). ATCC 4277 was able to degrade 93.3, 98.0, and 95.5 % of the DBT at 20, 80, and 100 % (v/v), respectively. The greatest percentage of DBT desulfurization (98%) and the highest specific rate of 2-HBP production (44 mM DBT/kg DCW/h) were obtained in a batch reactor using 80% (v/v).

Maass et al. (2015) reported the biodesulfurization and biodenitrogenation of heavy gas oil (62.5 µg/g S and 37.6 µg/g N) by Rhodococcus erythropolis ATCC 4277, in a batch reactor with different initial concentrations of HGO at 28 °C and 200 rpm, within 18 h. The best results were achieved for HGO ratios of 40, 20, and 60% (v/v), where the desulfurization rate of 148, 137, and 123 mg S/kg/h with sulfur percentage removal of 42.7, 39.4, and 35.5% and nitrogen percentage removal of 43.2, 40.2, and 31.2%, were recorded, respectively. Maass et al. (2015) examined the culture media of 40 and 100% (O/W) by optical microscopy in an attempt to presumably understand why 40% gave the best result and why it was completely inhibited in 100% (O/W). In a 40% culture, the presence of a considerable amount of refringent cells was observed, which is an indication that the cells of R. erythropolis ATCC 4277 were able to adapt well to the reaction medium containing 40% (v/v). Moreover, cells were well dispersed in the medium which is an indication that the medium provides favorable conditions for the microorganism. A good adaptation of the cells to this particular reaction medium was also observed in the result of minimum inhibitory concentration MIC-test by the macro-dilution method, while the cells were aggregated and there was no evidence of refringent cells in the 100% (O/W) culture. This was attributed to the nutritional deprivation growth condition which increases the cell surface hydrophobicity and acts as an ignition power for a cell–cell junction or biogranulation. Adhesion occurs when the electrostatic repulsion is overcome by van der Waals forces and hydrophobic interactions (Carvalho et al., 2009). Furthermore, the cells form an aggregate to protect cell population against the toxicity of hydrophilic compounds since this microorganism resistance decreases with the increase of the system toxicity, restraining the cells to maintain their viability under severe conditions (Carvalho et al., 2004, 2005, 2009; Carvalho and Da Fonseca, 2005; Heipieper et al., 2007). This, consequently, explained the recorded decrease in the BDS and BDN ability of R. erythropolis ATCC 4277, at a higher O/W phase ratio.

Nassar (2015) improved the BDS of diesel oil by using Fe₃O₄ MNPs coated cells, magnetically immobilized cells (alginate + Fe₃O₄ MNPs), and agar immobilized cells of Brevibacillus invocatus C19 and Rhodococcus erythropolis IGST8. The effect of microbial treatment on the BDS of the diesel oil at different ratios of oil to water (O/W) (10%, 25%, 50%, and 75% v/v) was studied using GC-FID and GC-FPD analysis. GC-FPD analysis was used to qualitatively and quantitatively evaluate the effects of free, coated, magnetically immobilized, and agar immobilized cells of C19 and IGTS8 on the sulfur contents of diesel oil as a model of real oil feed with an initial sulfur content of 8,600 ppm. The Fe₃O₄ MNPs coated cells of R. erythropolis IGTS8 showed good BDS efficiency (%BDS 78.26 %) in the case of a 10% (v/v) O/W phase ratio than that which occurred by free cells (%BDS 74.42%), magnetic immobilized cells (%BDS 57.26%), and agar immobilized cells (%BDS 58.98%). The desulfurization capability of DBT and BT by coated IGTS8 were 58.63% and 32.98%, respectively, at an optimum O/W phase ratio (10% v/v). The agar immobilized cells and coated cells of Brevibacillus invocatus C19 showed the highest BDS efficiency (%BDS 98.97% and 98.69%, respectively) at 25% (v/v) O/W phase ratio, compared to free cells (%BDS 91.31%) and magnetic immobilized cells (%BDS 89.99%). Thus, 10% and 25% (v/v) phase ratios were considered as the optimum O/W phase ratios of R. erythropolis IGTS8 and Brevibacillus invocatus C19, respectively. From a GC-FPD chromatogram (Figure 11.3), the BDS of DBT from diesel oil by agar immobilized C19 and coated C19 was 93.71% and 92.86%, respectively, and the BDS of BT was 91.42% and 88.76%, respectively, at 25% v/v optimum O/W phase ratio, while the BDS of DBT and BT by coated IGTS8 were 58.63% and 32.98%, respectively, at an optimum 10% v/v O/W phase ratio. The coated cells of IGTS8 showed a low capability to desulfurize 4-MDBT (%BDS 25.34%) and 4,6-DMDBT (%BDS 41.56%) from diesel oil compared to coated cells of C19 (%BDS 83.04%) and agar immobilized cells of C19 (%BDS 87.16%), as shown in Figure 11.3. The effect of microbial treatment on the hydrocarbon skeleton of the total resolvable components (TRP) of the diesel oil at different ratios of oil to water was also studied using GC-FID analysis.

The biodegradation capacity of total petroleum hydrocarbons (TPH) in diesel oil by agar immobilized C19 and coated C19 was 10.05% and 11.73% at an optimum O/W phase ratio (25% v/v), respectively, while the percent of degradation of TPH that occurred by coated IGTS8 was 34.98% at an optimum O/W phase ratio (25% v/v).

Adlakha et al. (2016) reported a 70% reduction in the initial S-content (50 ppm) of commercial diesel oil in a 5 L reactor using growing cells of Gordonia sp. IITR 100.

Shahaby and Essam El-din (2017) reported the isolation of Pseudomonas putida TU-S2, Bacillus pumilus TU-S5, and Rhodococcus erythropolis TU-S7 from petroleum hydrocarbons polluted soil for their ability to selectively desulfurize DBT to 2-HBP, where the BDS of different oil feeds (200 g/L) at 35 °C recorded 31, 21.1, 33.2, 21.2, and 21.6 for crude oil, diesel oil, kerosene, gasoline, and motor oil, respectively, using P. putida TU-S2, while 25, 19.3, 31, 25, and 29.0, respectively, were recorded using B. pumilus TU-S5 and, finally, recorded 26.1, 20.1, 29, 27, and 29%, respectively, using R. erythropolis TU-S7 within 24 h. Moreover, using a consortium of the three strains reported 90% within 72 h.

Fatahi and Sadeghi (2017) reported that the bio-modification of PVA by R. erythropolis produced biocatalysts with an active metabolism that facilitates the interaction of the bacterial strain with hydrophobic gasoline thiophenic compounds. According to GC-MS, thiophene and its 2-methyl, 3-methyl, and 2- ethyl derivatives had acceptable BDS efficiencies of about 26.67, 21.03, and 23.62%, respectively. Also, benzothiophene that has been detected in a gasoline sample had 38.89% BDS efficiency. The recovery of biocatalysts has been investigated and after three times of using in BDS activity, its biocatalytic ability had no significant decrement.

The BDS/BDN capability of R. erythropolis 4277 cells on different concentrations of HGO (10–90% w/w) was determined by using the best culture conditions: 6.15 g/L of yeast extract, 2.0 g/L of glucose, 5.0 g/L of malt extract, and 1.16 g/L of CaCO₃ at 23.7 °C and 180 rpm for 30 h (Todescato et al., 2017). The initial S and N contents of the used HGO were 6944 and 6549 µg/L, respectively. However, the BDN efficiency was 10 times higher than the BDS efficiency, where the presence of nitrogen in the yeast extract allowed the R. erythropolis ATCC 4277 cells to be more adapted to the nitrogen consume than to the sulfur. The presence of high amounts of nitrogen sources can be prejudice to BDS capability, as described by Porto et al. (2017), possibly because the high concentrations of nitrogen inhibit the formation of oxidoreductase and desulfinase enzymes. Such enzymes are extremely important in the sulfur degrading pathway of recalcitrant heterocyclic compounds (Monticello, 2000). Thus, BDS capability is not associated with optimal growth conditions, however the BDN was quite expressive, where 47% of nitrogen removal was achieved in the assay containing 10% (w/w) of HGO (Todescato et al., 2017).

11.4 BDS of Crude Oil and its Distillates by Thermophilic Microorganisms

Thermotolerant microorganisms are recommended for the BDS of real oil feed, as they will decrease the cost of cooling to ambient temperature if BDS is set down-stream to an HDS unit.

The facultative thermophilic bacterium, Mycobacterium goodie X7B, was reported to decrease the total sulfur content (TSC) of Liaoning Crude oil (1:9 O/W phase ratio) from 3,600 to 1,478 mg/L, within 72 h at 40 °C (Li et al., 2007a). In another study, the use of the thermophile Klebsiella sp. 13T resulted in 68.08% and 62.43% sulfur removal from heavy and light crude oil, respectively (Agarwal and Sharma, 2010).

Resting cells of Mycobacterium strain G3 were reported to desulfurize diesel oil form 116 ppm to 48 ppm within 24 h at 37 °C. When the desulfurization was carried out repeatedly using fresh cells, the sulfur concentration of diesel oil decreased to 44 mg/L (Okada et al., 2002). Upon comparing the initial desulfurization rate of diesel oil (i.e. with the first 2 h) with that of model oil (100 mM DBT in tetratdecane), it recorded 25 and 151 µmol S/g cell/h, respectively. This was attributed to the presence of various kinds of alkyl-DBTs that are assumed to comprise the major sulfur fraction in diesel oil and the desulfurization activity was low due to competitive inhibition.

The total sulfur content of diesel oil was reduced by 86%, using a facultative thermophilic bacterium, Mycobacterium sp. X7B, for 24 h at 45 °C (Li et al., 2003).

The thermophilic DBT-desulfurizing bacterium Mycobacterium pheli WU-F1 grew in a medium with three hydrodesulfurized light gas oils (LGOs) of different sulfur content (390ppm sulfur, 120 ppm sulfur, or 34 ppm sulfur) as a sole source of sulfur at 45 °C. BDS resulted in around a 60–70% reduction of sulfur content for all the three types of hydrodesulfurized LGOs. In addition, when resting cells incubated at 45 °C with hydrodesulfurized LGOs in the reaction mixtures containing 50% (v/v), BDS reduced the sulfur content from 390 to 100 ppm, from 120 to 42 ppm, and from 34 to 15 ppm. Analysis with GC/AED revealed that the peaks of alkylated DBTs, including 4-MDBT and 4,6-DMDBT, significantly decreased after BDS (Furuya et al., 2003).

Ishii et al. (2005) reported that with growing cells of Mycobacterium phlei WU-0103, total sulfur content in 12-fold-diluted straight-run LGO was reduced from 1000 to 475 ppm sulfur at 45 °C.

Li et al. (2005a) reported thermophilic and hydrocarbon tolerant Mycobacterium goodii X7B, which had been primarily isolated as a bacterial strain capable of desulfurizing dibenzothiophene to produce 2-hydroxybiphenyl via the 4S pathway, was also found to desulfurize benzothiophene to o-hydroxystyrene at 40 °C. This strain appeared to have the ability to remove organic sulfur from a broad range of sulfur species in gasoline at 40 °C and a 1/9 O/W phase ratio, where desulfurization of gasoline from 227 ppm to 71 ppm sulfur was achieved. The vast majority of biodesulfurization studies target diesel rather than gasoline, so this demonstration of gasoline desulfurization is important. Benzothiophenes are the most abundant organosulfur compounds in gasoline, whereas DBTs predominate in diesel. Gasoline is also considerably more toxic to bacteria than diesel. Thus, that achievement was a great addition to the BDS field.

Bhatia and Sharma (2012) assessed the ability of the thermophilic Klebsiella sp. 13T to desulfurize light (0.35% S) and heavy (2.63%) crude oil along with HDS-diesel (0.05% S) and diesel oil (0.18% S) in a batch BDS process of 10% (O/W) at 45 °C and 120 h. The recorded desulfurization percentage for the studied oil feed ranged between ≈ 22 and 53% in the following increasing order: HDS-diesel < diesel < heavy crude oil < light crude oil.

11.5 Application of Yeast and Fungi in BDS of Real Oil Feed

Few reports have been published about the application of yeast and fungi in the BDS of crude oil and its distillates

Bladi et al. (2003) reported the isolation of a yeast strain Rhodosporidium toruloides DBVPG 6662 that can grow on a variety of sulfur compounds. When this strain was grown on glucose in the presence of commercial emulsion of bitumen (Orimulsion: a bitumen amended with an emulsifying agent and water), 68% of the benzothiophene derivatives and dibenzothiophene derivatives were removed after 15 days of incubation.

El-Gendy et al. (2006) reported a biodesulfurization of crude oil by the halotolerant yeast, Candida parapsilosis NSh45, isolated from Egyptian hydrocarbon polluted sea water, in a batch process of 1/3 O/W phase ratio in 7 days at 30 °C with a mixing rate of 200 rpm. The NSh45 reduced the sulfur content of Belayim Mix crude oil (2.74 wt% sulfur) by 75%, with a decrease in the average molecular weight of asphaltenes by approximately 28% and dynamic viscosity by approximately 70%, compared to that of R. erythropolis IGTS8 which expressed total sulfur removal of approximately 64% and a decrease in average molecular weight of asphaltenes and crude dynamic viscosity of approximately 24% and 64%, respectively, under the same conditions (El-Gendy et al., 2006).

Torkamani et al. (2009) reported the isolation of the native fungus which has been identified as Stachybotrys sp. and is able to remove sulfur and nitrogen from heavy crude oil selectively at 30 °C. This fungus is able to desulfurize 76% and 64.8% of the sulfur content of heavy crude oil of the Soroush oil field and Kuhemond oil field in Iran (with the initial sulfur contents of 5 wt % and 7.6 wt %, respectively) in 72 and 144 h, respectively. This fungus strain has been isolated as a part of the heavy crude oil biodesulfurization project initiated by the Petroleum Engineering Development Company (PEDEC), a subsidiary of National Iranian Oil Company.

Adegunlola et al. (2010) investigated the ability of the immobilized spores of Aspergillus flavus to remove sulfur from crude oil. The spores of A. flavus were immobilized by mixing 80 g of the harvested spores in 1% (w/v) sterile alginate solution and then gelled into beads by dropping the suspension into a cold 15 g/L CaCl₂ solution. When 50 g of immobilized spores of A. flavus was added to crude oil for one, two, three, and seven days, the amounts of sulfur removed were 27.2%, 45.2%, 90.4%, and 91.7%, respectively. When 50 g of immobilized spores of A. flavus were introduced into the crude oil for 7 d at 35 °C, 40 °C, and 45 °C, the amount of sulfur removed was 63.2%, 55.3%, and 10.5%, respectively. Lastly, when 10 g, 50 g, and 100 g of immobilized spores of A. flavus were added to crude oil for 7 d, the amount of sulfur removed was 49.6%, 94.7%, and 53.9%, respectively.

11.6 Biocatalytic Oxidation

Microbial desulfurization of petroleum derivatives has two main problems: microbial activity is carried out in aqueous phase, thus, a two phase system reactor with intrinsic mass transfer limitations would be needed to metabolize the hydrophobic substrate and the microbial biocatalyst must have a broad substrate specificity for the various organosulfur compounds present in oil. These obstacles can be overcome by the utilization of broad specificity enzymes instead of whole microorganisms. An enzymatic oxidation process for the two-step desulfurization of fossil fuels involves (i) oxidation of diesel oil by chloroperoxidase, lignin peroxidase, manganese peroxidase, or cytochrome c, producing sulfoxide and sulfone, with a higher boiling point, leaving the majority of the hydrocarbons in their original form and (ii) distillation to remove the oxidized organosulfur compounds; other physicochemical processes can be used for the separation of the oxidized organosulfur compounds from the main hydrocarbon mixture, such as column chromatography, precipitation, and complexation with a solid support (Vazquez-Duhalt et al., 2002).

Enzymes are able to perform catalytic reactions in organic solvents (Dordick, 1989) with a reduction in mass transfer limitations. The solvent is the fuel itself. Enzymes can be applied under anhydrous conditions or at very low water activity, enzymes are generally more thermostable, and reactions could be performed at temperatures as high as 100 °C (Mozhaev et al., 1991).

Biocatalytic modification of complex mixtures from petroleum, such as asphaltenes, have been performed in organic solvents (Fedorak et al., 1993). Several enzymes have been reported for the oxidation of thiophenes and organosulfur compounds in vitro, including cytochromes P450 (Nastainzcyk et al., 1975; Fukushima et al., 1978; Takata et al., 1983; Mansuy et al., 1991; Alvarez and Ortiz de Montellano, 1992), lignin peroxidase from the white rot fungus Phanerochaete chrysosporium (Vazquez-Duhalt et al., 1994), lactoperoxidase (Doerge, 1986; Doerge et al., 1991), chloroperoxidase from Caldariomyces fumago (Alvarez et al., 1992; Kobayashi et al., 1986; Pasta et al., 1994), and horseradish peroxidase (Alvarez et al., 1992; Doerge, 1986; Doerge et al., 1991; Kobayashi et al., 1986). Non-enzymatic hemoproteins are also able to perform DBT oxidation in vitro, such as hemoglobin (Alvarez et al., 1992; Klyachko and Klibanov, 1992; Ortiz-Leon et al., 1995) and cytochrome c (Klyachko and Klibanov, 1992; Vazquez-Duhalt et al., 1993a,b). All the proteins mentioned above are hemoproteins and, in all cases, the products of the biocatalytic oxidations are the respective sulfoxides and/or sulfones.

Ayala et al. (1998) reported a biocatalytic oxidation of fuel oil as an alternative to biodesulfurization. The method includes a biocatalytic oxidation of organosulfides and thiophenes with hemoproteins to form sulfoxides and sulfones, followed by a distillation step in which these oxidized compounds are removed from the fuel. The reactions were successfully carried out in aqueous mixtures of diesel fuel (1.6 wt.%S) at room temperature that were biocatalytically oxidized with chloroperoxidase from Caldariomyces fumago in the presence of 0.25 mM hydrogen peroxide. The organosulfur compounds (OSCs) were effectively transformed to their respective sulfoxides and sulfones which were then removed by distillation at 50 °C. The resulting fraction after distillation contained only 0.27% sulfur and 71% of the total hydrocarbons were retained. Thus, a biocatalytic treatment of primary diesel fuel with chloroperoxidase from C. fumago, followed by a distillation, is able to reduce the sulfur content by 80%.

A simple and effective biochemical method for the desulfurization of bitumen has also been reported by Valentine (1999). A biological agent was used to remove oxidizable sulfur compounds from an emulsion of water and bitumen. Sulfur compounds were oxidized to water-soluble sulfates which could be physically or chemically removed, thus eliminating the SO_x production during combustion.

Vazquez-Duhalt et al. (2002) reported that oxidation using chloroperoxidase (CPO) in the presence of H₂O₂, followed by distillation at 50 °C, decreased the sulfur content of straight run diesel fuel from 1.6 to 0.27%, keeping 71% of the original hydrocarbons. A simple estimate of the cost of this technology has been reported; CPO showed a turnover number (i.e. number of substrate molecules that can be converted per molecule of enzyme before inactivation) of 500,000. Thus, 1 g of enzyme could reduce the sulfur content from 500 to 30 ppm of 0.81 tons of fuel (Ayala et al., 2007). Immobilized cytochrome c in poly(ethylene)glycol (PEG-Cyt) has been used to oxidize high sulfur content diesel oil (Zeynalov and Nagiev, 2015). In addition, the use of lipase NOVOZYM™ LC in the presence of H₂O₂ and carboxylic acid in absence of water or any co-factor, followed by furfural extraction of produced sulfoxide and sulfone, has been reported (Singh et al., 2009). The bio-catalyst is active up to 70 °C and preferably in the temperature range from 35 to 60 °C. Three types of diesel oil with different sulfur contents (6400, 1000 and 500 ppm) were used. The sulfur content decreased to 2300, 115, and 29 ppm, respectively.

11.7 Anaerobic BDS of Real Oil Feed

In 1961, ESSO Research and Engineering Co. patented an anaerobic process using Thiobacillus strain (ESSO Research and Engineering Company, 1961). Microbial conversion of organic sulfur in a crude petroleum slurry (10–20% water with added surfactant) was described. More than 90% removal of sulfur within a contact time of 10 min was claimed. VEB Petrolchemisches Kombinat Schwedt, in former East Germany, investigated an anaerobic route to crude oil desulfurization in 1974 (Eckart et al., 1980, 1982; Eckart, 1981). Uncharacterized mixed cultures obtained from oil-polluted soils and sludge were contacted with various oils in a 4-dm³ batch fermenter. Removal of sulfur was achieved, but at the expense of a considerable loss of hydrocarbon, mainly through metabolism of n-alkanes. In 1997, VEB Petrochemisches Kombinat Schwedt patented an anaerobic fermentation process. A 40% reduction in the organic sulfur content of Romaschkino crude oil was claimed after a 2–3 d anaerobic incubation. The sulfur was assumed to be released as H₂S and mercaptans were detected in the waste gases. Some 4–6% of the hydrocarbons were lost, probably through volatilization. Other problems included foaming, increasing viscosity of the crude oil through loss of light molecular weight components, and marked decrease in the activity of the biocatalyst after several days due to the mercaptans dissolved in the aqueous phase, rendering this process non-practical (Kohler et al., 1984).

Eckart et al. (1986) examined the anaerobic biodesulfurization of a Romashkino crude oil containing 1.8% sulfur. Mixed cultures grown with lactate and sulfate were employed in these experiments. Desulfovibrio spp. were predominant members of the microbial community. When the pH was controlled to maintain circumneutral conditions, desulfurization of 26 to 40% was noted in a few days.

Pifferi et al. (1990) performed a continuous anaerobic microbial desulfurization of crude oil, petroleum fractions, and petroleum products using sulfate reducing microorganisms in the presence of H₂ at ambient temperature and atmospheric pressure. The culture medium which was previously isolated from industrial effluents showed the presence of ferrous ions that gave considerable advantages since they enhanced the formation of a colloidal iron sulfide precipitate in the reaction mixture. The desulfurization tests of fuel oils with different sulfur content were conducted in a bench scale laboratory unit with a reactor capacity of 10 L. Desulfurization yield ranged from 25% to 90% for a fuel oil with an S-content 3 wt.%. The SO₄^2- ion was kept between 0.3–1.6 g/L. The results showed the effectiveness of the new culture in the desulfurization of the substrate with simultaneous controlled demolition of high molecular weight structure.

Kim et al. (1990b) reported that Desulfovibrio desulfuricans M6 removed up to 21% of the 3 wt.% sulfur in Kuwait crude oil within 6 days and M6 also reported up to 17 wt.% from other crude oils and their distillate products (Kim et al., 1995).

Some anaerobic microorganisms, such as Desulfomicrobium scambium and Desulfovibrio longreachii, have been reported to have the ability to desulfurize only about 10% of DBT dissolved in kerosene. The GC analyses of samples showed unknown metabolites, indicating that the bacteria had possibly followed a pathway different from common anaerobic pathways (Yamada et al., 2001).

Aribike et al. (2008) reported that Desulfobacterium indolicum isolated from oil contaminated soil exhibited very high desulfurizing ability towards kerosene at 30 °C in a 1/9 O/w phase ratio, resulting in reduction of sulfur from 48.68 ppm to 13.76 ppm over a period of 72 h with a significant decrease in thiophene and 2, 5-dimethyl thiophene. The GC/PFPD analysis revealed that kerosene contained 6.955 mg/L of thiophene and 41.724 mg/L of 2, 5-dimethyl thiophene and no benzothiophene or dibenzothiophene is detected in kerosene. The BDS-rate of Th was higher than that of 2, 5-dimethyl thiophene. At the end of 72 hours, 84% of thiophene has been desulfurized, while 70% of 2, 5–dimethyl thiophene was desulfurized. That was explained by the presence of the methyl substituents at positions 2 and 5 which would constitute a steric hindrance to the organism from reaching the sulfur atom in the thiophene ring.

Aribike et al. (2009) reported that Desulfobacterium anilini isolated from petroleum products-polluted soil showed a significant decrease of benzothiophene and dibenzothiophene in diesel with 82% removal of total sulfur after 72 h at 30 °C with a 1/9 O/W phase ratio.

Agarwal and Sharma (2010) reported the 63.29% and 61.40% biodesulfurization of two crude oil samples: heavy and light crude oils with sulfur contents of 1.88% and 0.378%, respectively, by Pantoea agglomerans D23W3. However, the use of P. agglomerans D23W3 under anaerobic conditions showed marginally better results than those under aerobic conditions.

Srivastava (2012) mentioned that some anaerobic microorganisms, such as Desulfomicrobium scambium and Desulfovibrio longreachii, have the ability to desulfurize about 10% of DBT dissolved in kerosene. Kareem et al. (2012) reported 81.5% anaerobic biodesulfurization of diesel oil with an initial sulfur concentration of 166.034 ppm using an isolated bacterial strain, Desulfobacterium indolicum within 72 h. The strict anaerobe, Gram negative Desulfatiglans aniline comb. nov., isolated from petroleum products contaminated soil, is reported for its efficient BDS capacity on kerosene obtained from a retail outlet in Lagos, Nigeria, as it reduced its S-content from 48.68 ppm to 12.32 ppm (representing 75% BDS efficiency) in a batch BDS process of 1:10 (O/W), at 180 rpm, pH7, over a period of 72 h at 30 °C. The gas chromatography analysis with a pulse flow photometric detector (GC/PFPD) analysis revealed that the peaks of thiophene and 2, 5–dimethyl thiophene were significantly decreased after biodesulfurization (Kareem et al., 2016).

The desulfurization of petroleum under anaerobic conditions would be attractive because it avoids costs associated with aeration, it has the advantage of liberating sulfur as a gas, and does not liberate sulfate as a byproduct that must be disposed by some appropriate treatment (Ohshiro and Izumi, 1999). However, due to low reaction rates, safety and cost concerns, and the lack of identification of specific enzymes and genes responsible for anaerobic desulfurization, anaerobic microorganisms effective enough for practical petroleum desulfurization have not been found yet, and an anaerobic BDS process has not been developed. Consequently, aerobic BDS has been the focus of the majority of research in BDS (Le Borgne and Quintero, 2003).

11.8 Deep Desulfurization of Fuel Streams by Integrating Microbial with Non-Microbial Methods

11.8.1 BDS as a Complement to HDS

One of the main drawbacks of HDS is that it is not equally effective in desulfurizing all classes of sulfur compounds present in fossil fuels (Chapter 2). The BDS process, on the other hand, has broad versatility on different organosulfur compounds (OSCs) (Pacheco, 1999). However, one of the main advantages of HDS (Chapter 2) is that its conditions not only desulfurize sensitive (labile) OSCs, but also (i) remove nitrogen and metals from organic compounds, (ii) induce saturation of at least some carbon–carbon double bonds, (iii) remove substances having an unpleasant smell or color, (iv) clarify the product by drying it, and (v) improve the cracking characteristics of the material (El-Gendy and Speight, 2016).

Therefore, with respect to these advantages, placing the BDS unit downstream of an HDS unit as a complementary technology to achieve ultra-deep desulfurization, rather than as a replacement, should also be considered.

Although some researchers are focusing on implementing the BDS process on a large scale, the BDS rates are still low when compared to HDS. This is due to the limitations that are faced in such processes. The main limitations include the need to enhance the thermal stability of desulfurization, the limited transport of the sulfur compounds from the oil to the membrane of the bacterial cell, and the limited ability to recover the biocatalyst (Kilbane, 2006). Most BDS processes are now focusing on using it as complementary steps for deep desulfurization, where the BDS is integrated with existing HDS units. BDS can be used either before or after the HDS unit. Kleshchev (1989) invented the HDS-BDS process, where HDS of crude oil is carried out as a first step to remove labile organic sulfides, then BDS of recalcitrant sulfur compounds, for example DBT, is performed by Rhodococcus rhodochrous (Figure 11.4a). Other researchers believe that employing BDS before HDS (Figure 11.4b) is more efficient for removing a major part of the hydro-treating resistant compounds. This will result in less hydrogen consumption in the HDS unit (Monticello, 2000).

Monti cello (1996) suggested a multistage process for desulfurization of fossil fuels. This method was based on subjecting vacuum gas oil to HDS prior to BDS in defined conditions. Pacheco (1999) reported that the Energy BioSystems Corporation (EBC) used BDS downstream of HDS. Fang et al. (2006) also showed that combination of HDS and BDS could reduce the sulfur content of catalytic diesel oil from 3358 to <20 mg/g. Thus, with respect to the advantages of both HDS and BDS, integrating BDS with HDS, for example, by placing a BDS unit downstream of an HDS unit as a complementary technology to achieve ultra-deep desulfurization, rather than as a replacement, is very promising and should be considered.

Rhodococcus erythropolis I-19, a genetically engineered bacterium and product of Energy Biosystem Corp, USA, was reported to reduce the S-content of hydrodesulfurized middle distillate from 1850 to 615 µg/mL, i.e. 67% BDS% (Folsom et al., 1999), where the initial desulfurization rate of the oil fraction was 2.5 µM/g DCW/min based on the change in total sulfur content in the oil phase.

Grossman et al. (2001) cultivated Rhodococcus sp. strain ECRD-1 in a medium with a hydrocarbon/water ratio of 1:4. The hydrocarbon phase was hydro-treated middle distillate oil with a total sulfur content of 669 ppm. The oil had only 5% of DBT and the majority of the remaining 95% was alkylated DBTs. The final sulfur concentration was detected to be 56 ppm after 7 d of cultivation in a rotary shaker at 25 °C, pH 7 (controlled), and 200 rpm.

The ability of Rhodococcus sp. P32C1 for desulfurization of n-hexadecane (n-C₁₆) containing DBT (model oil) was first studied and based on the optimum conditions determined from the BDS of model oil and its capacity for the BDS of real oil feed was studied. Two kinds of diesel oil, hydrodesulfurized diesel oil (HDS-diesel) and another diesel sample, with sulfur contents of 303 and 1000 ppm were used for BDS with Rhodococcus sp P32C1 in a batch system of 25% (O/W). About 48.5 and 23.7% of sulfur was removed in 24 h with desulfurization rates of about 0.6 and 1.0 mmol S/kg DCW/h, respectively (Maghsoudi et al., 2001).

A deeply hydrodesulfurized diesel oil containing significant amounts of 4,6-DMDBT was treated with Rhodococcus sp. IMP-S02 cells. Up to 60% of the total sulfur was removed at 30 °C and within 7 d and all the 4,6-DMDBT disappeared as a result of this treatment (Castorena et al., 2002).

Xu et al. (2002) investigated the BDS efficiency of hydrodesulfurized diesel oil with an initial S-content of 205 µg/mL in a batch BDS of 1:3 (O/W) and pH 7.5 using different bacterial isolates, where Rhodococcus sp. 1awq, IG, ZT, ZCR, and a mixture of isolates 5 and 6 recorded a BDS percentage of 80, 69.1, 17.5, 58.5, and 10.6%, respectively, at 30 °C, while isolate X7B expressed 90.3% BDS at 45 °C.

The desulfurization genes (dszABC) were cloned from Gordonia nitida (Jae et al., 2003). Nucleotide sequence similarity between the dszABC genes of G. nitida and those of Rhodococcus erythropolis IGTS8 was 89%. The similarities of deduced amino acids between the two were 86% for DszA, 86% for DszB, and 90% for DszC. The G. nitida dszABC genes were expressed in several different Escherichia coli strains under an inducible trc promoter. The metabolically engineered E. coli strains harboring the new desulfurization genes from G. nitida were able to efficiently desulfurize DBT and diesel oil. Deregulation of the transcription repression site by employing the inducible trc promoter and an artificial dszABC operon resulted in a relatively high efficiency of DBT conversion to 2-HBP. The maximum conversion of DBT to 2-HBP was 16% within 60 h. A hydrodesulfurized diesel oil with an S-content of 250 mg/L was examined for desulfurization (SK Corp., Daejeon, Korea). A batch BDS of 5% (v/v) diesel oil of 6 h after the induction with 0.5 mM IPTG at OD₆₀₀ of 0.7 recombinant E. coli W3110 (pTrcS1ExABC) removed 15% of sulfur in diesel oil, decreasing sulfur content from 250 to 212.5 mg/L in diesel oil within 60 h (Jae et al., 2003).

Five isolates belonging to the Rhodococcus/Gordonia cluster isolated by Abbad-Andaloussi et al. (2003a) from different soils exhibiting good growth characteristics and high BDS activities in both aqueous and organic media were studied for their abilities to desulfurize different types of diesel oils in order to better assess the potential of BDS, especially as a finishing step complementary to deep HDS. Actually, in spite of their taxonomic similarity, the five strains displayed different activities towards the diesel oil tested. BDS yield was also dependent upon the diesel oil used, especially its sulfur content. Some HDS-recalcitrant compounds, such as 4,6-DMDBT, could be completely removed, but highly alkylated DBTs were resistant to the action of the biocatalysts. Abbad-Andaloussi et al. (2003a) observed that BDS was more efficient on diesel oils with a low sulfur content and that biocatalysts were also active on LCO (light cycle oil, a gas-oil type fraction from catalytic cracking units). This result suggests that inhibitory or toxic effects by certain sulfur compounds may occur. A maximum BDS yield of 76% was obtained and it was possible to decrease the sulfur content of a previously deeply-hydrodesulfurized diesel oil down to 14 mg/kg. In addition, the sulfur removal rate was high. Furthermore, some HDS-recalcitrant compounds, such as 4,6-dimethyl DBT, could be totally desulfurized. The desulfurization kinetics were affected by the number and/or lengths of alkyl groups attached to the ring structure of DBT. From a technical point of view, these results showed that BDS could be quite suitable as a finishing step complementary to deep HDS because of its high selectivity. However, the action of the biocatalysts on the highly-alkylated DBTs remained low and these compounds would also have to be removed to decrease the sulfur content of diesel oil to levels as low as 10 mg/kg.

Labana et al. (2005) reported 1:3 (O/W) batch-BDS of two different feed diesel from an HDS unit: product diesel from an HDS unit (170 ppm S) and a high-speed diesel (70 ppm S), with resting cells of Rhodococcus sp. and Arthrobacter sulfureus, collected at mid-log phase. Those recorded BDS percentages of 50 and 53% for hydrodesulfurized diesel oil, respectively, within a 5 d incubation period at 30 °C and 200 rev/min, while Rhodococcus sp. recorded only 28% for the high-speed diesel oil sample, but A. sulfureus did not express any BDS potential on the sulfur content of that oil sample. The difference in BDS efficiencies was probably because of different chemical forms of sulfur. Nevertheless, it was concluded from that study that HDS and BDS probably have a synergistic effect in removing a broad spectrum of sulfur-containing compounds, as HDS enriches the HDS-refractory sulfur-bearing heterocycles, such as DBT, and depletes the HDS-labile organosulfur compounds (OSCs) (Monticello 1996). Thus, consequently, HDS followed by BDS could help meet the production of ultra-low sulfur fuel in accordance with the regulations.

Mukhopadhyaya et al. (2006) used a trickle bed reactor under a continuous mode for BDS of hydrodesulfurized diesel oil with an initial S-content of 200 ppm and pith balls have been used as an immobilizing matrix for Rhodococcus sp. NCIM2891, where approximately 99% of sulfur conversion occurred at an inlet diesel flow rate of 0.25 dm³/h. R. erythropolis XP was also reported for the BDS of a hydrodesulfurized diesel oil, where the S-content reduced by 94.5% from 259 to 14 ppm (Yu et al., 2006a). Another study by Nandi (2010) reported an internal loop airlift reactor for the BDS of hydrodesulfurized diesel from 500 ppm to almost zero level within 120 h using Rhodococcus sp. NCIM2891.

Ma et al. (2006) investigated methods to produce biodesulfurization catalysts and found that cultivation of Rhodococcus sp. in 1 mM dimethylsulfoxide was the most cost-effective. The microbial culture was then used in a bi-phasic system (1:9 O/W) and was found to remove 78% of sulfur (200 ppm initial concentration) from an HDS treated diesel oil.

Feng et al. (2006) reported the enhancement effect of Tween 80 on the BDS of a hydrodesulfurized FHD200 diesel oil (200 ppm S) with a 1/9 (O/W) in the presence of 2% glucose as a C-source. The BDS increased with increasing the surfactant concentration, reaching 90% at 0.5% Tween 80. Upon its application on another two diesel oils, FHD406 and KHD168 diesel oil, the results revealed 78.1% and 65.0%, respectively, compared to 56.6% and 52.9% without Tween 80.

Li et al. (2007b) reported the BDS of a hydrodesulfurized diesel oil with S-contents of 555 µg/g, using cell suspension and resting cells of R. erythropolis LSSE8–1 in a batch of 1/2 O/W phase ration at 30 °C and 170 rpm. This recorded BDS-rates of 59.7 and 65.6 µmol sulfur/g DCW/h, respectively, within 4 h of incubation. Upon the direct application of cell suspension obtained after growth for optimized growth for 72 h on another low S-content hydrodesulfurized diesel oil, the sulfur was reduced to 103 µg/g at a 1/2 O/W ratio in the first BDS treatment after 24 h. Then, upon a second treatment it reached 51 µg/g with a total S-removal of 79.4%. Since direct fermentation cell suspension and resting cells expressed high BDS efficiencies, it was concluded from that study that it is feasible that high cell density suspension containing the metabolites of a sulfur source can be directly used for diesel-BDS. Thus, the BDS process was apparently simplified, which had the advantage of saving cost. The simple process is convenient to treat a mass of petroleum fractions and a continuous operation might be applied as a complementary step after HDS to yield an ultra-low sulfur diesel oil (ULSD).

BDS of hydrodesulfurized diesel oils by a newly isolated strain of Rhodococcus erythropolis FSD-2 was investigated by Zhang et al. (2007). Two representative diesel oils were obtained after distillation and HDS to produce oils with sulfur contents of 666 and 198 µg/mL, respectively. The volumetric phase ratio of the aqueous phase to oil was 5/1. The specific desulfurization rate of diesel oil No. 1 and No. 2 was 0.78 mg S/g DCW/h and 0.26 mg S/g DCW/h, respectively. About 97% of the total sulfur content in the hydrodesulfurized diesel was removed by the two consecutive BDS processes with the majority (~94%) being removed in the first treatment, resulting in diesel with a sulfur content of 5.7 µg/mL.

Several studies have reported 47–94.5% BDS efficiencies of hydrodesulfurized diesel with an initial S-content ranging from 250 ppm to 3000 ppm (Mohebali and Ball, 2008).

Li et al. (2008) reported that a microbial consortium (2.2:1 w/w) of R. erythropolis DS-3, which is capable of desulfurizing DBT and its derivatives (Ma et al., 2002) and Gordonia sp. C-6 w, which is capable of desulfurizing BT and its derivatives (Li et al., 2006) could efficiently desulfurize 86% of the heterocyclic sulfur compounds (from 1260 to 180 ppm) in a hydrodesulfurized diesel oil after 3 cycles of BDS.

Application of chitosan flocculation and integration with cell immobilization onto celite for BDS of hydrotreated diesel oil (1/2 v/v) (O/W phase ratio) was performed. It was observed that the sulfur content of D304 diesel (304 µg/g) reduced by approximately 57% within 24 h by chitosan-celiteimmobilized cells. Moreover, the total sulfur of D123 diesel was reduced from 123 to 22 µg/g, corresponding to ≈ 82% BDS. It demonstrated that the flocculated and immobilized cells are capable of removing sulfur from hydrotreated diesel to yield ultra-low sulfur products (Li et al., 2011). BDS was noticed to reach a plateau after 8 h. The initial specific activities (30.6 – 25.6 µmol sulfur/g DCW/h) decreased quickly to between 18.9 – 3.5 and the average activity for the two diesel oils was about 24.8 and 14.6 µmol sulfur/g DCW/h, respectively, within 8 h. Thus, the residence times of the BDS process can be set at 6–8 h.

Bandyopadhyay et al. (2013a) reported the production of biosurfactant during the BDS of hydrodesulfurized diesel in a 2 dm³ B.Braun chemostat with a continuous stirred tank reactor (CSTR) with a working volume of 1.5 dm³, using Rhodococcus sp (NCIM 2891). The stirring rate and aeration rate were maintained at 100 rpm and 25 L/h, respectively. The diesel to aqueous phase ratio was maintained at 80:20 during all runs in the chemostat. The dilution rate was varied in the range of 0.03 to 0.1 and the sulfur concentration in feed diesel was varied from 200 to 540 ppm. Under each operating condition the reactor was operated for 4 days. A chemostat was operated to control/optimize the microbial growth rate, surfactant production rate, and the substrate utilization rate. These were achieved by adjusting the volumetric feed rate or dilution rate. After a certain operating period of 30 hours (>3τ), the concentrations of biomass, substrate, and product become invariant with reaction time. This may be considered to be the onset of a steady state. The concentrations of product and biomass decreased with dilution rate. This was attributed to the fact that as the dilution rate increases, the residence time in the reactor decreases and, consequently, the rates of outlet of both product and biomass outweigh the generation rates of the respective components resulting in an ultimate decreasing trend of concentration with dilution rate. The concentration of both 2-HBP and biomass expressed a decreasing trend with an increase in the dilution rate in the range of 0.03–0.07 h^-1. Moreover, both the produced biomass and 2-HBP concentrations at any dilution rate showed increasing trends with an increase of initial S-concentration. Thus, the generation rates increased with the increase of the studied substrate concentrations range. The surface tension of aqueous phase and oil phases of the reaction broth, interfacial tension, as well as the emulsification index of the broth obtained with an initial sulfur concentration of 330 ppm and diesel to aqueous ratio of 80:20 have been plotted with the operating time of the chemostat. It has been observed that the surface tension decreased and the value of E₂₄ increased with the increase in reaction time. The surface tension of the aqueous nutrient phase and diesel was observed to decrease from 71 dynes/cm to 30 dynes/cm and to 20 dynes/cm from 30 dynes/cm, respectively. Values of the emulsification index (E₂₄) were found to be varied from 18 to 58 over the growth period of 2 to 48 h in the chemostat. The critical micelle concentration (CMC) was found to be 200 mg/L. This was an indication of the formation of more biosurfactant, namely, 2-HBP and some triglycerides and polar and nonpolar lipids with the propagation of reaction. The increase of values of E₂₄ with reaction time also established the effectiveness of the biosurfactants produced as byproducts of the biodesulfurization of diesel. This came with the decrease in the sulfur concentration of the hydrotreated diesel to ≈ 20 ppm with a maximum conversion of 95%. In a similar study, Bandyopadhyay et al. (2013b) reported the production of ultra-low sulfur diesel (ULSD) and biosurfactants, namely, 2-hydroxybiphenyl and different lipids. The substituted benzothiophenes (BTs) and dibenzothiophenes (DBTs) in hydrotreated diesel get converted to 2-hydroxybiphenyl, which is a potential bio-surfactant. Kinetics of BDS of deep desulfurized diesel using Rhodococcus sp. (NCIM 2891) have been studied with special reference to the removal of OSCs in diesel and the production of 2-hydroxy biphenyl (2-HBP). The sulfur concentration of feed diesel was in the range of 200–540 mg/L. Aqueous phase to diesel ratios have been varied in the range of 9:1 to 1:9. The optimum ratio has been found to be 1:4 (O/W) based on both the ease of separation of the aqueous phase from diesel and the value of the specific microbial growth rate and a maximum conversion of sulfur at 95% has been achieved. A chemostat has been studied using an initial concentration of OSCs in diesel as a parameter. A mathematical model has been developed on the basis of kinetic parameters derived from shake flask analysis. The model has been validated by the comparison with the experimental data. Furthermore, in order to understand the reaction engineering behavior of the biodegradation process, a classical Monod type of a substrate uninhibited kinetic model (eq. 1) has been applied for simulation work. The experimental data generated from the studies in a chemostat have been used to determine the kinetic parameters based on a Monod model:

(1)

The rate equation of microbial growth:

(2)

The rate equation of S-removal:

(3)

Thus, the rate equation in terms of microbial growth kinetics:

(4)

where C_s and C_x are the S and biomass concentrations, respectively.

The mathematical model of the system has been developed on the basis of the following assumptions: influent stream of the bioreactor was sterile, there was no external mass transfer resistance present in the system, the OSCs of diesel were the only growth limiting substrates, and, finally, the microbial growth follows Monod Kinetics.

Thus, the system equations for batch mode were as follows:

Substrate (i.e. the S-content):

(5)

Biomass:

(6)

Product (i.e. the 2-HBP):

(7)

Taking into account that under a steady state, the mass balance equations for substrate, biomass, and product become:

(8)

(9)

(10)

Thus, the values of Monod kinetic parameters, the maximum specific growth rate, µ_max, half saturation constant, Ks, biomass yield, Yx/s, and 2-HBP yield, Yp/x, were determined by using batch type experimental data obtained with different initial sulfur concentrations (200 to 540 ppm) and the values recorded were 0.096 h^-1, 71 mg/L, 0.2, and 17 µmol/g DCW, respectively. Furthermore, the optimum ratio of diesel to aqueous medium was found to be 80:20 (Bandyopadhyay et al., 2013a), while the values of kinetic parameters, namely, µ_max and K_s of the microbial growth and yield coefficient of surfactant, with respect to biomass, have been determined to be 0.13 h^-1, 68 mg/L, and 18.5 µmol/g DCW, respectively, by conducting batch type experiments at the optimum ratio of oil to aqueous phase of 4:1 (W/O). A maximum conversion of 96% was achieved at a dilution rate of 0.03 h^-1 when the sulfur concentration in the feed diesel was 430 ppm. Moreover, the aqueous layer also contained extracellular biosurfactants (phospholipid, glycolipid, and rhamnolipid) in the range of 0.03 g/mL, probably resulting from the microbial secretion. The presence of polar and non-polar lipids was confirmed by thin layer chromatography (TLC) (Bandyopadhyay et al., 2013a). Bandyopadhyay et al. (2014) also reported the production of biosurfactant during the BDS of spent engine oil, which is a mixture of aliphatic and aromatic compounds, is one of the frequent environmental pollutants. When batch studies were conducted using Rhodococcus sp. (NCIM 2891) by varying the oil to aqueous medium ratio of 10:90 to 90:10, the results demonstrated that a maximum desulfurization of 80% was obtained in a batch-BDS at an oil to aqueous phase ratio of 70:30, pH 8.0, 160 rpm, and 28 °C, within a 30 h incubation period. Kinetic parameters of Monod type growth model, µ_max, Ks, and Yx/s recorded 0.12 h^–1, 73.5 mg/L, and 0.32, respectively, and those were determined by varying the initial sulfur content of spent oil in the range of 0.16 –1.05% (w/v) by diluting with hexadecane. The obtained results indicated a higher growth rate on diesel oil compared to that on spent oil. The biosurfactants, like glycolipids, phospholipids, and rhamnolipids, secreted by the microorganisms are preferentially transferred to the water phase to lower the surface tension. This, consequently, would facilitate the assimilation of nutrients by microorganisms from the aqueous phase (Bandyopadhyay et al. 2013a, 2014). Moreover, due to existence of the microorganisms only at the interface can the treated spent oil easily be separated from water phase and the interface containing microorganisms. However, it was observed that the microorganisms growing in the spent engine oil were smaller in size than those grown in the diesel phase (Bandyopadhyay et al., 2013b). This was attributed to the presence of metal contaminants present in the spent engine oil. The surface tensions of the aqueous nutrient phase and spent oil decreased with time from 69 dynes/cm to 24 dynes/cm and from 18 dynes/cm to 10 dynes/cm, respectively, while the E₂₄ increased from 14 to 50 and from 24 to 66 within 32 h of incubation period in the aqueous and spent linguine layers, respectively (Bandyopadhyay et al., 2014). This indicated that the production of surface active reagents in the oil phase and other extracellular lipids, like phospholipids, glycolipids, rhamnolipids, etc., occurred in the aqueous phase with the propagation of reaction and was associated with the microbial growth. Moreover, the GC/MS revealed the removal of the PASHs, 1,1-dimethyltetradecyl hydrosulfide, 2-nonadecanone-(2,4-dinitrophenyl-hydrazone), S-[2-((3-[2,4-Dihydroxy-3,3-dimethylbutanoyl)amino] propanyl)amino) ethyl]2tetradecynethioate, etc., which were predominantly present in the untreated spent engine oil, while the mono- and di-aromatic compounds, long, straight, and branched chain hydrocarbons (> C15) were not affected. Bandyopadhyay et al. (2014) recommended not only the production of useful biosurfactants during the BDS process, but also the generation of ultra-low sulfur diesel through pyrolysis of the biotreated spent engine oil. In another study, Rhodococcus sp (NCIM 2891) was also selected for the production of biosurfactant through biodesulfurization of hydrotreated diesel in a batch and continuous chemostat mode. The best BDS efficiency, cell growth, and biosurfactant production occurred at 80:20 O/W in a batch system. In a batch system, as the diesel proportion is increased, the availability of sulfur compounds, which is the energy source of the microorganism, as well as hydrocarbons, one of the carbon sources for growth, increases resulting in the increased trend in cell concentration up to a ratio of 80:20, where the S-content decreased from 350 ppm to 30 within 48 h at 28 °C. But beyond this OFP, the availability of oxygen might have been decreased at the interfacial zone due to formation of a thick oil layer. Thus causing a decline in the values of biomass concentration, sulfur conversion, and lowering of surface tension (Bandyopadhyay and Chowdhury, 2014).

Bordoloi et al. (2014) reported the BDS of hydrodesulfurized diesel oil with an S-content of 420 ppm by Achromobacter sp. resting cells with an age of 94 h grown on 0.5 M DBT at 37 °C in a batch process of 1/3 O/W, where the S-content decreased to 19 ppm after 24 h, which corresponds to a reduction of 7.1% of sulfur from the diesel oil /g DCW with a slight effect on the hydrocarbon skeleton of the oil feed.

The BDS of hydrodesulfurized diesel with an initial sulfur content of 70 ppm, using growing cells of Gordonia sp. IITR100 in a batch BDS-process of 1/3 O/W, led to a decrease in S-content, reaching to < 2 ppm at 30 °C and 250 rpm within 4 days’ incubation period, reached approximately 98%. This occurred with the observed disappearance of most of the peaks corresponding to the aromatic organosulfur compounds, including alkylated dibenzothiophenes and benzothiophenes, as analyzed by GC-SCD. This occurred without affecting the hydrocarbon skeleton of diesel oil as detected by GC-FID analysis. However, after 6 days of incubation, slight changes in the hydrocarbon skeleton occurred. This was explained by the possible depletion of the alternate carbon source in media after four days in the absence of which bacteria started to take up hydrocarbons (Adlakha et al., 2016).

11.8.2 BDS as a Complementary to ADS

The use of a biosorption agent to bind and separate sulfur compounds by forming a biosorption complex was discussed by Johnson et al. (1995). The invention included a procedure for the preparation of products resulting from biocatalytic sulfur oxidation such as 2-hydroxybiphenyl (2-HBP). The invention was tested in a two stage adsorption-conversion of 3% DBT and suspended Rhodococcus rhodochrous biocatalyst in hexadecane. The results showed rapid DBT adsorption and conversion to 2-HBP.

Li et al. (2005b) proposed a method to produce ultra-low sulfur diesel (ULSD) by integration adsorptive desulfurization (ADS) with biodesulfurization (BDS). Briefly, sulfur compounds (for example, dibenzothiophene DBT) are adsorbed on adsorbents and then the adsorbents are regenerated by microbial conversion. The π-complexation adsorbent, Cu(l)-Γ, was obtained by ion exchanging the Γ-type zeolite with Cu²⁺ and then auto-reduction in helium at 450 °C for 3 h was performed. The amounts of DBT desorbed and 2-HBP produced have been increased with addition of n-octane. DBT-BDS activity has been improved by increasing cell concentration and the ratio of water-to-adsorbent. The amount of 2-HBP produced was strongly dependent on the volume ratio of oil-to-water, cell concentration, and amount of adsorbent. Moreover, 89% of DBT desorbed from the adsorbents can be converted to 2-HBP within 6 h at almost 100% within 24 h when the volume ratio of oil-to-water was 1/5 mL/mL, the cell concentration was 60 g/L, and the ratio of adsorbent-to-oil was 0.03 g/mL. During the bio-regeneration process, desorption of DBT can be significantly improved by adding an oil phase. The adsorption capacity of the regenerated adsorbent is 95% that of the fresh one after being desorbed with Pseudomonas delafieldii R-8, washed with n-octane, dried at 100 °C for 24 h, and auto-reduced in He.

Li et al. (2009) studied ADS using different types of adsorbents, such as activated carbon (AC), NiY, AgY, alumina, 13X, and bio-regeneration properties of the adsorbents were studied with P. delafieldii R-8 (CGMCC 0570). The adsorption capacity of different adsorbents was tested using model oil (8.0 mmol/L DBT and 8.0 mmol/L naphthalene in n-octane) at ambient conditions. The ratio of oil to adsorbent was chosen as 100 mL/g. Hydrotreated diesel oil has also been used in this study. The regeneration system contained n-octane, aqueous phase, lyophilized cells, and spent adsorbents. All reactions were carried out in 100 mL flasks at 30 °C on a rotary shaker operated at 200 rpm. Adsorption–bio-regeneration properties were tested in an in-situ adsorption–bio-regeneration system which can conveniently be divided into two parts: adsorption and bio-regeneration (Figure 11.5). After the saturation of adsorbents, the adsorption system is shut down and the adsorption reactor was connected with the bioreactor. Then, the desorbed sulfur compounds were converted by R-8 cells. Finally, the desorbed adsorbents were treated with air at 550 °C to remove water from the adsorption system. The integrated system is able to efficiently desulfurize DBT and the ADS property of the bio-regenerated adsorbents is similar to fresh ones. The ADS capacity of DBT ranked in the following decreasing order: AC > NiY > AgY > alumina > 13X, while the selectivity of DBT over naphthalene followed the sequence: NiY > AgY > 13X ≈ alumina > AC. For the regeneration of the spent adsorbents, the interaction of the adsorbents with DBT was studied and revealed the following sequence: AC > NiY > AgY > alumina > 13X. The sequence of the DBT desorption ratio was 13X > alumina > AgY > NiY > AC. Since the interaction of DBT with AC was so strong, little DBT has been desorbed. Thus, that revealed that the stronger the interaction of DBT with the adsorbent, the more difficult to be desorbed.

Adsorption properties of NaY, MCM-41, and MAS towards hydrotreated diesel were also carried out at 30 °C in a ratio of 20 mL oil/ g adsorbent. The ADS properties of Ag-MAS were much better than AgMCM-41, while the ADS properties of Ag-MAS and Ag-MCM-41 were much better than those of Ag-Y. This was attributed to the large molecular compounds in diesel which can block the pores of Ag-Y, which, consequently, sharply decreases desulfurization efficiency. The bio-regeneration and recycling properties of Ag-MAS were also carried out with 79 g/g hydrotreated diesel in the presence of n-octane as a regeneration solvent (5 mL n-octane/g AgMAS). The spent Ag-MAS can be regenerated in the integrated system and regenerated adsorbents were found to have similar adsorption properties to the fresh ones.

In an attempt to reduce the accumulation of the inhibitory end product of DBT-BDS, 2-HBP and, consequently, increase BDS efficiency, Abin-Fuentes et al. (2013) studied the efficiency of different adsorbents: activated charcoal, molecular sieves (pore sizes, 4, 5, and 13 Å), Diaion HP-20, Dowex Optipore L-493, Dowex Optipore SD-2, biobeads, Amberlite XAD4, Amberlite IRC86, and Amberlite IRA958 to adsorb HBP from a hexadecane-water solution (1:1 v/v) that contained 10,000 M DBT and 10,000 M HBP with either 0.1 or 1.0 g of the studied resins. The specific loading of HBP (L_r) was determined by the following equation:

(11)

where C_HBP represents the total concentration of HBP (in oil and water), MMHBP is the molecular mass of HBP (170 g/mol), and t₀ and t_eq represent the times at initial and equilibrium conditions, respectively. Xr is the concentration of resin employed that was calculated as follows:

(12)

Where V_total is the total volume of the solution (oil plus water) and m_r is the mass of resin used. Dowex Optipore SD-2 resin was found to express the best results, that is the best HBP loading of 50 mg/g resin with the highest selectivity relative to DBT loading which was 2.5 times greater than that for DBT. The high affinity towards HBP was attributed to the strong hydrophobic interactions between the resins’ aromatic side groups and the biphenyl part of HBP. Then, it was applied in a BDS batch process using a resting cell suspension of R. erythropolis IGTS8. Ethanol was found to be the best extractant. The cell walls solubilization by treatment with an enzymatic mixture of lysozyme (100 mg/mL) and mutanolysin (5,000 U/mL) did not increase the concentration of DBT or HBP extracted from the cells. Sonication of the cells also did not increase extraction concentrations. Loadings (i.e. the adsorption) of HBP on the biocatalyst (Lc) and resin (Lr) were calculated from the expressions:

(13)

(14)

where C_extract,i is the concentration of HBP in the i^th extract, V_extract is the volume of the extract (constant for each fraction), m_cells is the mass of cells extracted, and m_r is the mass of resin extracted.

The partition coefficient of HBP between one component (component 1) and another component (component 2) in the four-component (oil, water, cells, and resin) mixtures of BDS experiments (P_comp1/comp2) was expressed as follows:

(15)

where C_HBP,comp1 is the HBP concentration measured in component 1 and C_HBP,comp2 is the HBP concentration measured in component 2. For example, P_R/C is the partition coefficient of HBP between the resin and the biocatalyst (cells). Similarly, P_O/W is the partition coefficient of HBP between hexadecane (oil) and water.

The resin Dowex Optipore SD-2 was found to have the highest affinity for HBP relative to the other components of the system. The resin’s affinity for HBP was about 100 times greater than the affinity of either the hexadecane oil phase or the biocatalyst. Furthermore, its affinity for HBP was about 10,000 times greater than that of the aqueous buffer. The partition coefficients of HBP between oil and the aqueous buffer (P_O/W) and between the biocatalyst and the aqueous buffer (P_C/W) were very similar. Also, the partition coefficient of HBP between the biocatalyst and oil (P_C/O) was calculated to be 1 in the absence of resin. Thus, the oil and biocatalyst components expressed very similar affinities for HBP. Moreover, both the oil and the biocatalyst have an affinity for HBP that is 50 to 60 times greater than that of the aqueous buffer in the absence of resin.

The partition coefficient P_C/W was defined as:

(16)

where C_HBP,water is the HBP concentration in the water phase and C_{HBP, intracellular} is the HBP concentration within the biocatalyst since the cell is composed of two components, the inner cytoplasmic space and the outer envelope/shell that encompasses the cytoplasm, which is the cell wall. The partition coefficient of HBP between the cytoplasm and water (P_cytoplasm/W) was estimated from solving its value in the expression:

(17)

where P_cell-wall/W is the partition coefficient of HBP between the cell wall and the water and it was estimated to be 550 and f_cell-wall and f_cytoplasm are the fractions of the total volume of a single cell that are occupied by the cell wall and cytoplasm, respectively. The values of f_cytoplasm and f_cell-wall were calculated as follows:

(18)

(19)

where W is the estimated cell wall thickness Rhodococcus species, which is approximately 10 nm (Sutcliff et al., 2010) and R_cell is its estimated radius, which is approximately 0.5 µm (Kilbane, 1992). The cytoplasmic HBP concentration (C_HBP,_cytoplasm), which decreased with the increase of X_r, was calculated as follows:

(20)

and recorded 1,100, 330, and 260 µM at resin concentrations (X_r) of 0, 10, and 50 g/L, respectively.

Although the resin was effective in reducing HBP retention within the cytoplasm of the biocatalyst, which is where the desulfurization enzymes are present and the corresponding HBP loadings on the biocatalyst (Lc) were calculated to be 1.6, 0.5, and 0.2 mg HBP/g DCW, despite the significant decrease in HBP retained within the cytoplasm, the total amount of HBP produced in the system did not increase with increasing resin concentration. Consequently, it was assumed that the biocatalyst and, in particular, the desulfurization enzymes might have been susceptible to cytoplasmic HBP concentrations of less than 260 µM.

In another study, Carvajal et al. (2017) studied the ADS efficiency of a model oil of 100 mg/L DBT and 100 mg/L 4,6-DMDBT on different adsorbents: alumina (Al), silica (Si), and sepiolite (Sep), where the highest removal occurred with particle sizes from 0.43 to 0.063 mm and it increased with the decrease of the particles’ size, which was attributed to the larger total surface area per volume and the shorter diffusion path for the adsorbate upon the usage of smaller particles. Comparison of the adsorption of both sulfur-containing organic molecules revealed higher adsorption of DBT on Si and Al, while no marked difference was observed in the case of Sep, which was attributed to the different specific areas and density and strength of acid sites of these materials. While higher adsorption of both sulfur-containing organic molecules on the Si support showed that the strength of acid sites is the main factor that affects the adsorption of recalcitrant sulfur-containing molecule, the adsorption capacity values revealed that the strongest interaction of DBT and 4,6-DMDBT was with Sep and Si, respectively. The removal of the adsorbed S-compounds for the recovery of the adsorbents has been done using free cells of R. erythropolis IGTS8 and the highest BDS efficiency was performed upon the usage of Sep and Si. The observed differences in BDS were attributed to the interactions between the cells and supports since both physical and biological factors can influence cell–support interactions, such as the size of the bacterial cells, ionic strength, and the support surface structure (Yee et al., 2000; Jeyachandran et al., 2006; Dinamarca et al., 2010). Moreover, the results showed higher activity in systems that had stronger interactions between the bacterial cells and support. There was also a recorded higher removal of DBT compared with that of 4,6-DMDBT. This was attributed to the difficulty of BDS of recalcitrant long-chain alkylated DBT (Bhatia and Sharma, 2010).

11.9 BDS of other Petroleum Products

The worldwide increase in the amount of rubber products comes with the rapid industrialization of the modern world. Generally, rubber materials are absolutely necessary for social production and social living. However, the annual waste of rubber is huge and it is hard to be naturally degraded because of the stable cross-linked three-dimensional network structure in vulcanized rubber that causes serious environmental pollution. Recycling rubber is a way to overcome the problem; the cross-links in rubber can be broken by mechanical energy, such as mechanical shearing (Fukumori et al., 2006), ultrasound (Sun and Isayev, 2009), microwave (Vega et al., 2008), and electron beam (Hassan et al., 2007). Chemical desulfurization reagents (De Debapriya et al. 2007; Rajan et al., 2007) can also cut the crosslink bonds or induce active groups on the surface of ground rubber. However, all these methods are expensive, consume a large amount of energy, and release toxic chemicals leading to a secondary pollution. In some developed countries, waste rubber is pulverized into ground rubber to be a replacement of virgin rubber. However, the cross-linked structure restricts the movement of molecular chains and adversely affects reprocessing performance, so the interfacial bonding strength is low when ground rubber is directly blended with raw rubber, resulting in a decrease of the mechanical properties of vulcanized rubber blends. The purpose of vulcanized rubber desulfurization is to break the sulfur crosslinks and allow the rubber to regain mobility for better reprocessability and remoldability. The desulfurization of vulcanized rubber can be obtained by both physical and chemical methods. The desulfurization of vulcanized rubber can significantly improve the coherence of the ground rubber and matrix and the performance of the blend. Microbial desulfurization of rubber gained attention (Holst et al., 1998; Kim and Park, 1999; Fliermans, 2002) for its ability to break sulfur crosslinks with low energy consumption, simple processes, low equipment requirement, and no pollution. However, the main problem in microbial desulfurization is that many microorganisms are sensitive to rubber additives. Moreover, rubber is lipophilic, so it cannot dissolve in an aqueous phase which allows microbes to stay on the rubber surface for a short time. In other words, the BDS effect still needs to improve. Therefore, it is important to increase the affinity between rubber and microorganism, raising the chance of microbial contacting with rubber.

The desulfurization effect of six bacterial strains (Thiobacillus ferrooxidans, Thiobacillus thiooxidans, Thiobacillus thioparus, Sulfolobus acidocaldarius, Rhodococcus rhodochrous, and ATCC 39327) on ground tire rubber (GTR) has been studied and S. acidocaldarius expressed the best effect (Romine and Snowdon-Swan, 1997; Romine and Romine, 1998). The “4S” pathway was proposed, through which the sulfur crosslinks were metabolized into sulfoxide/sulfone/sulfonate/sulfate. Meanwhile, researchers indicated that treated ground tire rubber (GTR) would achieve a good chemistry reactive if the bioprocess stayed at the first three steps. Thus, the modified GTR could be added into virgin rubber with high loading amounts and perform with good mechanical properties. Bredberg et al. (2001a, b) found that the thermophilic Pyrococcus furiosus could be used to desulfurize GTR, but the additives in rubber had some adverse effect on this bacterium. Löffler et al. (1993) evaluated the effects of T. thioparus, T. ferrooxidanshe, and T. thiooxidans on GTR, where T. thioparus expressed the best results and the particle size of GTR was found to have a large influence on the desulfurization effect. The fungal desulfurization of ground rubber, using a white rot basidiomycete, Ceriporiopsis subvermispora, to sulfate by selectively breaking the sulfur crosslinks in rubber has been reported (Sato et al., 2004), but microbial desulfurization breaks only the sulfur crosslinks on the surface of ground rubber because ground rubber and culture medium have no affinity and the contacting surfaces on which the desulfurization occurs are different two-phases. In another study, Ceriporiopsis subvermispora and Dichomitus squalens were cultivated separately in media with natural rubber (NR) for 0–250 days and found that C. subvermispora decreased the content of S-C bonds, but D. squalens expressed no obvious effect on the content of S-C bonds or S-S bonds (Sato et al., 2009).

Li et al. (2011) studied the microbial desulfurization of ground tire rubber (GTR) by Thiobacillus ferrooxidans, which was isolated from a soil of an iron mine in Xinlong, Hebei Province, China, and it was characterized by strong sulfur oxidizing capacity. GTR was immersed in 75% ethanol (v/v) for 24 h to kill the microorganisms attached to it and then filtered out and dried for use. T. ferrooxidans was cultured in flasks (500 mL with 200 mL medium) on a rotary shaker at 30 °C and the pH was adjusted to 2.5 with HCl. After 24 h, 5% (w/v) detoxified GTR and GTR flakes were added into the medium and desulfurized for 30 days. The biomass of the T. ferrooxidans and sulfate content in the medium were measured every 3 days. GTR was desulfurized in the modified Silverman medium during the cultivation of T. ferrooxidans for 30 days and T. ferrooxidans was able to maintain a high biomass. FTIR-ATR (Fourier-transform infrared spectroscopy-Attenuated total reflection) and XPS (X-ray photoelectron spectroscopy) spectra revealed the occurrence of a rupture of conjugated C=C bonds and a reduction of sulfur content on the surface of GTR during desulfurization. Compared with GTR, the carbon content of DGTR (Desulfurated ground tire rubber) surface slightly decreased from 94.38% to 93.43%, while the oxygen content significantly increased by 30%, from 4.73% to 6.15%, and the sulfur content substantially decreased by 52.8%, from 0.89% to 0.42%. The continuous increase of SO₄^2- in the medium indicated that sulfur on the surface of GTR was oxidized. Thus, this study proved that the selected T. ferrooxidans had strong effects on the metabolism of the sulfur element, as well as the cross-linked S, on the surface of GTR and expressed a good desulfurization effect on GTR. Microorganisms could cause conjugated C=C to break and changed some chemical structures on the surface of GTR. After desulfurization, the cross-linked bonds by S on the GTR surface had been partly ruptured and formed a sulfur oxide group. The sol fraction of GTR increased from its original 4.69% to 7.43%. Compared with GTR sheet, desulfurized ground tire rubber (DGTR) sheets had much smoother surfaces, better physical properties, and higher swelling values. As a result of microbial desulfurization and sulfur diffusion, the DGTR filled NR composites had better mechanical properties and lower crosslink density than GTR filled NR composites at the same loading. Compared with NR/GTR composites, NR/DGTR composites had lower internal friction losses of the molecular chain and better interface coherence between the DGTR and matrix.

The surface desulfurization of GTR was carried out via a biological treatment by Thiobacillus sp. with a strong sulfur oxidizing capacity (Li et al., 2012a). The bonding states and element content on the surface of GTR and desulfurized GTR (DGTR) were evaluated using an X-ray Photoelectron Spectroscopy (XPS). The contact angle of GTR was 120.5°, which was decreased down to 93.5° after treatment. The surface desulfurization of GTR was realized with biological treatment by Thiobacillus sp. for improving interfacial adhesion. After treatment, oxygen content on the surface of waste rubber increased by 30% and cross-linked sulfur bonds were partly cleaved and converted to sulfate or oxygen containing sulfur-based groups. The ration of S-S bonds and S-C bonds were respectively decreased by 18.3% and 42.3%.

The formation of S-O bonds was observed. The cure characteristics, swelling behavior, and crosslink density of natural rubber (NR)/GTR and NR/DGTR were examined. A lower cross-link density was obtained for NR/DGTR vulcanizates, which was attributed to the migration of sulfur being obstructed by GTR with cross-link structure and a lower sulfur content of DGTR. The improvement in mechanical properties was observed for NR/DGTR vulcanizates because of the enhanced interfacial interaction between the DGTR and NR matrix. The dynamic mechanical analysis (DMA) results showed that NR/DGTR vulcanizates had a reduction of molecular chain friction resistance during a glass transition region and scanning electron microscopy (SEM) studies further indicated a good coherency and homogeneity between the DGTR and NR matrix.

In another study by Li et al. (2012b), the BDS of GTR was performed by Sphingomonas sp. that was selected from coal mine soil in Sichuan Province, China and had sulfur oxidizing capacity. Before desulfurization, GTR was immersed in 75% ethanol (v/v) for 24 h in order to kill the microorganisms attached to it and remove harmful additives. Detoxified GTR was filtered out and dried in a sterile cabinet. After a 3 day incubation, GTR with glucose was added into the culture medium. The amount of ground rubber was 2.5% (w/v) of the medium. After desulfurization for 20 days, DGTR was filtered out and washed by distilled water several times. The results showed that GTR had low toxicity to Sphingomonas sp. and that it was able to maintain a high biomass. After desulfurization, not only a rupture of conjugated C=C bonds, but also a reduction of GTR sulfur content (22.9%) occurred. The sol fraction of GTR increased from its original 4.69% to 8.68% after desulfurization. The carbon content of DGTR (92.16%) was slightly smaller than that of GTR (96.41%), indicating the occurrence of a partial fracture in the carbon chain. The oxygen content of DGTR has obviously increased after desulfurization, indicating the production of some oxidation products on the surface of DGTR. The sulfur content reduced by 22.9% form GTR (0.96%) to DGTR (0.74%). DGTR sheets had better physical properties and higher swelling values than GTR sheets. Similar results were also obtained using other bacterium for rubber desulfurization. Sulfolobus acidocaldarius used for rubber desulfurization released 13.4% sulfur of rubber material during 7 d (Romine et al. 1997), the fungus could decrease the total sulfur content of the rubber by 29% in 200 d (Sato et al. 2004), and the sulfur content of natural rubber was reduced by up to 30% compared to that of the untreated rubber in the case of the 30 d microbial desulfurization by T. perometabolis (Jin and Jin, 1999).

The biodesulfurization process consists of three steps. Firstly, microbes grow on GTR surface. Secondly, it produces some desulfurized enzymes. Lastly, these enzymes reacted on the GTR surface. During the bioprocess, microorganisms contact with rubber particles in a bioreactor, for example, a shake flask or fermentation reactor. However, microbes cannot stay on a GTR particle surface for a long time because of incompatibility between these two matters. The effect of three non-ionic surfactants (Tween 20 Polyoxyethylene (20) sorbitan monolaurate C₅₈H₁₁₄O₂₆, Tween 60 Polyoxyethylene (20) sorbitan monostearate C₆₄H₁₂₈O_26, and Tween 80 Polyoxyethylene (20) sorbitan monooleate C₆₄H₁₂₄O₂₆) on the BDS of GTR by Sphingomonas sp. was examined by Hu et al. (2014). Tween 20, among these three surfactants, showed the best effect on the enhancement of BDS, as it has the shortest oil-soluble chain among the three surfactants. The SEM analysis proved that the Tween surfactants could effectively improve affinity between Sphingomonas sp. and GTR. After premixing with surfactant, the GTR particle surface was coated by the Tween molecule. Oil-soluble tails contacted with the surface of GTR, while the water-soluble ends extended to the culture medium and contacted with the microbes. The desulfurizing enzymes produced by microbes are water soluble and are needed to pass a surfactant molecule to react with sulfide bonds in GTR particles. Thus, the shorter the length of the hydrophobic part is, the easier for enzymes to reach the rubber surface. Moreover, the results of SEM-EDS (Scanning Electron Microscope-Energy Dispersive X-ray Spectrometer) showed that the amount of sulfur in a rubber surface layer of 0.4 µm was significantly decreased by 67%. XPS (X-ray photoelectron spectroscopy) analysis data showed that the area of S-S bonds and S-C bonds decreased while the S-O bonds obviously increased. The desulfurized effect was evaluated through measuring desulfurized depth, swelling value, and sulfur content of a desulfurized ground tire rubber (DGTR) sheet. The physical properties of desulfurized ground tire rubber/styrene butadiene rubber (DGTR/SBR) composite were measured and they were found to improve. Compared with the tensile strength and elongation of DGTR/SBR composite, those of DGTR₂₀/SBR significantly increased by 11.6% and 23.5%, respectively.

A microbe with desulfurizing capability, Alicyclobacillus sp., was selected by Yao et al. (2013) to recycle waste latex rubber (WLR). The growth characteristics of the microorganism and the technical conditions in the co-culture desulfurization process were studied. The results showed that the toxic effect of WLR on Alicyclobacillus sp. was not noticeable and the optimum addition amount of WLR was 5% (w/v) for desulfurization. The surfactant polysorbate 80 (Tween 80) had a toxic effect on Alicyclobacillus sp., but the growth of the microbe was vigorous if the proper technique was used; the mixing of WLR with Tween 80 was followed by the addition of the mixture into the culture media. After desulfurization by Alicyclobacillus sp., the contents of S and O elements on the surface of DWLR decreased by 62.5% and increased by 34.9%, respectively. The contents of S-C and S-S bonds decreased by 58.0% and 58.2%, respectively, and the content of S-O bonds increased by 79.0% on the surface of DWLR. Briefly, the results showed that the sulfur bonds on the surface of WLR were broken to form sulfones groups and the hydrophilic property of DWLR was improved.

Gordonia amicalisa was used by Hu et al. (2016) to treat two kinds of vulcanized synthetic rubbers, vulcanized isoprene rubber (v-IR) and vulcanized styrene butadiene rubber (v-SBR). The effects of microbial treatment on vulcanized rubbers were evaluated by changes of crosslinking density, sol fraction, morphology, element content, and chemical group of treated rubber samples. The results showed that G. amicalisa can break the C=C bond on v-IR and S-S bonds on v-SBR. After a microbial treatment of 20 days, the crosslink densities of v-IR and v-SBR decreased by 13.7% and 22.1%, respectively. The carbon content on the v-IR surface decreased by 9.1%. The FTIR analysis showed that C=C bonds transferred into C=O bonds, indicating the main chain scission. In regards to the v-SBR, sulfur content on its surface was decreased by 22.9 %, S-S bonds were broken, and S=O was produced, indicating sulfur cross-link scission. Furthermore, to elucidate the reason that G. amicalisa has different decross-linking mechanisms on v-SBR and v-IR, the inducing mechanism of the desulfurizing enzyme produced from G. amicalisa was investigated. The v-SBR and v-IR were used as enzyme inducers and DBT was used as a substrate reacting with the desulfurizing-enzyme. Then, the DBT, as a specific substrate, reacted with the cell culture solution, which was induced by different inducers. The concentration of DBT reacted with pure medium did not decrease, indicating that the contents in the medium have no effect on DBT. The reacted concentration of DBT with the control enzyme solution was unchanged, so the biodesulfurizing microorganisms (BDSM) cannot secrete the desulfurizing enzyme from the cell into the solution without the inducer. Regarding the enzyme solution with the inducers v-SBR (Enzyme_SBR) and v-IR (Enzyme_IR) respectively, DBT concentration in the Enzyme_SBR solution presented a decrease of 28.8%, while DBT in the Enzyme_IR solution remained unchanged, which indicated that the Enzyme_SBR solution has a desulfurizing enzyme which could metabolize DBT into other products. After a DBT reaction with Enzyme_SBR and Enzyme_IR solution for 72 h, the metabolic products of Enzyme_SBR and Enzyme_IR, standard solutions of DBT, and 2-HBP were detected by an ultraviolet spectrophotometer. DBT has specific bands at 310 nm and 323 nm and 2-HBP has characterized band at 287 nm; the 2-HBP band was detected and the areas of the DBT-band in Enzyme_SBR was smaller than those in standard DBT. This spectrum indicated that v-SBR can induce G. amicalisa to produce a desulfurizing enzyme, which can transfer DBT into 2-HBP. Reversely, Enzyme_IR solution only had DBT and no 2-HBP product, indicating that v-IR cannot induce a desulfurizing enzyme. The experiment results for v-IR and v-SBR vulcanized used as enzyme inducers showed that v-SBR can induce G. amicalisa to secrete a desulfurizing-enzyme from cell inside to outside and this desulfurizing-enzyme can transfer DBT into 2-HBP, while v-IR vulcanized has no such induced effect. This explained why G. amicalisa has different effects on v-IR and v-SBR vulcanized rubber.

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