Effects of nicotinamide and riboflavin on the biodesulfurization activity of dibenzothiophene by Rhodococcus erythropolis USTB-03

Rhodococcus erythropolis USTB-03 is a promising bacterial strain for the biodesulfurization of dibenzothiophene （DBT） via a sulfurspecific pathway in which DBT is converted to 2-hydroxybiphenyl （2HBP） as an end product. The effects of nicotinamide and riboflavin on the sulfur specific activity （SA） of DBT biodesulfurization by R. erythropolis USTB-03 were investigated. Both nicotinamide and riboflavin were found to enhance the expression of SA, which was not previously reported. When R. erythropolis USTB-03 was grown on a medium containing nicotinamide of 10.0 mmol or riboflavin of 50.0 μmol, SA was raised from 68.0 or so to more than 130 mmol 2HBP/（kg dry cells.h）. When R. erythropolis USTB-03 was grown in the presence of both nicotinamide of 5.0 mmol and riboflavin of 25.0 μmol, SA was further increased to 159.0 mmol 2HBP/（kg dry cells.h）. It is suggested that the biological synthesis of reduced form of flavin mononucleotide （FMNH2）, an essential coenzyme for the activities of biodesulfurization enzyme Dsz C and A, might be enhanced by nicotinamide and riboflavin, which was responsible for the increased SA of R. erythropolis USTB-03.

Biodesulfurization of vanadium-bearing titanomagnetite concentrates and pH control of bioleaching solution

Vanadium-bearing titanomagnetite concentrates were desulfurized with Acidithiobacillus ferrooxidans （A. ferrooxidans）. The sulfur content of the concentrates was reduced from 0.69wt% to 0.14wt% after bioleaching for 15 d with a 10% pulp density at 30℃. Maintaining a stable pH value during biodesulfurization was critical because of high acid consumption, resulting from a combination of nonoxidative and oxidative dissolution of pyrrhotite in acid solution. It is discovered that the citric acid-disodium hydrogen phosphate buffer of pH 2.0 can control the solution pH value smoothly in the optimal range of 2.0-3.0 for A. ferrooxidans growth. Using the buffer in the volume fraction range of 5.0%-15.0% stimulates A. ferooxidans growth and improves the biodesulfurization efficiency. Compared with the buffer-free control case, the maximum increase of biodesulfurization rate is 29.7% using a 10.0vol% buffer. Bioleaching provides an alternative process for desulfurization of vanadium-bearing titanomagnetite ores.

Selection of adsorbents for in-situ coupling technology of adsorptive desulfurization and biodesulfurization

In-situ coupling of adsorptive desulfurization and biodesulfurization is a new desulfurization technol- ogy for fossil oil. It has the merits of high-selectivity of biodesulfurization and high-rate of adsorptive desulfurization. It is carried out by assembling nano-adsorbents onto surfaces of microbial cells. In this work, In-situ coupling desulfurization technology of widely used desulfurization adsorbents of γ-Al2O3, Na-Y molecular sieves, and active carbon with Pseudomonas delafieldii R-8 were studied. Results show that Na-Y molecular sieves restrain the activity of R-8 cells and active carbon cannot desorb the sub- strate dibenzothiophene (DBT). Thus, they are not applicable to in-situ coupling desulfurization tech- nology. Gamma-Al2O3 can adsorb DBT from oil phase quickly, and then desorb it and transfer it to R-8 cells for biodegradation, thus increasing desulfurization rate. It is also found that nano-sized γ-Al2O3 increases desulfurization rate more than regular-sized γ-Al2O3. Therefore, nano- γ-Al2O3 is regarded as the better adsorbent for this in-situ coupling desulfurization technology.

Desulfurization of dibenzothiophene by a newly isolated Corynebacterium sp. ZD-1 in aqueous phase

Sulfur emission through fuel combustion is a global problem because it is a major cause of acid rain. Crud oil contains many heterocyclic organic sulfur compounds, among which dibenzothiophene(DBT) and DBTs bearing alkyl substitutions usually are representative compounds. A strain was isolated from refinery sludge and identified as Corynebacterium ZD-1. The behavior of DBT degradation by ZD-1 in aqueous phase was investigated. Corynebacterium ZD-1 could metabolize DBT to 2-hydroxybiphenyl(2-HBP) as the dead-end metabolite through a sulfur-specific pathway. In shake flask culture, ZD-1 had its maximal desulfurization activity in the late exponential growth phase and the specific production rate of 2-HBP was about 0.14(mmol·kg dry cell -1·min -1, mmol·KDC -1·min -1). Active resting cells for desulfurization should be prepared only in this period. 2-HBP inhibited the growth of strain ZD-1, the production of DBT degradation enzymes, and the activity of enzymes. Sulfate inhibited the production of dibenzothiophene(DBT) degradation enzymes but had no effect on the enzymes' activity. The production rates of 2-HBP at lower cell densities were higher and the maximum amount conversion of DBT to 2-HBP(0.067 mmol/L) after 8 h was gained at 9.2 g dry cell/L rather higher cell density. The results indicated that this newly isolated strain could be a promising biocatalyst for DBT desulfurization.

Effects of loading rate and hydraulic residence time on anoxic sulfide biooxidation

The optimal operation conditions in an anoxic sulfide oxidizing (ASO) bioreactor were investigated. The maximal removal rates for sulfide and nitrate were found to be 4.18 kg/(m3·d) and 1.73 kg/(m3·d), respectively. The volumetrical volumetric loading rates (LRs) observed through decreasing hydraulic retention time (HRT) at fixed substrate concentration are higher than those by increasing substrate concentration at fixed HRT. The sulfide oxidation in ASO reactor was partially producing both sulfate and sulfur; but the amount of sulfate produced was approximately one third that of sulfur. The process was able to tolerate high sulfide concentration, as the sulfide removal percentage always remained near 99% when influent concentration was up to 580 mg/L. It tolerated relatively lower nitrate concentration because the removal percentage dropped to 85% when influent con- centration was increased above 110 mg/L. The process can tolerate shorter HRT but careful operation is needed. Nitrate conversion was more sensitive to HRT than sulfide conversion since the process performance deteriorated abruptly when HRT was decreased from 3.12 h to 2.88 h. In order to avoid nitrite accumulation in the reactor, the influent sulfide and nitrate concentrations should be kept at 280 mg/L and 67.5 mg/L respectively. Present biotechnology is useful for removing sulfides from sewers and crude oil.

A Novel Method for Conversion of Valuable BDS Intermediates HPBS/Cx-HPBS from DBT/Cx-DBT

A simple recombinant PCR method was used to delete the dszB gene responsible for the slowest step of the Dsz pathway and allow the accumulation of hydroxyphenyl benzene sulfinate (HPBS) in the recombinant E. coli. Using GC/MS, HPBS accumulation was confirmed. The recombinant E. coli could also desulfurize Cx-DBT to corresponding Cx-HPBS. The result gave a new insight to BDS process and explored a new method to obtain valuable surfactants from cheap raw materials.

ISOLATION AND CHARACTERIZATION OF BACTERIA FOR REMOVAL OF SULFUR IN ORGANIC COMPOUNDS

Four bacterial strains have been isolated for their ability to growth on dibenzothiophene (DBT), dibenzothiophene sulfone (DBTO2). The results show that different bacteria strains oxidize the DBT to different products through different pathways. R-6 and R-16 metabolize DBT to DBTO2 and HBP; they are identified as species of Bacillus brevis and Bacillus sphaericus. For long time storage, R-9 identified as Nocardia globerula oxidizes DBT to DBT-sulfone (DBTO2) and benzenamine n-phenyl. The optimum temperature and pH for growth of R-16 are 32℃ and 7.02 respectively. The pH of broth decreases during the processes.

Simultaneous Removal of Thiophene and Dibenzothiophene by Immobilized Pseudomonas delafieldii R-8 cells

Biodesulfurization （BDS） is a promising technology for deep desulfurization. In this work, Pseudomonas delafieldii R-8 cells are immobilized in calcium alginate beads and used for BDS of transportation fuels. It is found that thiophene and dibenzothiophene （DBT） can be simultaneously metabolized by immobilized R-8 cells. The initial sulfur content in the model oil is 300 mg·kg-1 （thiophene ＂ DI3T= 1 ＂ 1）. After 10 h of treatment, the thiophene concentration is reduced by 40%, while DBT is reduced by 25%. The utilization rate of thiophene is faster than that of DBT. Moreover, the oil/water ratio of alginate immobilized cells is studied to reduce the water volume in desulfurization systems. Long-term recycling of BDS by alginate immobilized cells is carried out with oil/water ratio at 5 ： 1. The immobilized cells are successfully reused over 15 batch cycles. In the last batch, the desulfurization activity remains at least 75% of the first batch.

Microbial desulfurization of fuel oil

Culture conditions of desulfurization microbes were investigated with a bioreactor controlled by computer. Factors such as pH, choice of carbon source, optimal concentrations of carbon, nitrogen and sulfur sources were determined. The addition of carbon in a culture with a constant pH greatly improved the growth of Rhodococcus. Cells and cell debris from microbes rested using a sulfur-specific pathway were used to desulfurize diesel oil treated by hy-drodesulfurization (acquired from the Research Institute of Fushun Petroleum with total sulfur level at 205μg/mL). Strains lawq, IG, X7B, ZT, ZCR, and a mixture of No. 5 and No. 6, were used in the biodesulfurization process. The reduction of total sulfur was between 10.6% and 90.3%.

Desulfurization of dibenzothiophene(DBT) by a novel strain Lysinibacillus sphaericus DMT-7 isolated from diesel contaminated soil

A new bacterial strain DMT-7 capable of selectively desulfurizing dibenzothiophene（DBT） was isolated from diesel contaminated soil.The DMT-7 was characterized and identified as Lysinibacillus sphaericus DMT-7（NCBI GenBank Accession No.GQ496620） using 16S rDNA gene sequence analysis.The desulfurized product of DBT,2-hydroxybiphenyl（2HBP）,was identified and confirmed by high performance liquid chromatography analysis and gas chromatography-mass spectroscopy analysis respectively.The desulfurization kinetics revealed that DMT-7 started desulfurization of DBT into 2HBP after the lag phase of 24 hr,exponentially increasing the accumulation of 2HBP up to 15 days leading to approximately 60% desulfurization of the DBT.However,further growth resulted into DBT degradation.The induced culture of DMT-7 showed shorter lag phase of 6 hr and early onset of stationary phase within 10 days for desulfurization as compared to that of non-induced culture clearly indicating the inducibility of the desulfurization pathway of DMT-7.In addition,Lysinibacillus sphaericus DMT-7 also possess the ability to utilize broad range of substrates as sole source of sulfur such as benzothiophene,3,4-benzo DBT,4,6-dimethyl DBT,and 4,6-dibutyl DBT.Therefore,Lysinibacillus sphaericus DMT-7 could serve as model system for efficient biodesulfurization of diesel and petrol.

Preparation of microbial desulfurization catalysts

A Rhodococcus sp. lawq, a bacterium isolated from the soil cleaving the C-S bond of dibenzothiophene (DBT) via specific pathway, was investigated for cell growth and for its role in desulfurization. Clearly, the end product, 2-hydroxybiphenyl, inhibited the growth of the strain, the synthesis of the desulfurization enzymes, and the activity of the enzymes. The effects of sulfate on the DBT degradation enzymes were examined in the Rhodococcus sp. lawq growth system with DBT; the sulfate served, concurrently, as the sulfur source. The condition of the resting cells that were used in desulfurization, was also studied. The optimal concentration of the resting cells and the reaction conditions were determined. It was documented that there is no difference between desulfurization activity for resting cells cultured with sulfate as the sole sulfur source and that with the mixture of DBT and sulfate as the sulfur source.

Isolation and identification of nondestructive desulfurization bacterium

A nondestructive desulfurization microorganism has been isolated. The metabolism product analyses show that the strain can be a kind of biocatalyst to oxidize dibenzothiophene (DBT) into 2-hydroxydiphenyl (HBP), therefore the sulfur in DBT is removed selectively. The 16SrRNA information, cell wall analysis, physical, biochemical properties and morphological properties suggest that the isolated strain is Rhodococcus erythropolis. The strain can grow in the basal salts medium (BSM) that DBT concentration is no more than 10 mmol/L, and the optimal DBT concentration for growth is 1 mmol/L, however, the optimal DBT concentration for desulfurization is 0.5 mmol/L. The further research shows that the strain can also desulfur some other organosulfur-containing compounds such as thianaphthene, phenyl sulfide and 4,6-dimethyldiben- zothiophene (4,6-DMDBT).

Sulfur-Selective Desulfurization of Dibenzothiophene and Diesel Oil by Newly Isolated Rhodococcus erythropolis NCC-1

A dibenzothiophene （DBT）-desulfurizing bacteria strain was isolated from oil-contaminated soils and identified as Rhodococcus erythropolis NCC-1. Strain NCC-1 was found to convert DBT to hydroxybiphenyl （2-HBP） via the 4S pathway and also be able to use organic sulfur compounds other than DBT as a sole sulfur source. The strain could desulfurize 4,6-dimethyldibenzothiophene （4,6-DMDBT）, which is one of the most recalcitrant dibenzothiophene derivatives to hydrodesulfurization. When two type of oils, a model oil [n-hexadecane （n-C16） containing DBT] and a hydrodesulfurized diesel oil with various organic sulfur compounds, were treated with Rhodococcus erythropolis NCC-1 cells, the total sulfur content significantly decreased, from 150 to 20 mg/L for n-C16 and from 554 to 274 mg/L for diesel oil. The newly isolated strain NCC-1 is considered to have good potential for application in the biodesulfurization of fossil fuels.