Rongjiu Shi

Heterologous production of Pseudomonas aeruginosa rhamnolipid under anaerobic conditions for microbial enhanced oil recovery

The ex situ application of rhamnolipid to enhance oil recovery is costly and complex in terms of rhamnolipid production and transportation, while in situ production of rhamnolipid is restricted by the oxygen‐deficient environments of oil reservoirs. To overcome the oxygen‐limiting conditions and to circumvent the complex regulation of rhamnolipid biosynthesis in Pseudomonas aeruginosa, an engineered strain Pseudomonas stutzeri Rhl was constructed for heterologous production of rhamnolipid under anaerobic conditions.

Optimization of culture medium for anaerobic production of rhamnolipid by recombinant Pseudomonas stutzeri Rhl for microbial enhanced oil recovery

Response surface methodology was employed to enhance the anaerobic production of rhamnolipid by recombinant Pseudomonas stutzeri Rhl. Glycerol is a promising carbon source used to anaerobically produce rhamnolipid. In a Plackett–Burman design, glycerol, KH2PO4 and yeast extract were significant factors. The proposed optimized medium contained the following: 46·55 g l−1 glycerol; 3 g l−1 NaNO3; 5·25 g l−1 K2HPO4·3H2O; 5·71 g l−1 KH2PO4; 0·40 g l−1 MgSO4·7H2O; 0·13 g l−1 CaCl2; 1·0 g l−1 KCl; 1·0 g l−1 NaCl; and 2·69 g l−1 yeast extract. Using this optimized medium, we obtained an anaerobic yield of rhamnolipid of 3·12 ± 0·11 g l−1 with a 0·85‐fold increase. Core flooding test results also revealed that Ps. stutzeri Rhl grown in an optimized medium enhanced the oil recovery efficiency by 15·7%, which was 6·6% higher than in the initial medium. Results suggested that the optimized medium is a promising nutrient source that could effectively mobilize oil by enhancing the in situ production of rhamnolipid.

Production of biosurfactant by a Pseudomonas aeruginosa isolate and its applicability to in situ microbial enhanced oil recovery under anoxic conditions

Compared to ex situ application, in situ application of biosurfactants for microbial enhanced oil recovery (MEOR) is relatively cost-effective, and lack of oxygen in oil reservoirs is a bottleneck for in situ production of biosurfactants by mostly isolated biosurfactant-producing bacteria. Furthermore, few microorganisms can produce biosurfactants under anoxic conditions. A bacterial strain identified as Pseudomonas aeruginosa SG (GenBank accession number KJ995745) was isolated from Xinjiang oil field, and it can produce biosurfactant under anoxic conditions. Different organic substrates (glucose, sucrose, glycerol, corn steep powder, starch, molasses, soybean oil, sunflower oil) were tested to determine the optimal carbon source for anoxic production of biosurfactant by SG. Strain SG anaerobically grew well at temperatures (25–40 °C), pH (6.0–9.0), and salinity (0–30 g L−1 of NaCl), respectively. Thin layer chromatography and Fourier transform infrared spectroscopy revealed that the SG biosurfactant produced under anoxic conditions was similar to rhamnolipid. SG biosurfactant could reduce air–water surface tension from 71.6 to 33.3 mN m−1, and reduce oil–water interfacial tension from 26.1 to 2.14 mN m−1. And a critical micelle concentration value of 80 mg L−1 was obtained. Moreover, the biosurfactant displayed good emulsifying activity over hydrocarbons and crude oil. A core flooding test revealed that an extra 8.33% of original crude oil in the core was displaced through the in situ production of rhamnolipid by SG. The potential use of the isolated SG for in situ MEOR application was discussed. And bioaugmentation of SG in Xinjiang oil reservoirs will be a promising approach for in situ MEOR.

Simultaneous inhibition of sulfate-reducing bacteria, removal of H2S and production of rhamnolipid by recombinant Pseudomonas stutzeri Rhl: Applications for microbial enhanced oil recovery

Sulfate-reducing bacteria (SRB) are widely existed in oil production system, and its H2S product inhibits rhamnolipid producing bacteria. In-situ production of rhamnolipid is promising for microbial enhanced oil recovery. Inhibition of SRB, removal of H2S and production of rhamnolipid by recombinant Pseudomonas stutzeri Rhl were investigated. Strain Rhl can simultaneously remove S(2-) (>92%) and produce rhamnolipid (>136mg/l) under S(2-) stress below 33.3mg/l. Rhl reduced the SRB numbers from 10(9) to 10(5)cells/ml, and the production of H2S was delayed and decreased to below 2mg/l. Rhl also produced rhamnolipid and removed S(2-) under laboratory simulated oil reservoir conditions. High-throughput sequencing data demonstrated that addition of strain Rhl significantly changed the original microbial communities of oilfield production water and decreased the species and abundance of SRB. Bioaugmentation of strain Rhl in oilfield is promising for simultaneous control of SRB, removal of S(2-) and enhance oil recovery.

Microbial community characterization of an UASB treating increased organic loading rates of vitamin c biosynthesis wastewater

The microbial community of a mesophilic lab-scale upflow anaerobic sludge blanket (UASB) reactor treating vitamin C biosynthesis wastewater at gradually elevated organic loading rates (OLRs) was characterized using 16S rDNA-based polymerase chain reaction-DGGE (denatured gradient gel electrophoresis) analysis. The DGGE fingerprints suggested that the elevated OLRs did not cause any significant changes in the microbial community. The predominant bacterial bands were affiliated with the Firmicutes (Clostridiales, four bands), Proteobacteria (Deltaproteobacteria, six bands), Bacteroidetes, and Synergistetes, respectively. All the archaeal bands were very similar to already known methanogenic species: Methanobacterium formicicum (two bands), Methanomethylovorans hollandica (one band) and Methanosaeta concilli (two bands), which belonged to the divisions Methanobacteria and Methanomicrobia, respectively.

Microbial diversity and functionally distinct groups in produced water from the Daqing Oilfield, China

The microbial community structure and functionally distinct groups in three kinds of produced water samples from the shallow, mesothermic and low-salinity Daqing oil reservoir were systematically evaluated using both culture-dependent and culture-independent methods. Sequence analysis of the 16S rRNA genes indicated that the bacterial library was dominated by Acinetobacter and Arcobacter and the archaeal community was dominated by Methanosaeta and Methanolinea. Two isolated methanogens were closely related with Methanothermobacter thermautotrophicus and Methanoculleus receptaculi. The fermentative bacteria were identified as Pseudomonas, Haloanaerobium, Alcalibacter, Arcobacter, and Pannonibacter. The predominant nitrate-reducing bacteria fell within the genus Pseudomonas. The dominant members of the cultured hydrocarbon-oxidizing bacteria were phylogenetically associated with Micrococcus, Pseudomonas, and Bacillus. Enrichments of biosurfactants and biopolymer producing groups mainly yielded Pseudomonas, Bacillus, and Acenitobacter-related members. The functional groups related to polymer degradation were also affiliated with Pseudomonas and Bacillus. Results from this study provide the fresh insight into the diversity of microbial communities in Daqing petroleum reservoirs. The vast pool of functional strains retrieved in this study was presumed to include the promising strains that could be applied in microbial-enhanced oil recovery in future.

Enhanced treatment of wastewater from the vitamin C biosynthesis industry using a UASB reactor supplemented with zero-valent iron

The effects of zero-valent iron (Fe0) on the performance of a mesophilic upflow anaerobic sludge blanket (UASB) reactor treating high-strength wastewater from the vitamin C biosynthesis industry (VCW) was investigated during a 200-day period. The results showed that the chemical oxygen demand (COD) removal efficiency, CH4 content in biogas, specific methanogenic activity of sludge, and phosphate removal efficiency were significantly improved up to 81.8–96.1%, 76.5–79.6%, 1.71–2.87 g CH4-COD g−1 VSS d−1 and 68.5–85.2%, respectively, at elevated organic loading rates (OLRs) in the Fe0-amended reactor (RFe). In contrast, the corresponding values of 65.3–83.4%, 69.1–70.8%, 1.12–1.95 g CH4-COD g−1 VSS d−1 and 1.4–1.6%, respectively, were recorded in the control (R0). Elevated ferrous concentration of nearly 400 mg L−1 in sludge was detected in RFe, whereas in the effluent of both reactors it was low (<1.0 mg L−1). Batch tests further showed that Fe0 significantly enhanced the biodegradability of the VCW as shown by an increase in BOD/COD ratio from 0.41 to 0.65, and could serve as the electron donor for methanogenesis by anaerobic sludge, which were responsible for the differences between RFe and R0. The results suggest this integrated Fe0-microbial system is promising in facilitating the anaerobic digestion of VCW in UASB reactors.

Characterization and Evaluation of a Denitrifying and Sulfide Removal Bacterial Strain Isolated From Daqing Oilfield

Sulfate-reducing bacteria (SRB) and hydrogen sulfide were widely present in oil production systems, which are detrimental to oil exploitation industry. An anaerobic denitrifying and sulfide removal bacterial strain, identified as Pseudomonas stutzeri DQ1, was isolated from Daqing Oilfield. Starin DQ1 grew well at temperatures 20–40°C, pH 6.5–8.0, and salinity 0–50 g/L of NaCl. Strain DQ1 can completely remove sulfide less than 50.0 mg/L, and decrease the SRB numbers nearly four orders of magnitude. Bioaugmentation of strain DQ1 in oil production system will be a promising approach for simultaneous inhibition of SRB and removal of sulfide in Daqing Oilfield.

Bioaugmentation of oil reservoir indigenous Pseudomonas aeruginosa to enhance oil recovery through in-situ biosurfactant production without air injection

Considering the anoxic conditions within oil reservoirs, a new microbial enhanced oil recovery (MEOR) technology through in-situ biosurfactant production without air injection was proposed. High-throughput sequencing data revealed that Pseudomonas was one of dominant genera in Daqing oil reservoirs. Pseudomonas aeruginosa DQ3 which can anaerobically produce biosurfactant at 42 °C was isolated. Strain DQ3 was bioaugmented in an anaerobic bioreactor to approximately simulate MEOR process. During bioaugmentation process, although a new bacterial community was gradually formed, Pseudomonas was still one of dominant genera. Culture-based data showed that hydrocarbon-degrading bacteria and biosurfactant-producing bacteria were activated, while sulfate reducing bacteria were controlled. Biosurfactant was produced at simulated reservoir conditions, decreasing surface tension to 33.8 mN/m and emulsifying crude oil with EI24 = 58%. Core flooding tests revealed that extra 5.22% of oil was displaced by in-situ biosurfactant production. Bioaugmenting indigenous biosurfactant producer P. aeruginosa without air injection is promising for in-situ MEOR applications.

Anaerobic lipopeptide biosurfactant production by an engineered bacterial strain for in situ microbial enhanced oil recovery

Bacillus mojavensis JF-2 produces water-soluble lipopeptide under aerobic conditions, while Pseudomonas stutzeri DQ1 grows rapidly under anaerobic conditions. These bacteria were used to construct an engineered strain for anaerobic lipopeptide production by protoplast fusion for potential use in microbially enhanced oil recovery (MEOR). The resulting fusant strain FA-2 produced lipopeptide (382 mg l−1) anaerobically at temperatures up to 50 °C, across a pH range of 4.5–10.0, and at salt concentrations as high as 10% NaCl. Experimental results from a physical simulation core at 39 °C suggest that FA-2 has potential for use in MEOR.