Challenges and solutions for biofiltration of hydrophobic volatile organic compounds.

Rhamnolipids are the most extensively studied biosurfactants and has been successfully used in various areas from bioremediation to industrial fields. Rhamnolipids structural composition decide their physicochemical properties. Different physicochemical properties influence their application potential. Rhamnolipids can be produced at both aerobic conditions and anaerobic conditions by Pseudomonas aeruginosa. This study aims to evaluate the oxygen effects on the rhamnolipids yield, structural composition, physicochemical properties and the rhl-genes expression in P. aeruginosa SG. Results will guide researchers to regulate microbial cells to synthesize rhamnolipids with different activity according to diverse application requirements. Quantitative real-time PCR analysis revealed that rhlAB genes were down-regulated under anaerobic conditions. Therefore, strain P. aeruginosa SG anaerobically produced less rhamnolipids (0.68 g/L) than that (11.65 g/L) under aerobic conditions when grown in media containing glycerol and nitrate. HPLC–MS analysis showed that aerobically produced rhamnolipids mainly contained Rha-C8-C10, Rha–Rha-C10-C12:1 and Rha–Rha-C8-C10; anaerobically produced rhamnolipids mainly contained Rha-C10-C12 and Rha-C10-C10. Anaerobically produced rhamnolipids contained more mono-rhamnolipids (94.7%) than that (54.8%) in aerobically produced rhamnolipids. rhlC gene was also down-regulated under anaerobic conditions, catalyzing less mono-rhamnolipids to form di-rhamnolipids. Aerobically produced rhamnolipids decreased air–water surface tension (ST) from 72.2 to 27.9 mN/m with critical micelle concentration (CMC) of 60 mg/L; anaerobically produced rhamnolipids reduced ST to 33.1 mN/m with CMC of 80 mg/L. Anaerobically produced rhamnolipids emulsified crude oil with EI24 = 80.3%, and aerobically produced rhamnolipids emulsified crude oil with EI24 = 62.3%. Both two rhamnolipids products retained surface activity (ST   60.0%) under temperatures (4–121 °C), pH values (4–10) and NaCl concentrations less than 90 g/L. Oxygen affected the rhl-genes expression in P. aeruginosa, thus altering the rhamnolipids yield, structural composition and physicochemical properties. Rhamnolipids produced at aerobic or anaerobic conditions was structurally distinct. Two rhamnolipids products had different application potential in diverse biotechnologies. Although both rhamnolipids products were thermo-stable and halo-tolerant, aerobically produced rhamnolipids possessed better surface activity, implying its well wetting activity and desorption property; anaerobically produced rhamnolipids exhibited better emulsifying activity, indicating its applicability for enhanced oil recovery and bioremediation of petroleum pollution.

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Oxygen effects on rhamnolipids production by Pseudomonas aeruginosa

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

Aims

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.



Methods and Results

The rhlABRI genes for rhamnolipid biosynthesis were cloned into a facultative anaerobic strain Ps. stutzeri DQ1 to construct the engineered strain Rhl. Anaerobic production of rhamnolipid was confirmed by thin layer chromatography and Fourier transform infrared analysis. Rhamnolipid product reduced the air–water surface tension to 30·3 mN m−1 and the oil–water interfacial tension to 0·169 mN m−1. Rhl produced rhamnolipid of 1·61 g l−1 using glycerol as the carbon source. Rhl anaerobic culture emulsified crude oil up to EI24 ≈ 74. An extra 9·8% of original crude oil was displaced by Rhl in the core flooding test.



Conclusions

Strain Rhl achieved anaerobic production of rhamnolipid and worked well for enhanced oil recovery in the core flooding model. The rhamnolipid produced by Rhl was similar to that of the donor strain SQ6.



Significance and Impact of the Study

This is the first study to achieve anaerobic and heterologous production of rhamnolipid. Results demonstrated the potential feasibility of Rhl as a promising strain to enhance oil recovery through anaerobic production of rhamnolipid.

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

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.

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

The effect of biosurfactants on the solubility of tetrachloroethylene (PCE) and trichloroethylene (TCE) was studied in batch experiments pertaining to their use for solubilization and mobilization of such contaminants in surfactant enhanced aquifer remediation. Biosurfactants, rhamnolipid and surfactin used in solubility studies were synthesized in our laboratory by Pseudomonas aeruginosa (MTCC 2297) and Bacillus subtilis (MTCC 2423), respectively. The efficiency of the biosurfactants in solubilizing the chlorinated solvents was compared to that of synthetic surfactants. The Weight Solubilization Ratio (WSR) values for solubilization of PCE and TCE by biosurfactants were very high compared to the values obtained for synthetic surfactants. Surfactin proved to be a better surfactant over rhamnolipid. The WSR of surfactin on solubilization of PCE and TCE were 3.83 and 12.5, respectively, whereas the values obtained for rhamnolipid were 2.06 and 8.36. The solubility of the chlorinated solvents by biosurfactants was considerably affected by the changes in pH. The aqueous solubility of PCE and TCE increased tremendously with decrease in pH. The solubility of biosurfactants was observed to decrease with the pH, favoring partitioning of surfactants into the chlorinated solvents in significant amounts at lower pH. The excessive accumulation of biosurfactants at the interface facilitated interfacial tension reductions resulting in higher solubility of the chlorinated solvents at pH less than 7.

Effect of biosurfactants on the aqueous solubility of PCE and TCE

In-situ applications such as Microbial Enhanced Oil Recovery (MEOR) and remediation of contaminated sites demand production of biosurfactants in large quantities under oxygen limiting conditions. Few microorganisms have been isolated so far which can cater such need. In this paper, the characteristics of a biosurfactant produced under complete anaerobic conditions are presented. A novel biosurfactant producing microorganism, Pseudomonas sp ANBIOSURF-1 was isolated in our laboratory, from a microbial consortium enriched from municipal sewage sludge. The microorganism utilized vegetable oils and produced biosurfactant under complete anaerobic conditions. TLC tests revealed the presence of sugar and lipid as hydrophilic and hydrophobic moieties respectively. The biosurfactant synthesized under anaerobic condition thus belonged to class of glycolipids similar to that of rhamnolipids. The biosurfactant had a very low CMC value of 52 mg/l. The biosurfactant displayed good emulsifying activity over chlorinated solvents than the petroleum derivatives. The results suggest that Pseudomonas sp ANBIOSURF-1 could potentially be used for remediation of sites contaminated with chlorinated solvents through in-situ biosurfactant production.

John D. Albino

Papers

Effect of biosurfactants on the aqueous solubility of PCE and TCE

Partial Characterization of Biosurfactant Produced under Anaerobic Conditions by Pseudomonas sp ANBIOSURF-1