Jingcheng Xu

Evaluation of screening methods for demulsifying bacteria and characterization of lipopeptide bio-demulsifier produced by Alcaligenes sp.

In this paper, surface tension measurement, oil-spreading test and blood-plate hemolysis test were attempted in the screening of demulsifying bacteria. After the comparison to the screening results obtained in demulsification test, 50 mN/m of surface tension of culture was proposed as a preliminary screening standard for potential demulsifying bacteria. For the identification of efficient demulsifying strains, surface tension level was set at 40 mN/m. The detected strains were further verified in demulsification test. Compared to using demulsification test alone as screening method, the proposed screening protocol would be more efficient. From the screening, a highly efficient demulsifying stain, S-XJ-1, was isolated from petroleum-contaminated soil and identified as Alcaligenes sp. by 16S rRNA gene and physiological test. It achieved 96.5% and 49.8% of emulsion breaking ratio in W/O and O/W kerosene emulsion within 24h, respectively, and also showed 95% of water separation ratio in oilfield petroleum emulsion within 2h. The bio-demulsifier was found to be cell-wall combined. After soxhlet extraction and purification through silicon-gel column, the bio-demulsifier was then identified as lipopeptide biosurfactant by TLC and FT-IR.

Characterization and phylogenetic analysis of biodemulsifier-producing bacteria

Based on demulsification performance, twenty biodemulsifier-producing strains were isolated from various environmental sources. Five of them achieved nearly or over 90% of emulsion breaking ratio within 24 h. With the aid of biochemical and physiological tests and 16S rDNA analysis, these isolates were classified into eleven genera, in which six genera (Brevibacillus sp., Dietzia sp., Ochrobactrum sp., Pusillimonas sp., Sphingopyxis sp. and Achromobacter sp.) were firstly reported as demulsifying strains. Moreover, with data in this study and other literatures, a phylogenic tree was constructed, showing a rich diversity of demulsifying bacteria. Half of these bacteria belong to Actinobacterale order, which is famous for hydrocarbon degradation and biosurfactant biosynthesis. However, some strains in the same genera differed remarkably in demulsifying capability, surface properties and biochemical and physiological characteristics. This implied the biosynthesis and composition of biodemulsifier were more complicated than expected.

Iron and manganese removal by using manganese ore constructed wetlands in the reclamation of steel wastewater.

To reclaim treated steel wastewater as cooling water, manganese ore constructed wetland was proposed in this study for the removal of iron and manganese. In lab-scale wetlands, the performance of manganese ore wetland was found to be more stable and excellent than that of conventional gravel constructed wetland. The iron and manganese concentration in the former was below 0.05 mg/L at hydraulic retention time of 2-5 days when their influent concentrations were in the range of 0.16-2.24 mg/L and 0.11-2.23 mg/L, respectively. Moreover, its removals for COD, turbidity, ammonia nitrogen and total phosphorus were 55%, 90%, 67% and 93%, respectively, superior to the corresponding removals in the gravel wetland (31%, 86%, 58% and 78%, respectively). The good performance of manganese ore was ascribed to the enhanced biological manganese removal with the aid of manganese oxide surface and the smaller size of the medium. The presence of biological manganese oxidation was proven by the facts of good manganese removal in wetlands at chemical unfavorable conditions (such as ORP and pH) and the isolation of manganese oxidizing strains from the wetlands. Similar iron and manganese removal was later observed in a pilot-scale gravel-manganese-ore constructed wetland, even though the manganese ore portion in total volume was reduced from 100% (in the lab-scale) to only 4% (in the pilot-scale) for the sake of cost-saving. The quality of the polished wastewater not only satisfied the requirement for cooling water but also suitable as make-up water for other purposes.

Comparison between waste frying oil and paraffin as carbon source in the production of biodemulsifier by Dietzia sp. S-JS-1

In order to lower the production cost, waste frying oils were used in the biosynthesis of demulsifier by Dietzia sp. S-JS-1, which was isolated from petroleum contaminated soil. After 7 days of cultivation, the biomass concentration of the most suitable waste frying oil (WFO II) culture reached 3.78 g/L, which was 2.4 times the concentration of paraffin culture. The biodemulsifier produced with WFO II culture broke the emulsions more efficiently than that produced with paraffin culture, given the same volume ratio of carbon source in the culture medium and the same cultivation conditions. It achieved 88.3% of oil separation ratio in W/O emulsion and 76.4% of water separation ratio in O/W emulsion within 5 h. With the aid of thin layer chromatography (TLC) and Fourier transform infrared (FTIR) spectrometry, biodemulsifiers produced from both paraffin and WFO II were identified as a mixture of lipopeptide homologues. The subtle variation in the distribution of these homologues and high biomass concentration of WFO II cultures may account for the afore-mentioned good demulsification performance.

Optimization of biodemulsifier production from Alcaligenes sp. S-XJ-1 and its application in breaking crude oil emulsion

A biodemulsifier-producing strain of Alcaligenes sp. S-XJ-1, isolated from petroleum-contaminated soil of the Karamay Oilfield, exhibited excellent demulsifying ability. The application of this biodemulsifier significantly improved the quality of separated water compared with the chemical demulsifier, polyether, which clearly indicates that it has potential applications in the crude oil extraction industry. To optimize its biosynthesis, the impacts of carbon sources, nitrogen sources and pH were studied in detail. Paraffin, a hydrophobic carbon source, favored the synthesis of this cell wall associated biodemulsifier. The nitrogen source ammonium citrate stimulated the production and demulsifying performance of the biodemulsifier. An alkaline environment (pH 9.5) of the initial culture medium favored the strains growth and improved its demulsifying ability. The results showed paraffin, ammonium citrate and pH had significant effects on the production of the biodemulsifier. These three variables were further investigated using a response surface methodology based on a central composite design to optimize the biodemulsifier yield. The optimal yield conditions were found at a paraffin concentration of 4.01%, an ammonium citrate concentration of 8.08 g/L and a pH of 9.35. Under optimal conditions, the biodemulsifier yield from Alcaligenes sp. S-XJ-1 was increased to 3.42 g/L.