Xiangfeng Huang

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.

Analysis of biological demulsification process of water-in-oil emulsion by Alcaligenes sp. S-XJ-1

A demulsifying strain (S-XJ-1) was isolated from petroleum-polluted soil and identified as Alcaligenes sp. It showed emulsion breaking ratio of 81.3% for W/O emulsion within 24h when the cell concentration was 500mg/L. Evolution of water droplets during the biological demulsification process was investigated using a Turbiscan stability analyzer and microphotography. Further investigation focused on cell surface hydrophobicity and oil-water interfacial properties. The biological demulsification process began with rapid dispersal of the cells into the oil phase and adsorption onto the oil-water interface. This occurred due to high cell surface hydrophobicity and the presence of amphiphilic compounds in the cell walls. The cells had higher interfacial activity than the emulsifier molecules, and they displaced some of the emulsifier molecules, which effectively reduced the interfacial tension gradient. As a result, the interfacial film strength decreased, the water droplets coalesced and eventually phase separation occurred.

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.

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.

Culture strategies for lipid production using acetic acid as sole carbon source by Rhodosporidium toruloides

Rhodosporidium toruloides AS 2.1389 was tested using different concentrations of acetic acid as a low-cost carbon source for the production of microbial lipids, which are good raw materials for biodiesel production. It grew and had higher lipid contents in media containing 4-20 g/L acetic acid as the sole carbon source, compared with that in glucose-containing media under the same culture conditions. At acetic acid concentrations as high as 20 g/L and the optimal carbon-to-nitrogen ratio (C/N) of 200 in a batch culture, the highest biomass production was 4.35 g/L, with a lipid content of 48.2%. At acetic acid concentrations as low as 4 g/L, a sequencing batch culture (SBC) with a C/N of 100 increased biomass production to 4.21 g/L, with a lipid content of 38.6%. These results provide usable culture strategies for lipid production by R. toruloides AS 2.1389 when using diverse waste-derived volatile fatty acids.

Relationship of cell-wall bound fatty acids and the demulsification efficiency of demulsifying bacteria Alcaligenes sp. S-XJ-1 cultured with vegetable oils

Considering that the surface properties of demulsifying cells correlate with their demulsification efficiency, the demulsifying bacteria Alcaligenes sp. S-XJ-1 with various surface properties were obtained using different vegetable oils as carbon sources. The results show that better performance was achieved with demulsifying bacteria S-XJ-1 possessing a relatively high cell surface hydrophobicity (CSH) and total unsaturated degree for the cell-wall bound fatty acids. There also appeared to be a correlation between the specific cell-wall bound fatty acid components of the bacteria, in terms of carbon chain length or degree of unsaturation, and either CSH or demulsification efficiency. The fatty acids attached to the cell wall were mainly composed of palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3). C18:1 and C18:2 had a positive effect on the formation of CSH, while C18:0 and C18:3 had the opposite effect.

Separation and characterization of effective demulsifying substances from surface of Alcaligenes sp. S-XJ-1 and its application in water-in-kerosene emulsion.

The main goal of this work was to analyze the effect of surface substances on demulsifying capability of the demulsifying strain Alcaligenes sp. S-XJ-1. The demulsifying substances were successfully separated from the cell surface with dichloromethane-alkali treatment, and exhibited 67.5% of the demulsification ratio for water-in-kerosene emulsions at a dosage of 356mg/L. FT-IR, TLC and ESI-MS analysis confirmed the presence of a carbohydrate-protein-lipid complex in the demulsifying substances with the major molecular ions from mass-to-charge ratio (m/z) 165 to 814. After the substances separated, the cell morphology changed from aggregated to dispersed, and the concentration of cell surface functional groups decreased. Cell surface hydrophobicity and the ability of cell adhesion to hydrophobic surface of the treated cells was also reduced compared with original cell. It was proved that the demulsifying substances had a significant effect on cell surface properties and accordingly with demulsifying capability of Alcaligenes sp. S-XJ-1.

Application of waste frying oils in the biosynthesis of biodemulsifier by a demulsifying strain Alcaligenes sp. S-XJ-1.

Exploration of biodemulsifiers has become a new research aspect. Using waste frying oils (WFOs) as carbon source to synthesize biodemulsifiers has a potential prospect to decrease production cost and to improve the application of biodemulsifiers in the oilfield. In this study, a demulsifying strain, Alcaligenes sp. S-XJ-1, was investigated to synthesize a biodemulsifier using waste frying oils as carbon source. It was found that the increase of initial pH of culture medium could increase the biodemulsifier yield but decrease the demulsification ratio compared to that using paraffin as carbon source. In addition, a biodemulsifier produced by waste frying oils and paraffin as mixed carbon source had a lower demulsification capability compared with that produced by paraffin or waste frying oil as sole carbon source. Fed-batch fermentation of biodemulsifier using waste frying oils as supplementary carbon source was found to be a suitable method. Mechanism of waste frying oils utilization was studied by using tripalmitin, olein and tristearin as sole carbon sources to synthesize biodemulsifier. The results showed saturated long-chain fatty acid was difficult for S-XJ-1 to utilize but could effectively enhance the demulsification ability of the produced biodemulsifier. Moreover, FT-IR result showed that the demulsification capability of biodemulsifiers was associated with the content of C=O group and nitrogen element.

Mechanistic QSAR models for interpreting degradation rates of sulfonamides in UV-photocatalysis systems

Photocatalysis is one of the most effective methods for treating antibiotic wastewater. Thus, it is of great significance to determine the relationship between degradation rates and structural characteristics of antibiotics in photocatalysis processes. In the present study, the photocatalytic degradation characteristics of 10 sulfonamides (SAs) were studied using two photocatalytic systems composed of nanophase titanium dioxide (nTiO2) plus ultraviolet (UV) and nTiO2/activated carbon fiber (ACF) plus UV. The results indicated that the largest apparent SA degradation rate constant (Kapp) is approximately 5 times as large as that of the smallest one. Based on the degradation mechanism and the partial least squares regression (PLS) method, optimum Quantitative Structure Activity Relationship (QSAR) models were developed for the two systems. Mechanistic models indicated that the degradation rule of SAs in the TiO2 systems strongly relates to their highest occupied molecular orbital (Ehomo), the maximum values of nucleophilic attack (f(+)x), and the minimum values of the most negative partial charge on a main-chain atom (q(C)min), whereas the maximum values of OH radical attack (f(0)x) and the apparent adsorption rate constant values (kad) are key factors affecting the degradation rule of SAs in the TiO2/ACF system.