Production and partial characterization of biosurfactant produced by crude oil degrading bacteria

Production of biosurfactant by crude oil degrading bacteria for use in microbial enhanced oil recovery was investigated. Crude oil utilizing bacteria were isolated from soil by enrichment method on oil agar at 30 °C for 5 days. The isolates were identified and screened for biosurfactant production using blood haemolysis and emulsification tests. IR and GC–MS analyses were carried out to detect the type of biosurfactant. The biosurfactant was purified and its stability at various pH, temperature and salinity levels was studied. The organisms were identified as: Achromobacter xylosoxidans subspecies xylosoxidans, Bacillus licheniformis, Proteus vulgaris, Proteus mirabilis, Serratia marcescens, Sphingomonas paucimobilis and Micrococcus kristinae. Emulsification test (E₂₄) revealed that Serratia marcescens had the highest emulsification index of 87%. GC–MS indicated the biosurfactants as lipopeptides. The biosurfactant can be used in EOR under various environmental conditions.     

Biosurfactant production by Corynebacterium kutscheri from waste motor lubricant oil and peanut oil cake

AIM: Production and characterization of biosurfactant from renewable sources. METHODS AND RESULTS: Biosurfactant production was carried out in 3-l fermentor using waste motor lubricant oil and peanut oil cake. Maximum biomass (9.8 mg ml(-l)) and biosurfactant production (6.4 mg ml(-l)) occurred with peanut oil cake at 120 and 132 h, respectively. Chemical characterization of the biosurfactant revealed that it is a glycolipopeptide with chemical composition of carbohydrate (40%), lipid (27%) and protein (29%). The biosurfactant is able to emulsify waste motor lubricant oil, crude oil, peanut oil, kerosene, diesel, xylene, naphthalene and anthracene; the emulsification activity was comparatively higher than the activity found with Triton X-100. CONCLUSION: This study indicates the possibility of biosurfactant production using renewable, relatively inexpensive and easily available resources like waste motor lubricant oil and peanut oil cake. Emulsification activity found with the biosurfactant against different hydrocarbons showed the possibility of the application of biosurfactants against diverse hydrocarbon pollution. SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained from the study could be useful for large-scale biosurfactant production using economically cheaper substrates. Information obtained in emulsification activity and laboratory-scale experiment on bioremediation inferred that bioremediation of hydrocarbon-polluted sites may be treated with biosurfactants or the bacteria that produces it.     

Investigation of alkane biodegradation using the microtiter plate method and correlation between biofilm formation, biosurfactant production and crude oil biodegradation

Among 25 crude oil-degrading bacteria isolated from a marine environment, four strains, which grew well on crude oil, were selected for more study. All the four isolated had maximum growth on 2.5% of crude oil and strain BC (Pseudomonas) could remove crude oil by 83%. The drop collapse method and microtiter assay show that this strain produces more biosurfactant, and its biofilm formation is higher compared to other strains. Bacterial adhesions to crude oil for strains CS-2 (Pseudomonas), BC, PG-5 (Rhodococcus) and H (Bacillus) were 30%, 46%, 10% and 1%, respectively. Therefore, strain H with a low production of biosurfactant and biofilm formation had showed the least growth on these compounds. PCR analysis of these four strains showed that all isolates had alk-B genes from group (III) alkane hydroxylase. All isolate strains could utilize cyclohexan, octane, hexadecane, octadecan and diesel fuel oil; however, the microtiter plate assay showed that strain BC had more growth, respiration and biofilm formation on octadecan.     

Lacticin, a bacteriocin produced by Lactobacillus delbrueckii subsp. lactis

Twenty-one strains of Lactobacillus delbrueckii and L. helveticus were tested for bacteriocin production against each other. Lactobacillus delbrueckii subsp. lactis JCM 1106 and 1107 produced an inhibitory agent active against L. delbrueckii subsp. bulgaricus JCM 1002 and NIAI yB-62, L. delbrueckii subsp. lactis JCM 1248 and L. delbrueckii subsp. delbrueckii JCM 1012. Lactobacillus delbrueckii subsp. lactis JCM 1248 inhibited only the growth of L. delbrueckii subsp. bulgaricus NIAI yB-62. These agents were sensitive to proteolytic enzymes and heating (at 60°C for 10min). These agents were considered to be bacteriocins and designated lacticin A and B.     

Structure of the extracellular polysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus 291

The lactic acid bacterium Lactobacillus delbrueckii subsp. bulgaricus 291, when grown in skimmed milk, produced 80 mg/L exopolysaccharide with an average molecular mass of 1.4 x 10(3) kDa. Monosaccharide analysis, methylation analysis, MS, and 1D/2D NMR (1H and 13C) studies performed on the native polysaccharide, and on oligosaccharides obtained from a mild acid hydrolysate of the native polysaccharide, showed the polysaccharide to consist of branched pentasaccharide repeating units with the following structure: [structure: see text].     

Enhanced crude oil biodegradation in soil via biostimulation

Research on feasible methods for the enhancement of bioremediation in soil contaminated by crude oil is vital in oil-exporting countries such as Kuwait, where crude oil is a major pollutant and the environment is hostile to biodegradation. This study investigated the possibility of enhancing crude oil bioremediation by supplementing soil with cost-effective organic materials derived from two widespread locally grown trees, Conocarpus and Tamarix. Amendments in soils increased the counts of soil microbiota by up to 98% and enhanced their activity by up to 95.5%. The increase in the biodegradation of crude oil (75%) and high levels of alkB expression substantiated the efficiency of the proposed amendment technology for the bioremediation of hydrocarbon-contaminated sites. The identification of crude-oil-degrading bacteria revealed the dominance of the genus Microbacterium (39.6%), Sphingopyxis soli (19.3%), and Bordetella petrii (19.6%) in unamended, Conocarpus-amended, and Tamarix-amended contaminated soils, respectively. Although soil amendments favored the growth of Gram-negative bacteria and reduced bacterial diversity, the structures of bacterial communities were not significantly altered.     

Structural and physicochemical characterization of crude biosurfactant produced by Pseudomonas aeruginosa SP4 isolated from petroleum-contaminated soil

Pseudomonas aeruginosa strain SP4, isolated from petroleum-contaminated soil in Thailand, was used to produce a biosurfactant from a nutrient broth with palm oil as the carbon source. The key components of the crude biosurfactant were fractionated by using HPLC-ELSD technique. With the use of ATR-FTIR spectroscopy, in combination with (1)H NMR and MS analyses, chemical structures of the fractionated components of the crude biosurfactant were identified as rhamnolipid species. When compared to synthetic surfactants, including Pluronic F-68, which is a triblock nonionic surfactant containing poly(ethylene oxide) and poly(propylene oxide), and sodium dodecyl sulfate, the crude biosurfactant showed comparable physicochemical properties, in terms of the surface activities. The crude biosurfactant reduced the surface tension of pure water to 29.0 mN/m with a critical micelle concentration of approximately 200 mg/l, and it exhibited good thermal and pH stability. The crude biosurfactant also formed stable water-in-oil microemulsions with crude oil and various types of vegetable oils, but not with short-chain hydrocarbons.     

Porous biocarrier-enhanced biodegradation of crude oil contaminated soil

Soil contamination with crude oil from petrochemicals and oil exploitation is an important worldwide issue. Comparing available remediation techniques, bioremediation is widely considered to be a cost-effective choice; however, slow degradation of crude oil is a common problem due to the low numbers of bacteria capable of degrading petroleum hydrocarbons and the low bioavailability of contaminants in soil. To promote crude oil removal, biocarrier for immobilization of indigenous hydrocarbon-degrading bacteria was developed using porous materials such as activated carbon and zeolite. Microbial biomass reached 10¹⁰ cells g^?1 on activated carbon and 10⁶ cells g^?1 on zeolite. Total microbial and dehydrogenase activities were approximately 12 times and 3 times higher, respectively, in activated carbon than in zeolite. High microbial colonization by spherical and rod shapes were observed for the 5–20 μm thick biofilm on the outer surface of both biocarriers using electronic microscopy. Based on batch-scale experiments containing free-living bacterial cultures and activated carbon biocarrier into crude oil contaminated soil, biocarrier enhanced the biodegradation of crude oil, with 48.89% removal, compared to natural attenuation with 13.0% removal, biostimulation (nutrient supplement only) with 26.3% removal, and bioaugmentation (free-living bacteria) with 37.4% removal. In addition, the biocarrier increased the bacterial population to 10⁸ cells g^?1 dry soil and total microbial activity to 3.5 A₄₉₀. A hypothesis model was proposed to explain the mechanism: the biocarrier improved the oxygen, nutrient mass transfer and water holding capacity of the soil, which were the limiting factors for biodegradation of non-aqueous phase liquid (NAPL) contaminants such as crude oil in soil.Scientific relevanceThis study explored the role of biocarrier in enhancing biodegradation of hydrophobic contaminants such as crude oil, and discussed the function of biocarrier in improving oxygen mass transfer and soil water holding capacity, etc.     

Effects of nitrogen source on crude oil biodegradation

Summary The effects of NH₄Cl and KNO₃ on biodegradation of light Arabian crude oil by an oil-degrading enrichment culture were studied in respirometers. In poorly buffered sea salts medium, the pH decreased dramatically in cultures that contained NH₄Cl, but not in those supplied with KNO₃. The ammonia-associated pH decline was severe enough to completely stop oil biodegradation as measured by oxygen uptake. Regular adjustment of the culture pH allowed oil biodegradation to proceed normally. A small amount of nitrate accumulated in all cultures that contained ammonia, but nitrification accounted for less than 5% of the acid that was observed. The nitrification inhibitor, nitrapyrin, had no effect on the production of nitrate or acid in ammonia-containing cultures. When the culture pH was controlled, either by regular adjustment of the culture pH or by supplying adequate buffering capacity in the growth medium, the rate and extent of oil biodegradation were similar in NH₄Cl- and KNO₃-containing cultures. the lag time was shorter in pH-controlled cultures supplied with ammonia than in nitrate-containing cultures.     

生物表面活性剂在提高原油采收率方面的应用

生物表面活性剂和一般的化学表面活性剂一样,都拥有亲水和疏水基因,是微生物生长在水不溶的有机物中并以营养物而产生的代谢产物。在油田应用中,生物表面活性剂的作用是微生物提高采收率的重要机理之一,具有水溶性好、反应产物均一、安全无毒、驱油效果好等特点。本文从产生生物表面活性剂的菌种及生物表面活性剂的类型、生物表面活性剂的特性、实验研究、矿场实验及展望等五个方面综述了生物表面活性剂在提高原油采收率方面的应     

Detection and characterization of a novel antibacterial substance produced by a Lactobacillus delbrueckii strain 1043

A novel antibacterial substance produced by a strain isolated from Bulgarian yellow cheese was characterized. The producer strain was identified by molecular typing to belong to the species Lactobacillus delbrueckii , which is a rare producer of bacteriocins. The inhibitory agent was heat stable and active against lactic acid bacteria species and several food-borne pathogens : Listeria monocytogenes , Staphylococcus aureus , Enterococcus faecalis , Escherichia coli , Yersinia enterocolitica and Y. pseudotuberculosis . Its sensitivity to amylolitic enzymes and lipase suggested that a lipid and carbohydrate moiety could be important for the activity. The amino acid content of the purified bacteriocin was estimated to 29 amino acids. The bacteriocin was shown to be small (3·6–6 kDa) by three different methods : HPLC gel-filtration, SDS-PAGE and amino acid contents.     

Evidence for in situ crude oil biodegradation after the Prestige oil spill

In November 2002, the oil tanker Prestige sank off the Spanish coast after releasing approximately 17,000 tones of heavy fuel, coating several hundred kilometers of coastline in oil sludge. In December 2002 and February 2003, samples were collected from the shore of the Galician coast to analyse the indigenous population ability to carry out crude oil degradation in situ. Carbon isotopic ratio of the dissolved inorganic carbon (DIC) in seawater samples was used as a rapid method to directly assess activity of microbes on the oil components. 12CO2/13CO2 ratio in samples from certain locations along the coast revealed degradation of a very delta13C-negative source such as the Prestige crude oil (-30.6 per thousand). Putative biodegradation processes taking place at areas with high income of fresh seawater could not be detected with this technique. Laboratory-scale biostimulation processes carried out in samples with the highest oil biodegradation activity showed that N/P deficiency in seawater is a limiting factor for crude oil degradation. The most probable number (MPN) of crude oil component degraders was estimated for several aromatic compounds (naphthalene, anthracene, phenanthrene, pyrene) and for undecane. Our results clearly show that bacteria present in the contaminated water are readily able to transform components of the crude oil into inorganic carbon.     

Production of growth-inhibiting factors by Lactobacillus delbrueckii

AIMS: The detection of growth-inhibiting factors produced by Lactobacillus delbrueckii. METHODS AND RESULTS: A bioscreen assay was developed to study the effect of Lact. delbrueckii culture supernatant fluids on the growth of phylogenically or functionally related bacteria in broth cultures. Several growth-inhibiting factors could be distinguished based on differential effects on different test strains, separation by ultrafiltration and sensitivity to heat, proteinase treatment or catalase addition. CONCLUSION: Lactobacillus delbrueckii strain VI1007 was found to produce at least three growth-inhibiting factors, other than lactic acid, when grown under microaerobic conditions in MRS broth. These included H2O2 and a bacteriocin-like, heat- and proteinase-sensitive bactericidal molecule or complex with a molecular weight greater than 50 kDa. A third factor inhibited the growth of Streptococcus thermophilus. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay system used allows the detection of subtle interactions between strains, that are likely to be of ecological importance in mixed cultures but would go unnoticed in classical agar diffusion tests.     

Volatile hydrocarbon biodegradation by a mixed-bacterial culture during growth on crude oil

Volatile hydrocarbon biodegradation by a mixed-bacterial culture during growth on Bow River crude oil was investigated using solid phase microextraction (SPME). Inoculum treatments were examined in relation to C₅–C₁₁ hydrocarbon degradation. Up to 1600 mg/l biomass (dry weight) was tested without achieving significant volatile hydrocarbon partitioning and affecting analysis. Inoculum age rather than concentration had the most profound impact on biodegradation. When late log phase crude oil-grown inocula were used, C₅–C₁₁ biodegradation reached 55–60%; methylcyclohexane and other branched compounds eluting before n-C₈ were recalcitrant. Increasing the late log inoculum concentration from 0.63 to 63 mg/l resulted in a twofold increase in degradation rate without improving the substrate range. Methylcyclohexane recalcitrance was correlated with reduced levels of hydrocarbon-degrading bacteria and volatile hydrocarbon evaporation from the inoculum flasks. A decreased lag phase prior to degradation was observed when using early stationary phase cultures as inocula and most compounds up to C₁₁, including methylcyclohexane, were biodegraded. Journal of Industrial Microbiology & Biotechnology (2001) 26, 356–362. Received 16 November 2000/ Accepted in revised form 17 March 2001     

Effect of biosurfactant and fertilizer on biodegradation of crude oil by marine isolates of Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa

This study was conducted to investigate the effects of fertilizers and biosurfactants on biodegradation of crude oil by three marine bacterial isolates; Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa. Five sets of experiments were carried out in shake flask and microcosm conditions with crude oil as follows: Set 1-only bacterial cells added (no fertilizer and biosurfactant), Set 2-with additional fertilizer only, Set 3-with additional biosurfactant only, Set 4-with added biosurfactant + fertilizer, Set 5-with no bacterial cells added (control), all the above experimental sets were incubated for 168 h. The biosurfactant + fertilizer added Set 4, resulted in maximum crude oil degradation within shake flask and microcosm conditions. Among the three bacterial isolates, P. aeruginosa and biosurfactant produced by this strain resulted in maximum crude oil degradation compared to the other two bacterial strains investigated. Interestingly, when biosurfactant and bacterial cells were used (Set 3), significant oil biodegradation activity occurred and the difference between this treatment and that in Set 4 with added fertilizer + biosurfactant were only 4-5% higher degradation level in shake flask and 3.2-7% in microcosm experiments for all three bacterial strains used. It is concluded that, biosurfactants alone capable of promoting biodegradation to a large extent without added fertilizers, which will reduce the cost of bioremediation process and minimizes the dilution or wash away problems encountered when water soluble fertilizers used during bioremediation of aquatic environments.     

Structure of a neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B26

The neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B26 in skimmed milk was found to be composed of d-glucose and d-galactose in a molar ratio of 2:3. Linkage analysis and 1D/2D NMR ((1)H and (13)C) studies performed on the native polysaccharide, and on an oligosaccharide obtained from a partial acid hydrolysate of the native polysaccharide, showed the polysaccharide to consist of branched pentasaccharide repeating units with the following structure. [structure: see text]     

Structural characterisation of a highly branched exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB2074

Lactobacillus delbrueckii subsp. bulgaricus NCFB2074 when grown in skimmed milk secretes a highly branched exopolysaccharide. The exopolysaccharide has a heptasaccharide repeat unit and is composed of glucose and galactose in the molar ratio 3:4. Using chemical techniques and 1D and 2D NMR spectroscopy the polysaccharide has been shown to possess the following repeat unit structure: [carbohydrate structure: see text].     

Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis by SDS-PAGE of cell-wall proteins

AIMS: In the present study, a method based on SDS-PAGE fingerprinting of surface layer proteins was developed to identify Lactobacillus delbrueckii subsp. bulgaricus and subsp. lactis dairy isolates. METHODS AND RESULTS: The two subspecies, identified by species-specific PCR, were characterized by different SDS-PAGE cell-wall protein profiles; subspecies bulgaricus showed one band of about 31 kDa which, in some cases, was observed at a doublet, and subspecies lactis showed one band of about 21 kDa or 18 kDa. CONCLUSION: The sensitivity of this procedure for discriminating between the two subspecies was very high. The different types of SDS-PAGE profile for cell-wall proteins of the strains studied in this work did not seem to be correlated to the different dairies of origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The method appears to be an efficient taxonomic tool. It has the advantage of easy gel interpretation over fingerprinting of whole-cell protein extracts, and may be used as an alternative to established PCR-based techniques which, though rapid and safe, require expensive instruments and reagents.     

Modeling of crude oil biodegradation using two phase partitioning bioreactor

In this work, crude oil biodegradation has been optimized in a solid‐liquid two phase partitioning bioreactor (TPPB) by applying a response surface methodology based d ‐optimal design. Three key factors including phase ratio, substrate concentration in solid organic phase, and sodium chloride concentration in aqueous phase were taken as independent variables, while the efficiency of the biodegradation of absorbed crude oil on polymer beads was considered to be the dependent variable. Commercial thermoplastic polyurethane (Desmopan®) was used as the solid phase in the TPPB. The designed experiments were carried out batch wise using a mixed acclimatized bacterial consortium. Optimum combinations of key factors with a statistically significant cubic model were used to maximize biodegradation in the TPPB. The validity of the model was successfully verified by the good agreement between the model‐predicted and experimental results. When applying the optimum parameters, gas chromatography‐mass spectrometry showed a significant reduction in n‐alkanes and low molecular weight polycyclic aromatic hydrocarbons. This consequently highlights the practical applicability of TPPB in crude oil biodegradation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:797–805, 2014     

Lactic acid production from cellulosic waste by immobilized cells of Lactobacillus delbrueckii

Industrial waste corn cob residue (from xylose manufacturing) without pretreatment was hydrolyzed by cellulase and cellobiase. The cellulosic hydrolysate contained 52.4 g l⁻¹ of glucose and was used as carbon source for lactic acid fermentation by cells of Lactobacillus delbrueckii ZU-S2 immobilized in calcium alginate gel beads. The final concentration of lactic acid and the yield of lactic acid from glucose were 48.7 g l⁻¹ and 95.2%, respectively, which were comparative to the results of pure glucose fermentation. The immobilized cells were quite stable and reusable, and the average yield of lactic acid from glucose in the hydrolysate was 95.0% in 12 repeated batches of fermentation. The suitable dilution rate of continuous fermentation process was 0.13 h⁻¹, and the yield of lactic acid from glucose and the productivity were 92.4% and 5.746 g l⁻¹ h⁻¹, respectively. The production of lactic acid by simultaneous saccharification and fermentation (SSF) process was carried out in a coupling bioreactor, the final concentration of lactic acid was 55.6 g l⁻¹, the conversion efficiency of lactic acid from cellulose was 91.3% and the productivity was 0.927 g l⁻¹ h⁻¹. By using fed-batch technique in the SSF process, the final concentration of lactic acid and the productivity increased to 107.6 g l⁻¹ and 1.345 g l⁻¹ h⁻¹, respectively, while the dosage of cellulase per gram substrate decreased greatly. This research work should advance the bioconversion of renewable cellulosic resources and reduce environmental pollution.