Evaluation of bacterial surfactant toxicity towards petroleum degrading microorganisms

The acute toxicity of bacterial surfactants LBBMA111A, LBBMA155, LBBMA168, LBBMA191 and LBBMA201 and the synthetic surfactant sodium dodecyl sulfate (SDS) on the bioluminescent bacterium Vibrio fischeri was evaluated by measuring the reduction of light emission (EC(20)) by this microorganism when exposed to different surfactant concentrations. Moreover, the toxic effects of different concentrations of biological and synthetic surfactants on the growth of pure cultures of isolates Acinetobacter baumannii LBBMA04, Acinetobacter junni LBBMA36, Pseudomonas sp. LBBMA101B and Acinetobacter baumanni LBBMAES11 were evaluated in mineral medium supplemented with glucose. The EC(20) values obtained confirmed that the biosurfactants have a significantly lower toxicity to V. fischeri than the SDS. After 30 min of exposure, bacterial luminescence was almost completely inhibited by SDS at a concentration of 4710 mg L(-1). Growth reduction of pure bacterial cultures caused by the addition of biosurfactants to the growth medium was lower than that caused by SDS.     

Simultaneous phenanthrene and cadmium removal from contaminated soil by a ligand/biosurfactant solution

Surfactants and inorganic ligands are pointed as efficient to simultaneous removal of heavy metals and hydrophobic organic pollutants from soil. However, the biosurfactants are potentially less toxic to soil organisms than other chemical agents. Thus, in this study the efficiency of combinations of iodide (I⁻) ligand and surfactants produced by different bacterial species in the simultaneous removal of cadmium (Cd²⁺) and phenanthrene in a Haplustox soil sample was investigated. Four microbial surfactants and the synthetic surfactant Triton X-100 were tested with different concentrations of ligand. Soil samples contaminated with Cd²⁺ and phenanthrene underwent consecutive washings with a surfactant/ligand solution. The removal of Cd²⁺ increased with increased ligand concentration, particularly in solutions containing biosurfactants produced by the bacterial strains Bacillus subtilis LBBMA155 (lipopeptide) and Flavobacterium sp. LBBMA168 (mixture of flavolipids) and Triton X-100. Maximum Cd²⁺ removal efficiency was 99.2% for biosurfactant produced by Arthrobacter oxydans LBBMA 201 (lipopeptide) and 99.2% for biosurfactant produced by Bacillus sp. LBBMA111A (mixed lipopeptide) in the presence of 0.336 mol iodide l⁻¹, while the maximum efficiency of Triton X-100 removal was 65.0%. The biosurfactant solutions removed from 80 to 88.0% of phenanthrene in soil, and the removal was not influenced by the presence of the ligand. Triton X-100 removed from 73 to 88% of the phenanthrene and, differently from the biosurfactants, iodide influenced the removal efficiency. The results indicate that the use of a single washing agent, called surfactant-ligand, affords simultaneous removal of organic contaminants and heavy metals.     

Basis for formulating biosurfactant mixtures to achieve ultra low interfacial tension values against hydrocarbons

Biosurfactants could potentially replace or be used in conjunction with synthetic surfactants to provide for more cost-effective subsurface remediation. The design of surfactant formulations that are effective in lowering interfacial tension (IFT), which is necessary to mobilize entrapped hydrocarbons, requires information about the surface-active agent (surfactant) and the targeted non-aqueous phase liquids (NAPL). We hypothesized that biosurfactant and synthetic surfactant mixtures can be formulated to provide the appropriate hydrophobic/hydrophilic conditions necessary to produce low IFT against NAPLs, and that such mixtures will produce synergism that make them more effective than individual biosurfactants or synthetic surfactants. Our work tested the interfacial activity of biosurfactants from individual strains and mixtures of biosurfactants from different strains with and without a synthetic surfactant. Multiple regression analysis showed that, for lipopeptide biosurfactants produced by various Bacillus species, the interfacial activity against toluene depended on the relative proportions of 3-OH-C₁₄, C₁₅, C₁₆, and C₁₈ in the fatty acid tail. As the fatty acid composition became more heterogeneous the system produced lower IFT against toluene. In mixtures of lipopeptide biosurfactants with the more hydrophilic, rhamnolipid biosurfactant, the IFT against toluene decreased as the percentage of the 3-OH C₁₄ fatty acid increased in the lipopeptide. Mixtures of lipopeptide biosurfactants with the more hydrophobic synthetic surfactant, C12, C13-8PO SO₄Na, were able to produce low IFT against hexane and decane. In general, we found that lipopeptide biosurfactants with a heterogeneous fatty acid composition or mixtures of lipopeptide and rhamnolipid biosurfactants lowered the IFT against hydrophilic NAPLs. Conversely, mixtures of lipopeptide biosurfactants with a more hydrophobic synthetic surfactant lowered the IFT against hydrophobic NAPLs.     

Adding sodium dodecyl sulfate and Pseudomonas aeruginosa UG2 biosurfactants inhibits polycyclic aromatic hydrocarbon biodegradation in a weathered creosote-contaminated soil

  The effect of two anionic surfactants was assessed during biodegradation of 13 of the 16 USEPA priority polycyclic aromatic hydrocarbons (PAH) in a wood-preserving soil contaminated with creosote and pentacholorophenol for a period of at least 20 years. Sodium dodecyl sulfate (SDS) and biosurfactants from Pseudomonas aeruginosa UG2 were utilized at concentrations of 10, 100 and 500 μg/g soil. Because both surfactants are readily biodegradable, the microcosms received a fresh spike of surfactant every 2 weeks. Biodegradation of aged PAH residues was monitored by GC/MS for a period of 45 weeks. Results indicated that the biodegradation of the three-ring PAH was rapid and almost complete but was slowed by the addition of 100 μg/g and 500 μg/g chemical surfactant. Similarly, at the same concentrations, the two surfactants significantly decreased the biodegradation rate of the four-ring PAH. In this case, the inhibition was more pronounced with SDS. High-molecular-mass PAH (more than four rings) were not biodegraded under the test conditions. It was suggested that the preferential utilization of surfactants by PAH degraders was responsible for the inhibition observed in the biodegradation of the hydrocarbons. The high biodegradability and the inhibitory effect of these two surfactants would have a significant impact on the development of both above-ground and in situ site reclamation processes. Received: 22 February 1996 / Received revision: 31 May 1996 / Accepted: 16 June 1996     

Degradation of selected (bio-)surfactants by bacterial cultures monitored by calorimetric methods

The subjects of the article are investigations concerning the ability of both Rhodococcus opacus 1CP and mixed bacterial cultures to use selected surfactants as sole carbon and energy source. In a comparative manner the biosurfactants rhamnolipid, sophorolipid and trehalose tetraester, and the synthetic surfactant Tween 80 were examined. Particular emphasis was put on a combinatorial approach to determine quantitatively the degree of surfactant degradation by applying calorimetry, thermodynamic calculations and mass spectrometry, HPLC as well as determination of biomass. The pure bacterial strain R. opacus was only able to metabolize a part of the synthetic surfactant Tween 80, whereas the mixed bacterial cultures degraded all of the applied surfactants. Exclusive for the biosurfactant rhamnolipid a complete microbial degradation could be demonstrated. In the case of the other surfactants only primary degradation was observed.     

Isolation and characterisation of surface active compound-producing bacteria from hydrocarbon-contaminated environments

Bacteria able to produce surface active compounds (SACs) were isolated from hydrocarbon-contaminated environments. The phylogenetic diversity of the isolates was evaluated by 16S rRNA gene analysis. The production of bioemulsifiers and biosurfactants was determined on strains representative of 18 different bacterial genera. Cupriavidus sp. BSNC28C produced extracellular biosurfactants which reduce the surface tension into the culture medium up to 37.1 mN m⁻¹. Sixteen strains, belonging to 11 different genera, released extracellular emulsifiers able to stabilise oil–water emulsions. Among them, the strains Bradyrhizobium sp. BSNC30A and Bosea sp. BSNC5B showed emulsification activities comparable to those of synthetic surfactants. Overall, the novel SAC-producing strains characterised in this work display promising features for the future development of economically efficient industrial-scale biotechnological processes.     

Trophic interactions in soils as they affect energy and nutrient dynamics. IV. Flows of metabolic and biomass carbon

Flows of biomass and respiratory carbon were studied in a series of propylene-oxide sterilized soil microcosms. One-half of the microcosms received three pulsed additions of 200 ppm glucose-carbon to mimic rhizosphere carbon inputs. Biotic variables were: bacteria (Pseudomonas) alone, or amoebae (Acanthamoeba) and nematodes (Mesodiplogaster) singly, or both combined in the presence of bacteria.Over the 24-day experiment, respiration was significantly higher in the microcosms containing the bacterial grazers. Biomass accumulation by amoebae was significantly higher than that by nematodes. The nematodes respired up to 30-fold more CO₂ per unit biomass than did amoebae. Similar amounts of carbon flowed into both respiratory and biomass carbon in microcosms with fauna, compared with the bacteria-alone microcosms. However, partitioning of available carbon by the microfauna varied considerably, with little biomass production and relatively more CO₂-C produced in the nematode-containing microcosms. The amoebae, in contrast, allocated more carbon to tissue production (about 40% assimilation efficiency) and correspondingly less to CO₂.     

Bacterial biosurfactant increases ex situ biodiesel bioremediation in clayey soil

The contamination of soils by oily compounds has several environmental impacts, which can be reversed through bioremediation, using biosurfactants as auxiliaries in the biodegradation process. In this study, we aimed to perform ex situ bioremediation of biodiesel-contaminated soil using biosurfactants produced by Bacillus methylotrophicus. A crude biosurfactant was produced in a whey-based culture medium supplemented with nutrients and was later added to biodiesel-contaminated clayey soil. The produced lipopeptide biosurfactant could reduce the surface tension of the fermentation broth to 30.2 mN/m. An increase in the microbial population was observed in the contaminated soil; this finding can be corroborated by the finding of increased CO₂ release over days of bioremediation. Compared with natural attenuation, the addition of a lower concentration of the biosurfactant (0.5% w/w in relation to the mass of diesel oil) to the soil increased biodiesel removal by about 16% after 90 days. The added biosurfactant did not affect the retention of the contaminant in the soil, which is an important factor to be considered when applying in situ bioremediation technologies.

     

Respiratory activity of alginate-encapsulated Pseudomonas fluorescens cells introduced into soil

Summary Alginate-entrapped cells of Pseudomonas fluorescens were introduced into soil microcosms to evaluate their respiratory activity (O₂ consumption and CO₂ evolution) and survival during a 14-day incubation period at 20°C. Alginate-entrapped cells and cells resuspended in sterile distilled water and introduced into sterile soil exhibited relatively similar O₂ consumption/CO₂ evolution and survival over the 14-day period. The same treatments in non-sterile soil exhibited lower respiratory activity and a population density decrease of about 2.0 Log. cfu/g after 14 days. Alginate-entrapped bacterial cells may be a useful method for introducing genetically-engineered and non-engineered bacterial strains into the soil environment.     

Ectomycorrhizal exudates and pre-exposure to elevated CO2 affects soil bacterial growth and community structure

Ectomycorrhizal fungi produce low molecular weight organic compounds, supporting diverse microbial communities. To link mycorrhizal root exudation directly to bacterial responses, we used Scots pine exudates with (Suillus variegatus and Piloderma fallax) and without mycorrhiza as substrata for forest soil bacteria. Bacterial growth and vitality was monitored, and community composition determined using T-RFLP, cloning and sequencing. We investigated if the amount of organic acids in exudates explained bacterial growth, and whether bacterial communities were influenced by pre-exposure to elevated atmospheric CO₂. We demonstrated functional differences in bacterial growth rates related to CO₂. There was a shift in the bacterial community (e.g. Burkholderia sp. and gamma-proteobacteria) toward organisms better able to rapidly utilize exudates when pine microcosms were pre-exposed to elevated CO₂. Soil bacteria from all treatments tended to grow more abundantly and rapidly in exudates from Piloderma-colonized seedlings, suggesting that the organic acids and/or unidentified compounds present supported greater growth.     

Isolation,purification, and characterization of novel fengycin S from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> LSC04 degrading-crude oil

In this study, a biosurfactant-producing bacterial strain was isolated from oil-contaminated soil on the basis of its ability to degrade crude oil and tributyrin (C_4:0). LSC04 was identified as Bacillus amyloliquefaciens LSC04 via 16S rRNA gene analysis and partial gyrA gene sequence analysis. The biosurfactants were purified and structural analysis results showed that B. amyloliquefaciens LSC04 generated a lipopeptide biosurfactant. Two main ions of 1,086.9 and 1,491.2 were measured via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The m/z 1,491.2 was shown to correspond to the lipopeptide fengycin B, but the m/z 1,086.9 ion did not correspond to any known lipopeptide. As constituents of the peptides and the lipophilic portion of the m/z 1,491.2; 10 amino acids (Ile-Tyr-Gln-Pro-Val-Glu-Ser-Tyr-Orn-Glu); and β-hydroxy-C17 fatty acid were identified via ESI-MS/MS. Structurally, the lipopeptide of a molecular mass of 1,491.2 differed from fengycin B and fengycin A by a substitution of serine for the threonine residue in position 4, and the amino acid residue in position 6 was equal to that of fengycin A. The major compound, which had a molecular mass of 1,491.2 Da was designated “Fengycin S”.     

Lipopeptide produced from <Emphasis Type="Italic">Bacillus</Emphasis> sp. W112 improves the hydrolysis of lignocellulose by specifically reducing non-productive binding of cellulases with and without CBMs

Background

Surfactants have attracted increasing interest for their capability to improve the enzymatic hydrolysis of lignocellulosic biomass. Compared to chemical surfactants, biosurfactants have a broader prospect for industrial applications because they are more environmentally friendly and more effective in some researches. Commercial cellulase preparations are mainly composed of endoglucanases (EGs) and cellobiohydrolases (CBHs) that possess carbohydrate-binding modules (CBMs). However, the effects of lipopeptide-type biosurfactants on enzymatic saccharification of lignocellulose and adsorption behaviors of cellulases with CBMs remain unclear.

Results

In this study, we found that Bacillus sp. W112 could produce a lipopeptide-type biosurfactant from untreated biomass, such as wheat bran and Jerusalem artichoke tuber. The lipopeptide could enhance the enzymatic hydrolysis of dilute acid pretreated Giant Juncao grass (DA-GJG) by fungal and bacterial enzymes. The enhancement increased over a range of temperatures from 30 to 50 °C. Lipopeptide was shown to be more effective in promoting DA-GJG saccharification than chemical surfactants at low dosages, with a best stimulatory degree of 20.8% at 2% loading of the substrates (w/w). Lipopeptide increased the thermostability of EG and CBH in commercial cellulase cocktails. Moreover, the dual effects of lipopeptide on the adsorption behaviors of cellulases were found. It specifically lowered the non-productive binding of cellulases to lignin and increased the binding of cellulases to cellulose. In addition, we investigated the influence of lipopeptide on the adsorption behaviors of CBHs with CBMs for the first time. Our results showed that lipopeptide reduced the adsorption of CBM-deleted CBH to DA-GJG to a greater extent than that of intact CBH while the non-productive binding of intact CBH to lignin was reduced more, indicating that lipopeptide decreased the binding of CBMs onto lignin but not their combination with cellulose.

Conclusions

In this study, we found that lipopeptide from Bacillus sp. W112 promoted the enzymatic hydrolysis of DA-GJG at relative low loadings. The stimulatory effect could be attributed to increasing the cellulase thermostability, reducing non-productive adsorption of cellulases with CBMs caused by lignin and enhancing the binding of cellulases to cellulose.

     

Effect of surfactants on fluoranthene degradation by <Emphasis Type="Italic">Pseudomonas alcaligenes</Emphasis> PA-10

Two surfactants, Tween 80 and JBR, were investigated for their effect on fluoranthene degradation by a Pseudomonad. Both surfactants enhanced fluoranthene degradation by Pseudomonas alcaligenes PA-10 in shake flask culture. This bacterium was capable of utilising the synthetic surfactant and the biosurfactant as growth substrates and the critical micelle concentration of neither compound inhibited bacterial growth. The biosurfactant JBR significantly increased polycyclic aromatic hydrocarbon (PAH) desorption from soil. Inoculation of fluoranthene-contaminated soil microcosms with P. alcaligenes PA-10 resulted in the removal of significant amounts (45 ± 5%) of the PAH after 28 days compared to an uninoculated control. Addition of the biosurfactant increased the initial rate of fluoranthene degradation in the inoculated microcosm. The presence of a lower molecular weight PAH, phenanthrene, had a similar effect on the rate of fluoranthene removal.     

Potential application of cyclic lipopeptide biosurfactants produced by Bacillus subtilis strains in laundry detergent formulations

AIMS: Crude cyclic lipopeptide (CLP) biosurfactants from two Bacillus subtilis strains (DM-03 and DM-04) were studied for their compatibility and stability with some locally available commercial laundry detergents. METHODS AND RESULTS: CLP biosurfactants from both B. subtilis strains were stable over the pH range of 7.0-12.0, and heating them at 80 degrees C for 60 min did not result in any loss of their surface-active property. Crude CLP biosurfactants showed good emulsion formation capability with vegetable oils, and demonstrated excellent compatibility and stability with all the tested laundry detergents. CONCLUSION: CLP biosurfactants from B. subtilis strains act additively with other components of the detergents to further improve the wash quality of detergents. The thermal resistance and extreme alkaline pH stability of B. subtilis CLP biosurfactants favour their inclusion in laundry detergent formulations. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has great significance because it is already known that microbial biosurfactants are considered safer alternative to chemical or synthetic surfactants owing to lower toxicity, ease of biodegradability and low ecological impact. The present study provides further evidence that CLP biosurfactants from B. subtilis strains can be employed in laundry detergents.     

Evaluation of microbial surfactants for recovery of hydrophobic pollutants from soil

Summary Several microbially produced biosurfactants were evaluated for their ability to remove hydrophobic compounds from soil. The biosurfactants produced byPseudomonas aeruginosa UG2 andAcinetobacter calcoaceticus RAG-1 displayed the best results, with recovery of [¹⁴C]hexachlorobiphenyl from soil slurries of 48.0 and 41.9%, respectively.P. aeruginosa UG2 produced higher levels of extracellular biosurfactants than four otherP. aeruginosa strains.P. aeruginosa UG2 culture filtrate containing biosurfactants enhanced the recovery of several other individual hydrocarbons and polychlorinated biphenyl compounds, as well as several hydrocarbons in a mixture, from soil. The results, suggest that biosurfactants produced byP. aeruginosa UG2 have the potential for remediation of hydrophobic pollutants in soil environments.     

Biodegradation of petroleum industry oily-sludge using Jordanian oil refinery contaminated soil

The main objective of this study was to evaluate the effect of oily sludge concentration on its biodegradability in soil. Oily sludge was collected and applied to microcosms at full-, half-, or quarter-strength concentrations equivalent to 44.2, 22.2, and 11.1 g kg^?1 soil, respectively, of total petroleum hydrocarbons (TPH) contained in oily sludge. The biodegradability of oily sludge was evaluated by measuring CO₂ evolution and by measuring removal of TPH as well as its main composing fractions; namely; alkanes, aromatics, NSO-compounds, and asphaltenes. The collected soil contained 3.63 × 10⁶ cfu g^?1 soil of hydrocarbon-degrading bacteria, which is satisfactory to drive successful biodegradation of hydrocarbons in soil. These numbers increased significantly with oily sludge addition at a rate proportional to the added TPH reaching 3.35 × 10⁷ cfu g^?1 soil in the half-strength treatment. TPH mineralization rate followed the same pattern. However, TPH-mineralization efficiency was the greatest in quarter-strength treatment at 18.3%. TPH-removal efficiency was also highest in quarter-strength treatment at 30.9%. Nutrients addition caused mineralization inhibition. Since nutrients were added as a ratio of the added carbon, inhibition was the greatest with the highest TPH treatment. While alkanes were degraded, aromatics and asphaltenes were not, and NSO-compounds were enriched. Although SDS was completely biodegradable in soil, its addition promoted mineralization and removal of TPH from soil.     

Metal Removal from Contaminated Soils Through Bioleaching with Oxidizing Bacteria and Rhamnolipid Biosurfactants

The use of surfactants as a method for solubilization and removal of heavy metal contamination from soil has been reported before. Biosurfactants produced by some microorganisms are able to modify the surface of various metals and aggregate on interphases favoring the metal separation process from contaminated environments. We evaluated the feasibility of enhancing the removal of metal ions from mineral waste/contaminated soils using alternate cycles of treatment with rhamnolipid biosurfactants and bioleaching with a mixed bacterial culture of Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans. Bioleaching alone removed 50% Zn and 19% Fe. When rhamnolipids were used at low concentration (0.4 mg/mL), 11% Fe and 25% Zn were removed, while at 1 mg/mL 19% Fe and 52% Zn removal were achieved. When using a cyclic treatment combining bioleaching and biosurfactants, metal removal reached up to 36% for Fe and 63% to 70% for Zn.     

Diverse mechanisms for CO2 effects on grassland litter decomposition

The ongoing increase in atmospheric CO₂ concentration ([CO₂]) can potentially alter litter decomposition rates by changing: (i) the litter quality of individual species, (ii) allocation patterns of individual species, (iii) the species composition of ecosystems (which could alter ecosystem‐level litter quality and allocation), (iv) patterns of soil moisture, and (v) the composition and size of microbial communities. To determine the relative importance of these mechanisms in a California annual grassland, we created four mixtures of litter that differed in species composition (the annual legume Lotus wrangelianus Fischer & C. Meyer comprised either 10% or 40% of the initial mass) and atmospheric [CO₂] during growth (ambient or double‐ambient). These mixtures decomposed for 33 weeks at three positions (above, on, and below the soil surface) in four types of grassland microcosms (fertilized and unfertilized microcosms exposed to elevated or ambient [CO₂]) and at a common field site. Initially, legume‐rich litter mixtures had higher nitrogen concentrations ([N]) than legume‐poor mixtures. In most positions and environments, the different litter mixtures decomposed at approximately the same rate. Fertilization and CO₂ enrichment of microcosms had no effect on mass loss of litter within them. However, mass loss was strongly related to litter position in both microcosms and the field. Nitrogen dynamics of litter were significantly related to the initial [N] of litter on the soil surface, but not in other positions. We conclude that changes in allocation patterns and species composition are likely to be the dominant mechanisms through which ecosystem‐level decomposition rates respond to increasing atmospheric [CO₂].     

Isolation and characterization of bacteria from soil contaminated with diesel oil and the possible use of these in autochthonous bioaugmentation

Two bacterial species (isolates N and O) were isolated from a paddy soil microcosm that had been artificially contaminated with diesel oil to which extrinsic Pseudomonas aeruginosa strain WatG, had been added exogenously. One bacterial species (isolate J) was isolated from a similar soil microcosm that had been biostimulated with Luria–Bertani (LB) medium. Isolates N and O, which were tentatively identified as Stenotrophomonas sp. and Ochromonas sp., respectively, by sequencing of their 16 S rRNA genes had no ability to degrade diesel oil on their own in any liquid medium. When each strain was cocultivated with P. aeruginosa strain WatG in liquid mineral salts medium (MSM) containing 1% diesel oil, isolate N enhanced the degradation of diesel oil by P. aeruginosa strain WatG, but isolate O inhibited it. In contrast, isolate J, which was tentatively identified as a Rhodococcus sp., degraded diesel oil contained not only in liquid LB and MSM, but also in paddy soil microcosms supplemented with LB medium. The bioaugmentation capacity of isolate J in soil microcosms contaminated with diesel oil was much higher than that of P. aeruginosa strain WatG. The possibility of using isolate J for autochthonous bioaugmentation is discussed.