Isolation and Characterization of Biosurfactant-and Bioemulsifier-Producing Bacteria from Petroleum Contaminated Sites in Western Canada

Biosurfactant-producing bacteria were isolated from two petroleum contaminated sites in western Canada. Seven potential biosurfactant/bioemulsifier-producing isolates were screened and characterized. All of the seven isolates were able to form emulsions. Emulsion-stabilizing capacity was also measured up to 48 hrs. Strain C-111-2 and C-203-2 would lead to highly reduced surface tension. For strain C-203-2, the optimum conditions that supported bacteria growth and production were investigated. The influences of carbon sources, medium pH values, and temperature were taken into account. The experimental results indicated that the crude oil and glucose were promising carbon sources for biosurfactants production; the isolated strains produced a maximum concentration of biosurfactant in a neutral pH environment and showed a higher surface activity under the temperature level of 35°C than that under 10°C. To further optimize the carbon and nitrogen source for biosurfactant production, response surface methodology (RSM) was applied to explore the favorable concentration of two carbon sources: glucose, crude oil, and one nitrogen source, NaNO₃. The optimal concentration of 8.1g/L, 4% and 3.9 g/L for glucose, crude oil, and NaNO₃, respectively, which can be obtained through RSM analysis.     

Optimization of submerged fermentation medium for citrinin-free monascin production by Monascus

Microbial fermentation of citrinin-free Monascus pigments is in favor in the development of food industry. This study investigated the influences of carbon source, nitrogen source, and mineral salts on the cell growth, monascin (MS), and citrinin (CT) production in Monascus M9. A culture medium composition was established for maximizing the production of citrinin-free MS in submerged culture, as follows: 50?g/L Japonica rice powder, 20?g/L NH₄NO₃, 3?g/L NaNO₃, 1.5?g/L KH₂PO₄, 1?g/L MgSO₄?·?7H₂O, 0.2?g/L MnSO₄. Under these conditions, no CT was detectable by high performance liquid chromatography. The yield of MS reached 14.11?mg/g, improving approximately 30% compared with before optimization.     

Isolation and characterization of a biosurfactant‐producing Fusarium sp. BS‐8 from oil contaminated soil

This study reports characterization of a biosurfactant‐producing fungal isolate from oil contaminated soil of Missa Keswal oil field, Pakistan. It was identified as Fusarium sp. BS‐8 on the basis of macroscopic and microscopic morphology, and 18S rDNA gene sequence homology. The biosurfactant‐producing capability of the fungal isolates was screened using oil displacement activity, emulsification index assay, and surface tension (SFT) measurement. The optimization of operational parameters and culture conditions resulted in maximum biosurfactant production using 9% (v/v) inoculum at 30°C, pH 7.0, using sucrose and yeast extract, as carbon and nitrogen sources, respectively. A C:N ratio of 0.9:0.1 (w/w) was found to be optimum for growth and biosurfactant production. At optimal conditions, it attained lowest SFT (i.e., 32 mN m^?1) with a critical micelle concentration of ≥ 1.2 mg mL^?1. During 5 L shake flask fermentation experiments, the biosurfactant productivity was 1.21 g L^?1 pure biosurfactant having significant emulsifying index (E₂₄, 70%) and oil‐displacing activity (16 mm). Thin layer chromatography and Fourier transform infrared spectrometric analyses indicated a lipopeptide type of the biosurfactant. The Fusarium sp. BS‐8 has substantial potential of biosurfactant production, yet it needs to be fully characterized with possibility of relatively new class of biosurfactants. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1065–1075, 2014     

A Biosurfactant-Producing Pseudomonas aeruginosa Strain

A Pseudomonas aeruginosa strain producing an extracellular surfactant (biosurfactant) was isolated. The growth of this strain, referred to as 50.3, on a mineral glycerol-containing medium produces an emulsifying activity (60%) and decreases the surface tension of the culture liquid by a factor of 2.8 (to 25 mN/m). The optimum conditions for its growth and production of biosurfactants are intense aeration, pH 7.0–8.0, and the presence of Mg²⁺. The optimum biosurfactant properties were achieved when glucose was used as the only source of carbon and energy and NH₄Cl was used as a source of nitrogen. The biosurfactant was isolated from the culture liquid by extraction and precipitation.     

CULTURE AND PHYSIOLOGICAL REQUIREMENTS OF A BACTERIZED CLADOPHORA GLOMERATA (L.) KUTZ FROM LAKE MICHIGAN

A defined medium was developed for a bacterized Gladophora glomerata from Lake Michigan yielding 0.8 g dry weight/m²/20 days and a doubling time of 1.2 days. Best growth was at 25 C under continuous aeration and 200–500 ft-c light. Aeration with 2% CO₂, with or without THIS buffer, inhibited growth. Calcium nitrate, urea, and NaNO₃ were all utilized as nitrogen sources; however, more NO₃ nitrogen was required than urea or Ca nitrate N for maximum yields. Germination of zoospores through zoosporangial walls was observed.     

Distillery and curd whey wastes as viable alternative sources for biosurfactant production

Biosurfactant production from synthetic medium and industrial waste, viz. distillery and whey wastes was investigated by using an oily sludge isolate Pseudomonas aeruginosa strain BS2. In synthetic medium separately supplemented with glucose and hexadecane as water-soluble and -insoluble carbon sources, respectively, strain BS2 reduced the surface tension of the fermentation broth from 57 to 27 mN/m. The culture produced biosurfactant during the stationary growth phase and its yield was 0.97 g/l. The culture utilized distillery and whey wastes for its growth, as maximum cell counts reached to 54 × 10⁸ and 64 × 10⁹ c.f.u./ml from an initial inoculum size of 1 × 0⁵ c.f.u./ml, respectively, within 48 h of incubation and in these wastes the yields of biosurfactant obtained were 0.91 and 0.92 g/l, respectively. In synthetic medium, distillery and whey wastes, strain BS2 produced a crystalline biosurfactant which belonged to the category of secondary metabolites and its maximum production occurred after the onset of nitrogen-limiting conditions. After recovering biosurfactant from the fermented waste, the chemical oxygen demand (COD) of distillery and whey wastes was significantly reduced by 81 and 87%, respectively. Total acids, nitrogen and phosphate levels in distillery waste were reduced by 90, 92 and 92%, respectively, while in case of whey waste the concentration of these nutrients was reduced by 88, 95 and 93%, respectively. The isolated biosurfactant possessed potent surface active properties, as it effectively reduced the surface tension of water from 72 to 27 mN/m and formed 100% stable emulsions of a variety of water-insoluble compounds such as hydrocarbons, viz. hexadecane, crude oil, kerosene and oily sludge and pesticides, viz. dichlorodiphenyltrichloroethane (DDT) and benzene hexachloride (BHC). The effectiveness of biosurfactant was also evident from its low critical micellar concentration (CMC) which was 0.028 mg/ml.     

Antioxidant property and production of exopolysaccharide from Armillaria mellea in submerged cultures: Effect of culture aeration rate

Effects of culture aeration rate on production and antioxidant property of exopolysaccharide (EPS) by Armillaria mellea were investigated in a 5‐L stirred‐tank bioreactor where an optimal biomass aeration rate of 1.2 vvm with 0.22 g/g cell yield and 0.6 vvm EPS formation rate with 7.66 mg/g product yield were achieved. A two‐stage aeration process to maximize the biomass and EPS productions proceeded with a 1.55‐fold enhancement (from 4.28 to 6.65 g/L) in biomass formation and a 2.68‐fold enhancement (from 86.9 to 233.2 mg/L) in the EPS production, as compared with those from the aeration rate of 0.3 vvm. The molecular weights of EPS in cultures of different aeration rates are closely correlated with their protein/polysaccharide ratios (R²=0.830) and EC₅₀ (EC₅₀, the effective concentration where the antioxidant property is 50%) values in antioxidant activity (R²=0.960), reducing power (R²=0.894) and chelating ability (R²=0.954). EPS from the two‐stage aeration rate culture shows a strong antioxidant property by the conjugated diene method, reducing power and chelating ability on ions. Therefore, we present results to regulate and to optimize A. mellea cultures to efficiently produce biomass and EPS. The fermented EPS has the potential to be used as for antioxidant‐related functional foods and pharmaceutical industries.     

Emulsifying and surface active agents from Corynebacterium hydrocarboclastus

A Corynebacterium hydrocarboclastus culture isolated in our laboratory (see, Zajic and Knettig, Developments in Industrial Microbiology, 1971, p. 87) has been shown to produce an extracellular biopolymer with emulsifying properties when grown on a mixture of linear hydrocarbons. This microorganism was found to grow well on a variety of carbohydrates and hydrocarbons. However, the best substrates were pure linear hydrocarbons and particularly, n-C₁₂, n-C₁₃, and n-C₁₄. The substrates supporting good growth gave good polymer production. Maximum cell mass of 10–11 g/liter and a maximum amount of polymer of 5–6 g/liter were recorded. The polymers recovered from the different substrates were found to be complex molecules or mixtures with a protein, a lipid, and a carbohydrate moiety. All the polymers are surface active and have two critical micelle concentrations.     

High lipids accumulation in <Emphasis Type="Italic">Rhodosporidium toruloides</Emphasis> by applying single and multiple nutrients limitation in a simple chemically defined medium

Microbial oil produced by the oleaginous yeast Rhodosporidium toruloides ATCC 204091 (formerly referred to as Rhodotorula glutinis) has a similar fatty acid composition to the vegetable oils and represents a potential alternative for biodiesel production. Finding strategies to improve the oil production by this yeast is desirable, as it is one of this nutrient’s limitations during the accumulation phase, as well as one of the main factors influencing the process. Therefore, the effect of single or combined nutrient limitation on lipid accumulation by R. toruloides was investigated. Biomass production and lipid accumulation by R. toruloides was improved using experimental designs in a two-step batch culture on a chemically-defined culture medium with high initial glucose concentration. For the first culture step, a Box–Behnken design was applied to optimize the main medium components’ concentrations, while maintaining a high biomass production. A biomass concentration of 44.3 g/L was reached with a medium composed of (g/L): glucose, 100; KH₂PO₄, 4.6; NaNO₃, 13.4; MgSO₄ ^.7H₂O, 0.2; and CaCl₂ ^.2H₂O, 0.11. For the second culture step, the biomass was transferred to lipid accumulation media. A 2³ factorial experimental design was conducted to investigate the effect of N, P and S limitations (individually or jointly) on lipid production from glucose (100 g/L). Lipid accumulation on dry cell mass was 77.04, 65.42, 70.13 and 69.84% for N, P, S and simultaneous nutrients’ limitations, respectively.     

Rhamnolipid production using Shewanella seohaensis BS18 and evaluation of its efficiency along with phytoremediation and bioaugmentation for bioremediation of hydrocarbon contaminated soils

Abstract

This study reports the combined use of a rhamnolipid type biosurfactant (BS) along with phytoremediation and bioaugmentation (BA) for bioremediation of hydrocarbon-contaminated soils. Bacterial isolates obtained from hydrocarbon contaminated soil were screened for rhamnolipid production and isolate BS18, identified as Shewanella seohaensis, was selected for bioremediation experiments. Growth of BS18 in mineral salt medium (MSM) with diesel oil as the carbon source showed a maximum biomass of 8.2?g L^?1, rhamnolipid production of 2.2?mg g^?1 cell dry weight, surface tension reduction of 28.6?mN/m and emulsification potential (EI₂₄%) of 65.6. Characterization of rhamnolipid based on Fourier transmittance infrared (FTIR) analysis confirmed the presence of OH, CH₂/CH₃, C=O, and COO stretching vibrations, respectively, which are distinctive features of rhamnolipid type BSs. In bioremediation experiments, the lowest hydrocarbon concentration of 2.1?mg g^?1 of soil for non-sterilized soil and 4.3?mg g^?1 of soil for sterilized soil was recorded in the combined application of rhamnolipid, phytoremediation, and BA. This treatment also yielded the highest hydrocarbon degrading bacterial population (6.4 Log Cfu g^?1 of soil), highest plant biomass (8.3?g dry weight plant^?1), and the highest hydrocarbon uptake (512.3?mg Kg^?1 of plant).     

Expression of Vitreoscilla hemoglobin in Gordonia amarae enhances biosurfactant production

The gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb) was electroporated into Gordonia amarae, where it was stably maintained, and expressed at about 4 nmol VHb g⁻¹ of cells. The maximum cell mass (OD₆₀₀) of vgb-bearing G. amarae was greater than that of untransformed G. amarae for a variety of media and aeration conditions (2.8-fold under normal aeration and 3.4-fold under limited aeration in rich medium, and 3.5-fold under normal aeration and 3.2-fold under limited aeration in mineral salts medium). The maximum level of trehalose lipid from cultures grown in rich medium plus hexadecane was also increased for the recombinant strain, by 4.0-fold in broth and 1.8-fold in cells under normal aeration and 2.1-fold in broth and 1.4-fold in cells under limited aeration. Maximum overall biosurfactant production was also increased in the engineered strain, by 1.4-fold and 2.4-fold for limited and normal aeration, respectively. The engineered strain may be an improved source for producing purified biosurfactant or an aid to microorganisms bioremediating sparingly soluble contaminants in situ.     

Statistical model based optimization of spore production by solid-state culture of Beauveria bassiana

A significant enhancement of spore production by solid-state culture of Beauveria bassiana was achieved via a statistical modeling and optimization approach. A two-level Plackett–Burman design was implemented to screen the significant medium components for spore production. Among the six variables tested, sucrose, NaNO₃, and H₃BO₃ were identified as the most significant factors (confidence levels above 95%) for spore production. The steepest ascent experiments were adopted to determine the optimal region of the medium composition. The developed second-order polynomial model predicted that the maximal spore production was 6.24×10⁹/g with the following optimal culture conditions: sucrose 34.7 g/L, NaNO₃ 52.8 g/L, and H₃BO₃ 79.6 µg/L. Finally the verification experiments were carried out and the maximal spore production reached 6.221×10⁹/g at the above optimal conditions. The spore production in the verification experiments was very close to that predicted by the model, indicating that the constructed second-order model had superior modeling and optimization ability and can be applied in the optimal spore production of Beauveria bassiana YP9 as a biological control agent.     

Statistical optimization of culture media for production of phycobiliprotein by Synechocystis sp. PCC 6701

Synechocystis sp. PCC 6701 has a brilliantly colored pigment, phycobiliprotein containing phycoerythrin. Culture medium was optimized by sequential designs in order to maximize phycobiliprotein production. The observed fresh weights after 6 days were 0.58 g/L in BG-11, 0.83 g/L in medium for Scenedesmus sp. and 0.03∼0.52 g/L in the other tested media. Medium for Scenedesmus sp. was selected to be optimized by fractional factorial design and central composite design since the medium maintained a more stable pH within a desirable range due to higher contents of phosphate. The fractional factorial design had seven factors with two levels: KNO₃, NaNO₃, NaH₂PO₄, Na₂HPO₄, Ca(NO₃)₂, FeEDTA, and MgSO₄. From the result of fractional factorial design, nitrate and phosphate were identified as significant factors. A central composite design was then applied with four variables at five levels each: nitrate, phosphate, pH, and light intensity. Parameters such as fresh weight and phycobiliprotein contents were used to determine the optimum value of the four variables. The proposed optimum media contains 0.88 g/L of nitrate, 0.32 g/L of phosphate under 25 μE·m⁻²·s⁻¹ of light intensity. The maximum phycobiliprotein contents have been increased over 400%, from 4.9 to 25.9 mg/L after optimization.     

Production of Inosine from Hydrocarbons by Corynebacterinm petrophilum

Using the adenine auxotroph of hydrocarbonoclastic microorganism, Corynebacterium petrophilum, the effects of glucose on the inosine productivity were investigated. The mutant did not produce inosine from glucose as the sole source of carbon. Production of inosine in n-C₁₆ medium was found to be inhibited by the addition of glucose. To obtain information on such effect of glucose, several characters were compared between the cells grown in glucose medium and those grown in n-C₁₆ medium. Intracellular content of UV-absorbing materials of the glucose-cells was higher than that of hydrocarbon-cells. The glucose-celle could not grow in media containing adenosine or 5′-AMP. On the other hand, hydrocarbon-cells were able to achieve growth, with adenine, adenosine and 5′-AMP contained in the hydrocarbon medium, but, in the case of glucose medium, the cells could grow only in the presence of adenine. Furthermore, the growth of this mutant in n-C₁₆ medium was found to be inhibited by a larger amount of adenine than that required for the maximum growth, and this inhibition was overcome by the addition of guanine. The significance of the effect of guanine was discussed.     

Improving lysine production through construction of an Escherichia coli enzyme-constrained model

Microbial cell factories are widely used for the production of high-value chemicals. However, maximizing production titers is made difficult by the complicated regulatory mechanisms of these cell platforms. Here, k_cat values were incorporated to construct an Escherichia coli enzyme-constrained model. The resulting ec_iML1515 model showed that the protein demand and protein synthesis rate were the key factors affecting lysine production. By optimizing the expression of the 20 top-demanded proteins, lysine titers reached 95.7 ± 0.7 g/L, with a 0.45 g/g glucose yield. Moreover, adjusting NH₄⁺ and dissolved oxygen levels to regulate the synthesis rate of energy metabolism-related proteins caused lysine titers and glucose yields to increase to 193.6 ± 1.8 g/L and 0.74 g/g, respectively. The ec_iML1515 model provides insight into how enzymes required for the biosynthesis of certain products are distributed between and within metabolic pathways. This information can be used to accurately predict and rationally design lysine production.     

Synergistic extraction using sweep-floc coagulation and acidification of rhamnolipid produced from industrial lignocellulosic hydrolysate in a bioreactor using sequential (fill-and-draw) approach

Sequential fill-and-draw fermentation strategy provides an approach to increase the productivity by replenishing nutrients and minimizing the toxic effects of by-products. In the present work, the same strategy was adopted using lignocellulosic industrial rice-straw C₆ hydrolysate stream to produce rhamnolipids from Achromobacter sp. (PS1) in a 6 L bioreactor with a working-volume of 2 L. The production results showed overall rhamnolipid production of 22.03 g/L in 15 days observed at par with 19.35 g/L obtained under shake flask conditions in 18 days. At each sequential feed (2 % sugars), a rise in dissolved oxygen (D.O) concentration was observed in the range between 60–53 % which declined to 47–39 % with consecutive depletion in sugar concentration under no D.O control. For maximum extraction of rhamnolipids from culture broth, the synergistic effect of sweep floc-coagulation using FeCl₃ at 0.4 % (w/v) followed by its acidification and solvent extraction was adopted which resulted in maximum recovery of 97.5 % compared to 89.05 % recovery obtained in simply acidification followed by solvent extraction. The characterization of partially purified biosurfactant using tandem-MS revealed six-congeners, Rha-C₁₀-C₁₀ and Rha-Rha-C₁₀-C₁₀ being the most abundant. Oil recovery of 92.21 % from motor-oil impregnated sand using crude rhamnolipid further added the value to the biosurfactant.     

Effects of biosurfactants produced by Candida antarctica on the biodegradation of petroleum compounds

The effects of biosurfactants on the biodegradation of petroleum compounds were investigated. Candida antarctica T-34 could produce extracellular biosurfactant mannosylerythritol lipids (MELs) when it was cultured in vegetable oil. In addition, in our previous study, it was found that this strain could also produce a new type of biosurfactant while it grew on n-undecane (C₁₁H₂₄), and the biosurfactant was named as BS-UC. In flask culture of Candida antarctica, the addition of BS-UC could improve the biodegradation rate of some n-alkanes (e.g. 90.2% for n-decane, 90.2% for n-undecane, 89.0% for dodecane), a mixture of n-alkanes (82.3%) and kerosene (72.5%). By comparing the effects of the biosurfactants BS-UC and MEL and chemical surfactants on the biodegradation of crude oil, it was found that biosurfactants could be used to enhance the degradation of petroleum compounds instead of chemical surfactants. In a laboratory scale immobilized bioreactor, the addition of biosurfactant improved not only the emulsification of kerosene in simulated wastewater but also its biodegradation rate. The highest degradation rate of kerosene by addition of MEL and BS-UC reached 87 and 90% at 15 h, respectively. The results showed that the biosurfactant BS-UC was highly promising for work on biodegradation of hydrophobic contaminants.     

15N-nmr spectroscopy. 33. Assignments of isotactic and syndiotactic sequences in poly(D,L-amino acids)

¹⁵N-enriched (D ,L -Leu)_n, (γ-OMe-D ,L -Glu)_n, (D ,L -Val)_n, and (D ,L -Phe)_n were prepared, 40.55-MHz ¹⁵N-nmr spectra were measured in various solvents. The signal patterns depend strongly on the nature of the solvent, yet in most cases at least four signals are resolved, representing the four enantiomeric pairs of triads L -L -L (D -D -D ), L -D -L (D -L -D ), L -L -D (D -D -L ), and D -L -L (L -D -D ). Numerous copolypeptides of the general structure (A)_n-B*-(A)_m (the asterisk denotes 40–50% ¹⁵N enrichment) were synthesized and measured as models for syndiotactic sequences in the spectra of poly(D ,L -amino acids). In this way unambiguous assignments for both isotactic and syndiotactic trials were obtained. A spectroscopic rule was established: “isotactic sequences absorb downfield of syndiotactic ones.” Furthermore, the spectra of various types of stereocopolypeptides such as (L -Leu/L -Val)_n and (L -Leu/D -Val)_n were investigated, including the ternary systems (L -Leu/L -Ala/D -Ala)_n (L -Leu/L -Ala/Gly)_n, (L -Leu/D -Ala/Gly)_n, (L -Val/L -Ala/Gly)_n, and (L -Val/D -Ala/Gly)_n. All copolymerization of D - and L -amino acid NCAs investigated in this work showed a low degree of stereoselectivity.     

Selective production of bikaverin in a fluidized bioreactor with immobilized Gibberella fujikuroi

The best culture medium composition for the production of bikaverin by Gibberella fujikuroi in shake-flasks, i.e. 100 g glucose l^–1; 1 g NH₄Cl l^–1; 2 g rice flour l^–1; 5 g KH₂PO₄ l^–1 and 2.5 g MgSO₄ l^–1, was obtained through a fractional factorial design and then scaled-up to a fluidized bioreactor. The effects of carbon and nitrogen concentrations, inoculum size, aeration, flow rate and bead sizes on batch bikaverin production using immobilized G. fujikuroi in a fluidized bioreactor were determined by an orthogonal experimental design. Concentrations of up to 6.83 g bikaverin l^–1 were obtained when the medium contained 100 g glucose l^–1 and 1 g NH₄Cl l^–1 with an inoculum ratio of 10% v/v, an aeration rate of 3 volumes of air per volume of medium min^–1, and a bead size of 3 mm. Based on dry weight, the bikaverin production was 30–100 times larger than found in submerged culture and approximately three times larger than reported for solid substrate fermentation.