Optimization of low-cost biosurfactant production from agricultural residues through response surface methodology

Biosurfactants are surface-active compounds capable of reducing surface tension and interfacial tension. Biosurfactants are produced by various microorganisms. They are promising replacements for chemical surfactants because of biodegradability, nontoxicity, and their ability to be produced from renewable sources. However, a major obstacle in producing biosurfactants at the industrial level is the lack of cost-effectiveness. In the present study, by using corn steep liquor (CSL) as a low-cost agricultural waste, not only is the production cost reduced but a higher production yield is also achieved. Moreover, a response surface methodology (RSM) approach through the Box–Behnken method was applied to optimize the biosurfactant production level. The results found that biosurfactant production was improved around 2.3 times at optimum condition when the CSL was at a concentration of 1.88 mL/L and yeast extract was reduced to 25 times less than what was used in a basic soybean oil medium (SOM). The predicted and experimental values of responses were in reasonable agreement with each other (Pred-R² = 0.86 and adj-R² = 0.94). Optimization led to a drop in raw material price per unit of biosurfactant from $47 to $12/kg. Moreover, the biosurfactant product at a concentration of 84 mg/L could lower the surface tension of twice-distilled water from 72 mN/m to less than 28 mN/m and emulsify an equal volume of kerosene by an emulsification index of (E24) 68% in a two-phase mixture. These capabilities made these biosurfactants applicable in microbial enhanced oil recovery (MEOR), hydrocarbon remediation, and all other petroleum industry surfactant applications.     

The application of a high throughput analysis method for the screening of potential biosurfactants from natural sources

The presence of biosurfactants in growth media can be evaluated by a variety of methods, none of which are suitable for high throughput studies. The method described here is based on the effect of meniscus shape on the image of a grid viewed through the wells of a 96-well plate. The efficacy of the method was demonstrated by the selection of a bacterium (producing a biosurfactant able to reduce the surface tension of pure water from 72 to 28.75 mN m^− 1) from a culture collection isolated from aviation fuel-contaminated land. The assay was found to be more sensitive, rapid and easy to perform than other published methods. It does not need specialised equipment or chemicals and excludes the bias which results from the surfactant properties of medium used for bacterial growth.     

Optimization of biosurfactant production using waste from biodiesel industry in a new membrane assisted bioreactor

The main byproduct of biodiesel production is glycerol. Here, crude glycerol – byproduct of biodiesel industry – was evaluated as sole carbon source in rhamnolipids production by Pseudomonas aeruginosa. The optimal concentration of crude glycerol and sodium nitrate was assessed using response surface methodology, resulting in about 40–50 mg/L.h of rhamnolipids, which was about four times higher than previously reported in the literature. Fermentation parameters were similar to those observed with commercial glycerol as sole carbon source. The optimized medium was suitable for production using simple (22.9 mg/L.h) and fed-batch (32.4 mg/L.h) fermentation in oxygen-controlled bioreactor without foaming formation. Composition and relative abundance of rhamnolipid congeners showed that crude glycerol had little effect on metabolic pathways involved in their production. CMC values were approximately 130 mg/L and 230–260 mg/L for rhamnolipids from crude and commercial glycerol fermentation, respectively, which were about 2–6 times lower than CMC values of synthetic surfactants.     

Ethanol production from syngas by Clostridium strain P11 using corn steep liquor as a nutrient replacement to yeast extract

The feasibility of replacing yeast extract (YE) by corn steep liquor (CSL), a low cost nutrient source, for syngas fermentation to produce ethanol using Clostridium strain P11 was investigated. About 32% more ethanol (1.7 g L⁻¹) was produced with 20 g L⁻¹ CSL media in 250-mL bottle fermentations compared to media with 1 g L⁻¹ YE after 360 h. Maximum ethanol concentrations after 360 h of fermentation in a 7.5-L fermentor with 10 and 20 g L⁻¹ CSL media were 8.6 and 9.6 g L⁻¹, respectively, which represent 57% and 60% of the theoretical ethanol yields from CO. Only about 6.1 g L⁻¹ of ethanol was obtained in the medium with 1 g L⁻¹ YE after 360 h, which represents 53% of the theoretical ethanol yield from CO. The use of CSL also enhanced butanol production by sevenfold compared to YE in bottle fermentations. These results demonstrate that CSL can replace YE as the primary medium component and significantly enhance ethanol production by Clostridium strain P11.     

Conditions of Phenol Extraction of RNA from Rat Liver Subcellular Cytoplasmic Fractions I. Effect of a Precipitation of Microsomal RNA on its Purity

Purification of RNA extracted with phenol from rat liver microsomes by precipitation in sodium benzoate and phenol solutions resulted in the removal of a readily measurable amount of proteins and polysaccharides. The stabilization of RNA was observed by changes in the profiles of the centrifugation gradients. This effect was the same over a wide range of yields in RNA obtained with extraction in the presence of buffers with varying pH levels.     

Optimization of polyhydroxybutyrate production by <Emphasis Type="Italic">Bacillus</Emphasis> sp. CFR 256 with corn steep liquor as a nitrogen source

Polyhydroxyalkanotes (PHAs), the eco-friendly biopolymers produced by many bacteria, are gaining importance in curtailing the environmental pollution by replacing the non-biodegradable plastics derived from petroleum. The present study was carried out to economize the polyhydroxybutyrate (PHB) production by optimizing the fermentation medium using corn steep liquor (CSL), a by-product of starch processing industry, as a cheap nitrogen source, by Bacillus sp. CFR 256. Response surface methodology (RSM) was used to optimize the fermentation medium using the variables such as corn steep liquor (5–25 g l⁻¹), Na₂HPO₄ 2H₂O (2.2–6.2 g l⁻¹), KH₂PO₄ (0.5–2.5 g l⁻¹), sucrose (5–55 g l⁻¹) and inoculum concentration (1–25 ml l⁻¹). Central composite rotatable design (CCRD) experiments were carried out to study the complex interactions of the variables. The optimum conditions for maximum PHB production were (g l⁻¹): CSL-25, Na₂HPO₄ 2H₂O-2.2, KH₂PO₄ − 0.5, sucrose − 55 and inoculum − 10 (ml l⁻¹). After 72 h of fermentation, the amount of PHA produced was 8.20 g l⁻¹ (51.20% of dry cell biomass). It is the first report on optimization of fermentation medium using CSL as a nitrogen source, for PHB production by Bacillus sp.     

Biosurfactant production and growth kinetics of bacteria in a designer marine medium: improved physiochemical properties

A combinatorial screening strategy was adopted for the development of a suitable medium for enhanced biosurfactant production by a marine strain. As a result, a modified marine medium (MMM) was developed, which contained urea and strontium chloride besides other salts important for the growth of marine bacteria. This medium supported growth, evident from a higher maximum growth rate value of 0.42 h(-1) and an enhanced biosurfactant production of 2.58 g/L. The critical micelle concentration (CMC) was determined for the biosurfactants obtained from all tested media combinations. The biosurfactant produced with this medium was stable at high temperature (100 °C), a wide range of pH (5-11) and salt concentration of 5-35%. The emulsifying activity and stability of the biosurfactant obtained using MMM was better than the biosurfactant obtained using conventional media. This biosurfactant with improved physiochemical properties is suitable for a wide range of applications in industry and for marine environmental cleaning.     

Batch and continuous cultures of<Emphasis Type="Italic"> Mannheimia succiniciproducens</Emphasis> MBEL55E for the production of succinic acid from whey and corn steep liquor

Mannheimia succiniciproducens MBEL55E isolated from bovine rumen is able to produce a large amount of succinic acid in a medium containing glucose, peptone, and yeast extract. In order to reduce the cost of the medium, whey and corn steep liquor (CSL) were used as substrates for the production of succinic acid by M. succiniciproducens MBEL55E. Anaerobic batch cultures of M. succiniciproducens MBEL55E in a whey-based medium containing CSL resulted in the production of succinic acid with a yield of 71% and productivity of 1.18 g/l/h, which are similar to those obtained in a whey-based medium containing yeast extract (72% and 1.21 g/l/h). Anaerobic continuous culture of M. succiniciproducens MBEL55E in a whey-based medium containing CSL resulted in a succinic acid yield of 69% and a succinic acid productivity as high as 3.90 g/l/h. These results show that succinic acid can be produced efficiently and economically by M. succiniciproducens MBEL55E from whey and CSL.     

High-density spore production of a <Emphasis Type="Italic">B</Emphasis>. <Emphasis Type="Italic">cereus</Emphasis> aquaculture biological agent by nutrient supplementation

Previous studies have demonstrated the efficacy of our Bacillus cereus isolate (NRRL 100132) in reducing concentrations of nitrogenous wastes and inhibiting growth of fish pathogens. In vivo efficacy and tolerance to a range of physiological conditions in systems used to rear Cyprinus carpio make this isolate an excellent candidate for aquaculture applications. Production cost is an important consideration in development of commercially relevant biological products, and this study examines the optimization of nutrient supplementation, which has an impact on high-density production of spores by fermentation. Corn steep liquor (CSL) was identified as a lower cost and more effective nutrient source in comparison to conventional nutrient substrates, in particular yeast extract and nutrient broth. The improved sporulation performance of B. cereus could be related to the increased availability of free amino acids, carbohydrates, and minerals in CSL, which had a positive effect on sporulation efficiency. The impact of nutrient concentration on spore yield and productivity was modeled to develop a tool for optimization of nutrient concentration in fermentation. An excellent fit of the model was confirmed in laboratory fermentation studies. A cost comparison revealed that production using liquid phytase and ultrafiltered-treated CSL was less expensive than spray-dried CSL and supported cultivation of B. cereus spores at densities higher than 1 × 10¹⁰ CFU ml⁻¹.     

Production Kinetics and Tensioactive Characteristics of Biosurfactant from a Pseudomonas aeruginosa Mutant Grown on Waste Frying Oils

Various waste frying oils (WFOs) were evaluated as substrates for rhamnolipid production by Pseudomonas aeruginosa mutant EBN-8 in the presence or absence of rhamnolipid precursor, under single-/batch-fed conditions. Soybean WFO was the best substrate, producing 9.3 g rhamnolipid l⁻¹ with the specific product yield of 2.7 g g⁻¹ h, under batch-fed cultivation with the addition of rhamnolipid precursor. The surface tension of the cell-free culture broth (CFCB) was 29.1 mN m⁻¹ and the interfacial tension against n-hexadecane was <1 mN m⁻¹. The hydrocarbon/ CFCB systems showed the relative emulsion stability to be in the range of 89.7–92.3.     

Biochemical and kinetic evaluation of lipase and biosurfactant assisted ex novo synthesis of microbial oil for biodiesel production by Yarrowia lipolytica utilizing chicken tallow

The present study explores the production of biodiesel, a sustainable replacement for depleting fossil fuel by utilizing microbial oil, which was procured from Yarrowia lipolytica employing chicken tallow as the carbon substrate. Chicken tallow, yeast extract, and MgSO₄·7H₂O were screened for biomass production through Plackett–Burman design. Further, Box–Behnken design analysis was performed, and the optimal concentration of the medium variables was found to be 20 g/L of chicken tallow, 7.0 g/L of yeast extract, and 0.45 g/L of MgSO₄·7H₂O.The various parameters viz., pH (6), temperature (30 °C), RPM (150), inoculum volume (5%, v/v), and C/N ratio (100) were optimized for maximal biomass and lipid yield, and lipid content. Nile red-stained cells were observed for intracellular lipid bodies using fluorescence microscopy, and its fluorescence intensity was measured bythe flow cytometer. The dimorphic transition and substrate assimilation of Y. lipolytica were analyzed using scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). Batch kinetic studies revealed the concomitant synthesis of microbial lipid (4.16 g/L), lipase (43 U/mL), and biosurfactant (1.41 g/L). The GC-MS analysis of microbial oil presented the fatty acid profile as oleic acid (49.15%), palmitic acid (29.83%), stearic acid (11.43%), linoleic acid (3.83%), palmitoleic acid (3.77%), and myristic acid (1.32%).     

Improvement of glycerol production by Candida krusei in batch and continuous cultures using corn steep liquor

No fermentation parameter was affected at phosphate concentration above 0.4 g l^–1 when KH₂PO₄ was used as phosphate source and the glucose consumption rate was difficult to control when corn steep liquor (CSL) was adopted as the phosphate source. However, if CSL was supplemented as a source of growth factors instead of as the phosphate source, not only glucose uptake and glycerol was improved, but also fermentation became easy to control and a steady state of continuous culture was easily obtained.     

Screening for tyrosinase inhibitors from actinomycetes; identification of trichostatin derivatives from Streptomyces sp. CA-129531 and scale up production in bioreactor

In the course of a primary screening of 614 microbial actinomycete extracts for the discovery of tyrosinase inhibitors, the EtOAc extract of the fermentation broth of the strain Streptomyces sp. CA-129531 isolated from a Martinique sample, exhibited in cell free and cell-based assays the most promising activity (IC₅₀ value of 63 μg/mL). Scaled-up production in a bioreactor led to the isolation of one new trichostatic acid analogue, namely trichostatic acid B (1), along with six known trichostatin derivatives (2–7), four diketopiperazines (8–11), two butyrolactones (12–13) and one hydroxamic acid siderophore (14). Among them, trichostatin A (4) showed a Ki value of 6.1 μM and six times stronger anti-tyrosinase activity (IC₅₀ 2.18 μΜ) than kojic acid (IC₅₀ 14.07 μΜ) used as a positive control. Deoxytrichostatin A (6) displayed also strong inhibitory activity against tyrosinase (IC₅₀ 19.18 μΜ). Trichostatin A production in bioreactor started together with the exponential phase of growth (day 4) and the maximum concentration was reached at day 9 (2.67 ± 0.13 μg/mL). Despite the cytotoxicity of some individual components, the EtOAc extract showed no cytotoxic effect on HepG2, A2058, A549, MCF-7 and MIA PaCa-2 cell lines, (IC₅₀ >2.84 mg/mL) and against BG fibroblasts at the concentrations where the whitening effect was exerted, reassuring its safety and great tyrosinase inhibitory potential.     

Efficient long-term and high-yielded production of a recombinant proteoglycan in eukaryotic HEK293 cells using a membrane-based bioreactor

Standard culture systems of eukaryotic cells generally failed to deliver sufficient amounts of recombinant proteins without increasing the costs of production. We here showed that membrane-based bioreactors, initially developed for the production of monoclonal antibodies, can be very useful for the production using engineered HEK293 cells, of a recombinant proteoglycan called endocan, with achievement of high level expression and efficient long-term production. When compared to standard procedures, the growth in suspension and at high density of these cells in one bioreactor promoted a 60-fold increase of the concentration of the soluble recombinant endocan. These culture conditions did not affect cell viability, stable expression, recognition by specific monoclonal antibodies or electrophoretic profile of the recombinant endocan. Such an easy to scale up system to produce recombinant protein should open soon new opportunities to study structure and functions of endocan or any other glycosylated cell products newly investigated.     

Improved guggulsterone production from sugars, precursors, and morphactin in cell cultures of Commiphora wightii grown in shake flasks and a bioreactor

Cell cultures of Commiphora wightii (Arnott.) Bhandari were grown in shake flasks and a bioreactor and an increase in guggulsterone accumulation up to 18 μg l⁻¹ was recorded in cells grown in the production medium containing a combination of sucrose:glucose (4% total), precursors (phenylalanine, pyruvic acid, xylose, and sodium acetate), morphactin, and 2iP. A yield of 10 g l⁻¹ biomass and ∼200 μg l⁻¹ guggulsterone was recorded in a 3-l flask and in a 2-l stirred tank bioreactor compared with 6.6 g biomass and 67 μg l⁻¹ guggulsterone in 250-ml flasks. Increased vessel size was correlated with increased biomass and guggulsterone accumulation. 2iP alone was not effective for biomass and guggulsterone accumulation in cell cultures of C. wightii.     

转瓶培养与生物反应器微载体培养乙脑病毒的比较

分别用15L转瓶与15L生物反应器微载体(2.5g/L CytodexⅢ)系统培养Vero细胞并接种乙型脑炎病毒(简称乙脑病毒)。转瓶培养Vero细胞7~8d,细胞数最高能达到8×108;当单层细胞长至3.0~4.5×108时接种乙脑病毒,病毒滴度能达到6.5~6.98 lg PFU/ml,并能够连续收获4~5次;采用微载体系统培养Vero细胞,细胞密度最高能达到170×108;当单层细胞长至60~70×108时接种乙脑病毒,病毒滴度能达到7~7.5 lg PFU/ml,并能够连续收获13~15次。两种方式培养的乙脑病毒收获液分别经灭活、浓缩、柱层析纯化后制备Vero细胞乙脑纯化疫苗,各项检定指标均符合《中国药典》的相关要求。     

Monolayers assembled from a glycolipid biosurfactant from <Emphasis Type="Italic">Pseudozyma</Emphasis> (<Emphasis Type="Italic">Candida</Emphasis>) <Emphasis Type="Italic">antarctica</Emphasis> serve as a high-affinity ligand system for immunoglobulin G and M

A carbohydrate ligand system has been developed which is composed of self-assembled monolayers (SAMs) of mannosylerythritol lipid-A (MEL-A) from Pseudozyma antarctica, serving for human immunoglobulin G and M (HIgG and HIgM). The estimated binding constants from surface plasmon resonance (SPR) measurement were K _a = 9.4 × 10⁶ M⁻¹ for HIgG and 5.4 × 10⁶ M⁻¹ for HIgM, respectively. The binding site was not in the Fc region of immunoglobulin but in the Fab region. Large amounts of HIgG and HIgM bound to MEL-A SAMs were directly observed by atomic force microscopy.     

Microbiological characterization of a coke oven contaminated site and evaluation of its potential for bioremediation

The soil microbial population of a coke oven site was investigated in order to evaluate its potential for bioremediation. The study was carried out in soil samples with distinct polynuclear aromatic hydrocarbon (PAH) contamination levels, comparing the population profiles constituted by total heterotrophic and PAH-utilizing strains. Isolation of degrading strains was performed with phenanthrene or pyrene as sole carbon sources. The ability to degrade other PAHs, such as anthracene, fluorene and fluoranthene was also investigated. The results showed a reduction of 30% in species diversity and microbial density drops one order of magnitude in contaminated samples. Furthermore, the number of PAH-utilizing colonies was higher in the contaminated area and about 20% of the isolates were able to degrade phenanthrene and pyrene, while this value decreased to 0.15% in uncontaminated samples. Three PAH-degrader strains were identified as: CDC gr. IV C-2, Aeromonas sp. and Pseudomonas vesicularis. The ability of these strains to degrade other PAHs was also investigated.     

Recombinant production of serine hydroxymethyl transferase from Streptococcus thermophilus and its preliminary evaluation as a biocatalyst

The glyA gene encoding a serine hydroxymethyl transferase (SHMT) with threonine aldolase activity was isolated from Streptococcus thermophilus YKA-184 chromosomal DNA. This aldolase is a pyridoxal 5′-phosphate-dependent enzyme that stereospecifically catalyzes the interconversion of l-threonine to glycine and acetaldehyde. The enzyme was overexpressed in Escherichia coli M15 as a recombinant protein of 45 kDa with a His6-tag at its N-terminus. The recombinant enzyme was purified to homogeneity by a single chromatographic step using Ni-nitrilotriacetic acid affinity, obtaining a high activity-recovery yield (83%). Lyophilized and precipitated enzymes were stable at least for 10 weeks when stored at −20°C and 4°C. It was observed that the K _m for l-allo-threonine was 38-fold higher than that for l-threonine, suggesting this enzyme can be classified as a specific l-allo-threonine aldolase. The optimum pH range of threonine aldolase activity for the recombinant SHMT was pH 6–7. When tested for aldol addition reactions with non-natural aldehydes, such as benzyloxyacetaldehyde and (R)-N-Cbz-alaninal, two possible β-hydroxy-α-amino acid diastereoisomers were produced, but with moderate stereospecificity. The enzyme showed potential as a biocatalyst for the stereoselective synthesis of β-hydroxy-α-amino acids.     

Surface plasmon resonance studies and biochemical evaluation of a potent peptide inhibitor against cyclooxygenase-2 as an anti-inflammatory agent

Cyclooxygenase (COX) is a key enzyme in the biosynthetic pathway leading to the formation of prostaglandins, which are mediators of inflammation [D.L. Dewitt, W.L. Smith, Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence, Proc. Natl. Acad. Sci. USA 85 (1988) 1412-1416, 1]. It exists mainly in two isoforms COX-1 and COX-2 [A. Raz, A. Wyche, N. Siegel, P. Needleman, Regulation of fibroblast cyclooxygenase synthesis by interleukin-1, J. Biol. Chem. 263 (1988) 3022-3028, 2]. The conventional non-steroidal anti-inflammatory drugs (NSAIDs) have adverse gastrointestinal side-effects, because they inhibit both isoforms [T.D. Warner, F. Guiliano, I. Vojnovic, A. Bukasa, J.A. Mitchell, J.P. Vane, Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis, Proc. Natl. Acad. Sci. USA 96 (1999) 7563-7568, 3; L.J. Marnett, A.S. Kalgutkar, Cyclooxygenase 2 inhibitors: discovery, selectivity and the future, Trends Pharmacol. Sci. 20 (1999) 465-469, 4; J.R. Vane, NSAIDs, Cox-2 inhibitors, and the gut, Lancet 346 (1995) 1105-1106, 5]. Therefore drugs which selectively inhibit COX-2, known as coxibs were developed. Recent reports on the harmful cardiovascular and renovascular side-effects of the anti-inflammatory drugs have led to the quest for a novel class of COX-2 selective inhibitors. Keeping this in mind, we have used the X-ray crystal structures of the complexes of the COX-1 and COX-2 with the known inhibitors for a rational, structure based approach to design a small peptide, which is potent inhibitor for COX-2. The peptides have been checked experimentally by in-vitro kinetic studies using surface plasmon resonance (SPR) and other biochemical methods. We have identified a tripeptide inhibitor which is a potential lead for a new class of COX-2 inhibitor. The dissociation constant (K(D)) determined for COX-2 with peptide WCS is 1.90x10(-10)M, the kinetic constant (K(i)) determined by spectrophotometry is 4.85x10(-9)M and the IC(50) value is 1.5x10(-8)M by ELISA test.