Production of sophorolipids in high concentration from deproteinized whey and rapeseed oil in a two stage fed batch process using Candida bombicola ATCC 22214 and Cryptococcus curvatus ATCC 20509

High concentrations of 422 g sophorolipids l–1 were produced using a two-stage cultivation process: first deproteinized whey concentrate (DWC) containing 110 g lactose l–1 was used for cultivation of the yeast Cryptococcus curvatus ATCC 20509, resulting in 34 g dry weight l–1, 20 g single-cell oil l–1 and reducing the chemical oxygen demand (COD) from 159 g l–1 to 35 g oxygen l–1. Afterwards cells were disrupted by passing the cell suspension directly through a high pressure laboratory homogeniser. After autoclavation, the resulting crude cell extract containing the single-cell oil served as substrate for growth of Candida bombicola ATCC 22214 and for sophorolipid production in a second stage. When the single-cell oil was consumed, repeated feeding of 400 g rapeseed oil l–1 was started increasing the yield of sophorolipids to 422 g l–1. A simple technique for product isolation, sedimentation, could be used to harvest the crude sophorolipids. © Rapid Science Ltd. 1998     

Production of Sophorolipids from Candida bombicola ATCC 22214 Using Turkish Corn Oil and Honey

The ability of Candida bombicola ATCC 22214 to produce sophorolipids using Turkish corn oil and honey was investigated. Shake flask experiments were carried out both with and without the addition of glucose as the second carbon source. The organism could produce sophorolipids under both conditions but higher production was obtained when corn oil was combined with glucose. The 3 L bioreactor was first operated in batch mode, using both corn oil and glucose. When all the glucose was consumed, ¹/₄th of the broth was pumped out and was replaced by freshly prepared medium containing 10 % [w/v] of cheap market honey as the sole carbon source. Feed was comprised of corn oil. High concentrations of sophorolipids (> 400 g/L) were produced. The crude products obtained from the batch cultivation could be solidified as very light brown solids when unused oil was removed by hexane, while the products of the two‐stage cultivation remained as viscous, honey‐like liquids after identical treatments.     

The effect of medium composition on the structure and physical state of sophorolipids produced by Candida bombicola ATCC 22214

Candida bombicola was grown using a variety of lipophilic carbon substrates. Most of the hydrocarbon and carboxylic acid substrates resulted in a mixture of sophorolipids consisting of free acids and the more desirable lactones. The ratio of diacylated lactone to free acid in these mixtures was a maximum when produced using hexadecane and heptadecane. All of the other lipophilic substrates resulted in significant amounts of free acids being produced. These lactone products were unique in that they precipitated as crystals, which were easily separated from the culture medium. All of the other products were isolated as oils as is usually reported in the literature. Finally, the amounts of these crystals recovered were significantly higher than those observed for any of the oily products. It was possible to determine the degree of direct incorporation of the lipophilic substrates into the sophorolipids for a homologous series of alkanes. The amount of direct incorporation increased with increasing chain length to a maximum for pentadecane, hexadecane and heptadecane. As the length of the alkane substrate increased further, the amount of direct incorporation then decreased until there was no apparent incorporation for eicosane.     

Utilization of sweetwater as a cost-effective carbon source for sophorolipids production by Starmerella bombicola (ATCC 22214)

Biosurfactants are microbially synthesized surfactants that are environmental friendly due to low toxicity. Sophorolipid is one of the simplest biosurfactants with well-defined structure produced by Starmerella bombicola(ATCC 22214) on glucose and vegetable oil as the carbon source. The raw material cost accounts for 10-30% of the overall cost. Glycerol is readily available from a commercial fat-splitting process as sweetwater at a very low cost. Sophorolipids was synthesized using glycerol and sweetwater as a cost-effective carbon source. The glycerol was further replaced with sweetwater as a source of glycerol. Optimum glycerol concentration was 15% w/v with 10% w/v sunflower oil, giving 6.6 g/L of sophorolipids. The crude sophorolipid contains two major components; both of them were lactonic sophorolipids as analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC), liquid chromatography-mass spectroscopy (LC-MS), and nuclear magnetic resonance ((1)H-NMR).     

Optimization of the fermentation media for sophorolipid production from Candida bombicola ATCC 22214 using a simplex centroid design

This article describes the use of a simplex centroid mixture experimental design to optimize the fermentation medium in the production of sophorolipids (SLs) using Candida bombicola. In the first stage, 16 media ingredients were screened for the ones that have the most positive influence on the SL production. The sixteen ingredients that were chosen are five different carbohydrates (fructose, glucose, glycerol, lactose, and sucrose), five different nitrogen sources (malt extract, peptone extract, soytone, urea, and yeast extract), two lipid sources (mineral oil and oleic acid), two phosphorus sources (K₂HPO₄ and KH₂PO₄), MgSO₄, and CaCl₂. Multiple regression analysis and centroid effect analysis were carried out to find the sugar, lipid, nitrogen source, phosphorus source, and metals having the most positive influence. Sucrose, malt extract, oleic acid, K₂HPO₄, and CaCl₂ were selected for the second stage of experiments. An augmented simplex centroid design for five ingredients requiring 16 experiments was used for the optimization stage. This produced a quadratic model developed to help understand the interaction amongst the ingredients and find the optimal media concentrations. In addition, the top three results from the optimization experiments were used to obtain constraints that identify an optimal region. The model together with the optimal region constraints predicts the maximum production of SLs when the fermentation media is composed of sucrose, 125 g/L; malt extract, 25 g/L; oleic acid, 166.67 g/L; K₂HPO₄, 1.5 g/L; and CaCl₂, 2.5 g/L. The optimal media was validated experimentally and a yield of 177 g/L was obtained. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Production of sophorolipids with eicosapentaenoic acid and docosahexaenoic acid from Wickerhamiella domercqiae var. sophorolipid using fish oil as a hydrophobic carbon source

Sophorolipids (SLs) were synthesized by Wickerhamiella domercqiae var. sophorolipid CGMCC 1576 grown on fish oil and glucose. They were purified using preparative HPLC and their structures were identified by MS/MS. The yields of total and lactonic SLs were 47 and 19 g l^?1 in shake-flasks when fish oil 4 % (v/v) was used with glucose in the medium. The composition of SL mixture contained more than 20 SL molecules. Several unconventional SL molecules with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) including zero-, mono- and di-acetylated acidic SLs with EPA and a di-acetylated acidic SL with DHA were obtained. Two unconventional lactonic SL molecules, non-acetylated lactonic SL with 22:3 and non-acetylated lactonic SL with 20:0, were also obtained.     

Using cheese whey for hydrogen and methane generation in a two-stage continuous process with alternative pH controlling approaches

This study focuses on the exploitation of cheese whey as a source for hydrogen and methane, in a two-stage continuous process. Mesophilic fermentative hydrogen production from undiluted cheese whey was investigated at a hydraulic retention time (HRT) of 24 h. Alkalinity addition (NaHCO₃) or an automatic pH controller were used, to maintain the pH culture at a constant value of 5.2. The hydrogen production rate was 2.9 ± 0.2 L/Lreactor/d, while the yield of hydrogen produced was approximately 0.78 ± 0.05 mol H₂/mol glucose consumed, with alkalinity addition, while the respective values when using pH control were 1.9 ± 0.1 L/Lreactor/d and 0.61 ± 0.04 mol H₂/mol glucose consumed. The corresponding yields of hydrogen produced were 2.9 L of H₂/L cheese whey and 1.9 L of H₂/L cheese whey, respectively. The effluent from the hydrogenogenic reactor was further digested to biogas in a continuous mesophilic anaerobic bioreactor. The anaerobic digester was operated at an HRT of 20d and produced approximately 1 L CH₄/d, corresponding to a yield of 6.7 L CH₄/L of influent. The chemical oxygen demand (COD) elimination reached 95.3% demonstrating that cheese whey could be efficiently used for hydrogen and methane production, in a two-stage process.     

Co-production of hydrogen and methane from potato waste using a two-stage anaerobic digestion process

Hydrogen and methane co-production from potato waste was examined using a two-stage process of anaerobic digestion. The hydrogen stage was operated in continuous flow under a pH of 5.5 and a HRT of 6h. The methane stage was operated in both continuous and semi-continuous flows under HRTs of 30 h and 90 h, respectively, with pH controlled at 7. A maximum gas production rate of 270 ml/h and an average of 119 ml/h were obtained from the hydrogen stage during the operation over 110 days. The hydrogen concentration contained in the gas was 45% (v/v), on average. The maximum and average gas production rates observed from methane reactor during the 74 days of semi-continuous flow operation were 187 and 141 ml/h, respectively, with an average methane concentration of 76%. Overall, 70% of VS, 64% of total COD in the feedstock were removed. The hydrogen and methane yields from the potato waste were 30 l/kg TS (with a maximum of 68 l/kg) and 183 l/kg TS (with a maximum of 225 l/kg), respectively. The total energy yield obtained was 2.14 kW h/kg TS, with a maximum of 2.74 kW h/kg TS.     

Production of penicillin acylase by a recombinant Escherichia coli using cheese whey as substrate and inducer

Cheese whey (CW) is the major subproduct from cheese manufacturing and it is considered as a waste pollutant since its high content of lactose. In this work a fermentation process for the production of penicillin acylase (PA) by a recombinant Escherichia coli and using CW as unique carbon source and inducer was developed. A design factorial 3(2) was used to evaluate the influence of independent variables (dissolved oxygen and CW concentration) on the ability of E. coli W3110/pPA102 to produce PA. Maximum specific PA activity of 781 U g(-1) was attained at 5 g L(-1) of CW and 3% dissolved oxygen. The results showed that CW can be used successfully as unique carbon source and inducer for the production of recombinant proteins using constructions driven by the lac promoter and this way reducing the discharges of that pollutant to the environment.     

Operation of a bioelectrochemical system as a polishing stage for the effluent from a two-stage biohydrogen and biomethane production process

Anaerobic bioenergy production processes including fermentative biohydrogen (BioH₂), anaerobic digestion (AD) and bioelectrochemical system have been investigated for converting municipal waste or various biomass feedstock to useful energy carriers. However, the performance of a microbial fuel cell (MFC) fed on the effluent from a two-stage biogas production process has not yet been investigated extensively in continuous reactor operation on complex substrates. In this study we have investigated the extent to which a microbial fuel cell (MFC) can reduce COD and recover further energy from the effluent of a two-stage biohydrogen and biomethane system. The performance of a four-module tubular MFC was determined at six different organic loadings (0.036–6.149 g sCOD L⁻¹ d⁻¹) in terms of power generation, COD removal efficiency, coulombic efficiency (CE) and energy conversion efficiency (ECE). A power density of 3.1 W m⁻³ was observed at the OLR = 0.572 g sCOD L⁻¹ d⁻¹, which resulted in the highest CE (60%) and ECE (0.8%), but the COD removal efficiency decreased at higher organic loading rates (35.1–4.4%). The energy recovery was 92.95 J L⁻¹ and the energy conversion efficiency, based on total influent COD was found to be 0.48–0.81% at 0.572 g sCOD L⁻¹ d⁻¹. However, the energy recovery by the MFC is only reported for a four-module reactor and improved performance can be expected with an extended module count, as chemical energy remained available for further electrogenesis.     

Production of biofuel from waste cooking palm oil using nanocrystalline zeolite as catalyst: process optimization studies

The catalytic cracking of waste cooking palm oil to biofuel was studied over different types of nano-crystalline zeolite catalysts in a fixed bed reactor. The effect of reaction temperature (400-500 °C), catalyst-to-oil ratio (6-14) and catalyst pore size of different nanocrystalline zeolites (0.54-0.80 nm) were studied over the conversion of waste cooking palm oil, yields of Organic Liquid Product (OLP) and gasoline fraction in the OLP following central composite design (CCD). The response surface methodology was used to determine the optimum value of the operating variables for maximum conversion as well as maximum yield of OLP and gasoline fraction, respectively. The optimum reaction temperature of 458 °C with oil/catalyst ratio=6 over the nanocrystalline zeolite Y with pore size of 0.67 nm gave 86.4 wt% oil conversion, 46.5 wt% OLP yield and 33.5 wt% gasoline fraction yield, respectively. The experimental results were in agreement with the simulated values within an experimental error of less than 5%.     

Differential protein composition of bovine whey: a comparison of whey from healthy animals and from those with clinical mastitis

During clinical mastitis in dairy cows, the quantity of milk produced decreases and the composition of the milk is altered. As the severity of inflammation associated with the disease increases, the chemical composition of milk approaches that of blood as a consequence of increased permeability of the blood mammary barrier, or de novo intramammary synthesis, as has been suggested for mammary associated serum amyloid A3. A better understanding of these events may provide new approaches for the diagnosis and treatment of mastitis. The objective of this study was to document the changes in the protein composition of milk during clinical mastitis using a proteomic approach, with the objective of identifying new diagnostic markers of mastitis. Whey from dairy cows with clinical mastitis was compared to whey from healthy animals by two-dimensional gel electrophoresis (2-DE) with colloidal Coomassie staining and matrix-assisted desorption/ionization mass spectrometry. Increases in the concentrations of proteins of blood serum origin, including serotransferrin and albumin, were identified in mastitic whey compared to normal whey, while concentrations of the major whey proteins alpha-lactalbumin and beta-lactoglobulin were reduced in mastitic whey. Mass spectrometry subsequently confirmed the location of albumin, alpha-lactalbumin and beta-lactoglobulin on the 2-DE gels at M(r)/pI of 69 294/5.8, 14 200/4.5 and 19 883/4.9 respectively.     

Purification of the two major proteins from whey concentrate using a cation‐exchange selective adsorption process

The packed‐bed adsorption and elution of aqueous solutions of whey concentrate powders were investigated at pH 3.7 using a 5‐mL SP Sepharose FF column to separate and isolate two major proteins namely, α‐lactalbumin (ALA) and β‐lactoglobulin (BLG) from these solutions. ALA displaced and eluted BLG from the column in a pure form. Pure ALA could then be eluted with good recovery. A novel consecutive two‐stage separation process was developed to separate ALA and BLG from whey concentrate mixtures. Almost all of the BLG in the feed was recovered, with 78% being recovered at 95% purity and a further 20% at 86% purity. In addition, 67% of ALA was recovered, 48% at 54% purity and 19% at 60% purity. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Carbonic anhydrase inhibitors: inhibition of the beta-class enzymes from the fungal pathogens Candida albicans and Cryptococcus neoformans with simple anions

The catalytic activity and inhibition of the beta-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Candida albicans (Nce103) and Cryptococcus neoformans (Can2) with inorganic anions such as halogenides, pseudohalogenides, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, perchlorate, sulfate were investigated. The two enzymes showed appreciable CO(2) hydrase activity (k(cat) in the range of (3.9-8.0)x10(5)s(-1), and k(cat)/K(m) in the range of (4.3-9.7)x10(7)M(-1)s(-1)). Can2 was weakly inhibited by cyanide and sulfamic acid (K(I)s of 8.22-13.56 mM), while all other anions displayed more potent inhibition. Nce103 was strongly inhibited by cyanide and carbonate (K(I)s of 10-11 microM), and weakly inhibited by sulfate, phenylboronic, and phenyl arsonic acid (K(I)s of 14.15-30.85 mM). These data demonstrate that pathogenic, fungal beta-CAs may be targets for the development of antifungals that have a novel mechanism of action.     

Highly Enantioselective Production of Chiral Secondary Alcohols with Candida zeylanoides as a New Whole Cell Biocatalyst

The increasing demand for biocatalysts in synthesizing enantiomerically pure chiral alcohols results from the outstanding characteristics of biocatalysts in reaction, economic, and ecological issues. Herein, fifteen yeast strains belonging to three food originated yeast species Candida zeylanoides, Pichia fermentans, and Saccharomyces uvarum were tested for their capability for asymmetric reduction of acetophenone to 1‐phenylethanol as biocatalysts. Of these strains, C. zeylanoides P1 showed an effective asymmetric reduction ability. Under optimized conditions, substituted acetophenones were converted to corresponding optically active secondary alcohols in up to 99% enantiomeric excess and at high yields. The preparative scale asymmetric bioreduction of 4‐nitroacetophenone ( 1m ) by C. zeylanoides P1 gave (S)‐1‐(4‐nitrophenyl)ethanol ( 2m ) with 89% yield and > 99% enantiomeric excess. Compound 2m has been obtained in an enantiomerically pure and inexpensive form. Additionally, these results indicate that C. zeylanoides P1 is a promising biocatalyst for the synthesis of chiral alcohols in industry.     

Use of wastes for sophorolipids production as a transition to circular economy: state of the art and perspectives

Chemical processes and petroleum-based chemicals are being substituted by biological processes and bioproducts. Surfactants and biosurfactants are an example of this trend. Among the biosurfactants, sophorolipids (SLs) have excellent surface and interfacial tension properties, which make them ideal to be used in a wide variety of applications. SLs are produced at full scale through submerged fermentation of pure substrates (glucose and oleic acid). However, research trends suggest that there is a lot of interest to produce SLs from waste effluents and other low-cost substrates, both in submerged and solid-state fermentation processes. This study reviews the current research in the production of SLs via fermentation processes, focusing on those using wastes, by-products, or low-cost substrates (liquids or solids). It details the substrates, process variables, microorganisms, and use of supplementary media for batch, fed-batch, and continuous submerged or solid-state fermentation processes. Sophorolipids production based on industrial by-products and waste effluents presents huge potential for its application at an industrial scale in a more economical and environmentally friendly process, boosting the necessary change to circular economy.

     

Structural studies of the capsular polysaccharide of a non-neoformans Cryptococcus species identified as C. laurentii, which was reclassified as Cryptococcus flavescens, from a patient with AIDS

Cryptococcus flavescens, a strain originally identified as C. laurentii, was isolated from the cerebrospinal fluid of an AIDS patient, and the soluble capsular polysaccharide of the yeast was investigated. Glucuronoxylomannan (GXM) was obtained from C. flavescens under conditions similar to those used to obtain C. neoformans polysaccharide. However, the GXM differed from C. neoformans polysaccharide in the decreased O-acetyl group content. The structure of GXM was determined by methylation analysis, partial acid hydrolysis, NMR analyses, and controlled Smith degradation. These analyses indicated that GXM has the following structure: an alpha-(1-->3)-D-mannan backbone with side chains of beta-D-glucuronic acid residues bound to the C-2 position of the mannose residue. The C-6 position of the mannose is substituted with D-man-beta-(1-->4)-D-xyl-beta-(1--> disaccharide. Furthermore, the existence of side chains containing more than two xylose residues was suggested. This mannosylxylose side chain is a novel structure in polysaccharides of C. neoformans and other Cryptococcus species.     

2,3-Butanediol production using Klebsiella oxytoca ATCC 8724: Evaluation of biomass derived sugars and fed-batch fermentation process

For this study, 2,3-butanediol (BD) fermentation from pure and biomass-derived sugar were optimized in shake-flask and 5-L bioreactor levels using Klebsiella oxytoca ATCC 8724. The results showed that 70 g/L of single sugar (glucose or xylose) and 90 g/L of mixed-sugar (glucose:xylose = 2:1) were optimum concentrations for efficient 2,3-BD fermentation. At optimum sugar concentrations, 2,3-BD productivities were 1.03, 0.64 and 0.50 gL⁻¹ h⁻¹, and yields were 0.43, 0.36 and 0.35 g/g in glucose, xylose and mixed-sugar medium, respectively. The lack of simultaneous utilization of glucose and xylose led to the lowest productivity in the mixed-sugar medium. Detoxification of biomass hydrolyzates was necessary for efficient 2,3-BD fermentation when sugar concentrations in the medium was 90 g/L or higher, but not with sugar concentrations of 30 g/L or less. A fed-batch fermentation using glucose medium led to an increase 2,3-BD titer to 79.4 g/L and yields 0.47 g/g, while productivity decreased to 0.79 gL⁻¹ h⁻¹. However, the fed-batch process was inefficient using mixed-sugar and biomass hydrolyzates because of poor xylose utilization. These results indicated that appropriate biomass processing technologies must be developed to generate separate glucose and xylose streams to produce high 2,3-BD titer from biomass-derived sugar using a fed-batch process.     

Biotin protein ligase from Candida albicans: expression, purification and development of a novel assay

Biotin protein ligase (BPL) is an essential enzyme responsible for the activation of biotin-dependent enzymes through the covalent attachment of biotin. In yeast, disruption of BPL affects important metabolic pathways such as fatty acid biosynthesis and gluconeogenesis. This makes BPL an attractive drug target for new antifungal agents. Here we report the cloning, recombinant expression and purification of BPL from the fungal pathogen Candida albicans. The biotin domains of acetyl CoA carboxylase and pyruvate carboxylase were also cloned and characterised as substrates for BPL. A novel assay was established thereby allowing examination of the enzyme’s properties. These findings will facilitate future structural studies as well as screening efforts to identify potential inhibitors.