Enhanced sophorolipid production by feeding-rate-controlled fed-batch culture

To develop the easier control method for fed-batch culture of sophorolipid production, we chose rapeseed oil as the most productive oil and compared their productivities in relation to different concentrations of glucose. The optimal concentration of glucose was 30 g/L for sophorolipid production. A fed-batch method was conducted using Candida bombicola ATCC 22214 with rapeseed oil as a secondary substrate. The feeding rate of rapeseed oil was dependent on pH and was calculated by the consumption rate of NaOH and rapeseed oil. The glucose concentration was constantly maintained between 30 and 40 g/L. As a result, we have produced a crude sophorolipid up to 365 g/L for 8 days through a feeding-rate-controlled fed-batch process.     

Vegetable oil enhances sophorolipid production by <Emphasis Type="Italic">Rhodotorula bogoriensis</Emphasis>

The yeast Rhodotorula bogorensis produces sophorolipids of different structures to those produced by Candida bombicola. However, the yield is very low. To improve sophorolipid production by R. bogoriensis, vegetable oil was supplemented to the medium as a hydrophobic substrate: with rapeseed oil the sophorolipid yield was 1.26 g/l but without oil was 0.33 g/l. Cultures with meadowfoam oil produced 0.77 g sophorolipids/l. Lipase-treated meadowfoam oil, however, gave no significant increase in sophorolipid production. Possible explanations for the enhanced sophorolipid synthesis are discussed.     

Sophorolipids-induced cellulase production in cocultures of Hypocrea jecorina Rut C30 and Candida bombicola

Sophorose is a potent but expensive inducer for cellulase production. In this study the feasibility of using sophorolipids, natural lipids that contain sophorose, for cellulase induction was investigated. Enhanced cellulase production by Hypocrea jecorina Rut C30 grown on glycerol, a substrate without cellulase-inducing ability, was first confirmed by addition of the crude sophorolipids collected from Candida bombicola fermentation. Cocultures of H. jecorina Rut C30 and C. bombicola were then employed to evaluate the effects of coculture conditions: the cell concentration ratio between the two cultures, the concentration of vegetable oil (as lipid precursor for sophorolipid synthesis, in addition to glycerol as the primary carbon source), the presence of nitrogen source for growth, and the substitution of glucose for glycerol as the primary carbon source. Specific cellulase productivity of H. jecorina Rut C30 was significantly higher under the conditions that promoted sophorolipid production by C. bombicola. The ability of H. jecorina Rut C30 to degrade sophorolipids was also confirmed. The results of the study indicated that the sophorolipids produced by C. bombicola can be degraded by H. jecorina Rut C30 and the sophorose generated from the degradation can effectively induce the fungal cellulase synthesis.     

Production of 1,3-propanediol by Klebsiella pneumoniae from glycerol broth

Broth containing 152 g glycerol l⁻¹ from Candida krusei culture was converted to 1,3-propanediol by Klebsiella pneumoniae. Residual glucose in the broth promoted growth of K. pneumoniae while acetate was inhibitory. After desalination treatment of glycerol broth by electrodialysis, the acetate in the broth was removed. A fed-batch culture with electrodialytically pretreated broth as␣substrate was developed giving 53 g 1,3- propanediol l⁻¹ with a yield of 0.41 g g⁻¹ glycerol and a productivity of 0.94 g l⁻¹ h⁻¹.     

Utilization of vegetable oil in the production of clavulanic acid by <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis>ATCC 27064

Production of clavulanic acid (CA) by Streptomyces clavuligerus ATCC 27064 in shake-flask culture (28 °C, 250 rev min^–1) was evaluated, with media containing different types and concentrations of edible vegetable oil. Firstly, four media based on those reported in the literature were examined. The medium containing soybean oil and starch as carbon and energy source gave the best production results. This medium, with the starch replaced by glycerol, and with various soybean oil concentrations (16, 23 and 30 g l^–1) was utilized to further investigate CA production. Medium containing 23 g l^–1 led to the highest CA productivity (722 mg l^–1 in 120 h) and that one containing 30 g l^–1 gave the highest CA titre (753 mg l^–1 in 130 h). Also, substitution of corn and sunflower edible oils furnished similarly good results in terms of CA titre and productivity. It can be concluded that easily available vegetable oil is a very promising substrate for CA production, since it is converted slowly to glycerol and fatty acids, which are the main carbon and energy source for the microorganism.     

Integrated sophorolipid production and gravity separation

A novel method for the integrated gravity separation of sophorolipid from a fermentation broth has been developed, enabling removal of a sophorolipid phase of either higher or lower density than the bulk fermentation broth, while cells and other media components are recirculated and returned to the bioreactor. The capability of the separation system to recover an enriched sophorolipid product phase was demonstrated on three sophorolipid producing fed batch fermentations using Candida bombicola, giving an 11% reduction in fermenter volume required whilst maintaining sophorolipid production. Sophorolipid recoveries of up to 86% (280 g) of the total produced over a whole fermentation were achieved at an enrichment of up to 9. Furthermore, the broth viscosity reduction achieved by removal of the sophorolipid phase enabled a 34% reduction in mixing power to maintain the same dissolved oxygen level by the end of the fermentation, with a 9% average reduction over the course of the fermentation. Fermentation duration could be extended to 1023 h, allowing production of 623 g sophorolipid from 1 l initial batch volume. These benefits could lead to a substantial decrease in the cost of sophorolipid production, making high volume applications such as enhanced oil recovery economically feasible.     

Metabolic Interactions Between Glucose, Glycerol, Alanine and Acetate in Leishmania braziliensis panamensis Promastigotes

¹³C-nuciear magnetic resonance (NMR) spectroscopy was used to investigate the products of glycerol and acetate metabolism released by Leishmania braziliensis panamensis promastigotes and also to examine the interaction of each of these substrates with glucose or alanine. The NMR data were supplemented by measurements of the rates of oxygen consumption and substrate utilization, and of ¹⁴CO₂ production from ¹⁴C-labeIed substrate. Cells incubated with [2-¹³C]glycerol released acetate, succinate and D-lactate in addition to CO₂. Cells incubated with acetate released only CO₂. More succinate C-2/C-3 than C-l/C-4 was released from both [2-¹³C]glycerol and [2-¹³C]glucose, indicating that succinate was formed predominantly by CO₂ fixation followed by reverse flux through part of the Krebs cycle. Some redistribution of the position of labeling was also seen in alanine and pyruvate, suggesting cycling through pyruvate/oxaloacetate/phosphoenolpyruvate. Cells incubated with combinations of 2 substrates consumed oxygen at the same rate as cells incubated with 1 or no substrate, even though the total substrate utilization had increased. When promastigotes were incubated with both glycerol and glucose, the rate of glucose consumption was unchanged but glycerol consumption decreased about 50%, and the rate of ¹⁴CO₂ production from [l,(3)-¹⁴C]glycerol decreased about 60%. Alanine did not affect the rates of consumption of glucose or glycerol, but decreased ¹⁴CO₂ production from these substrates by increasing flow of label into alanine. Although glucose decreased alanine consumption by 70%, it increased the rate of ¹⁴CO₂ production from [U-¹⁴C]- and [l-¹⁴C]alanine by about 20%. This is consistent with rapid equilibration of alanine with pyruvate derived from glucose and yet little decrease in the specific activity of the large alanine pool.     

Heterotrophic growth and lipid production of Chlorella protothecoides on glycerol

During the production of biodiesel, a significant amount of glycerol is generated which currently has little commercial value. A study on the growth and lipid production of Chlorella protothecoides using glycerol as the carbon source was performed to demonstrate the utility of recycling crude glycerol created during biodiesel production. Glycerol was examined as both the sole carbon source and in combination with glucose. Algae cultures grown on only glycerol in shake flasks showed a specific growth rate and final lipid yield of 0.1/h and 0.31 g lipid/g substrate, respectively. The values were similar to those observed on pure glucose, 0.096/h and 0.24 g lipid/g substrate. When the media contained a mixture of glycerol and glucose, simultaneous uptake of the two substrates was observed. Due to the difference in rates of lipid storage, lipid production was 0.077 g lipid/(l h) during growth on glycerol, while growth on glucose had a productivity of 0.096 g lipid/(l h). During growth on the 9:1 mixture of both glucose and glycerol, lipid productivity was 0.098 g lipid/(l h). In order to simulate the use of waste glycerol from biodiesel production the experiments were repeated and similar growth rates, yields, and lipid productivities were achieved. Therefore, we have demonstrated the promise for simultaneous high growth rates and lipid yields of C. protothecoides heterotrophically grown on mixtures of glycerol.     

Improved n-butanol production by a non-acetone producing Clostridium pasteurianum DSMZ 525 in mixed substrate fermentation

The kinetics of growth, acid and solvent production in batch culture of Clostridium pasteurianum DSMZ 525 were examined in mixed or mono-substrate fermentations. In pH-uncontrolled batch cultures, the addition of butyric acid or glucose significantly enhanced n-butanol production and the ratio of butanol/1,3-propanediol. In pH-controlled batch culture at pH?=?6, butyric acid addition had a negative effect on growth and did not lead to a higher n-butanol productivity. On the other hand, mixed substrate fermentation using glucose and glycerol enhanced the growth and acid production significantly. Glucose limitation in the mixed substrate fermentation led to the reduction or inhibition of the glycerol consumption by the growing bacteria. Therefore, for the optimal growth and n-butanol production by C. pasteurianum, a limitation of either substrate should be avoided. Under optimized batch conditions, n-butanol concentration and maximum productivity achieved were 21 g/L, and 0.96 g/L?×?h, respectively. In comparison, mixed substrate fermentation using biomass hydrolysate and glycerol gave a n-butanol concentration of 17 g/L with a maximum productivity of 1.1 g/L?×?h. In terms of productivity and final n-butanol concentration, the results demonstrated that C. pasteurianum DSMZ 525 is well suitable for n-butanol production from mixed substrates of biomass hydrolysate and glycerol and represents an alternative promising production strain.     

Glycerol increases growth,protein production and alters the fatty acids profile of Spirulina (Arthrospira) sp LEB 18

Most of the crude glycerol produced globally is generated by biodiesel production, which makes this byproduct an environmental responsibility of the biofuel industries. Among the forms of this compound in use, microalgae cultivation is a promising alternative that may generate a reduction in crude glycerol treatment costs via using it as an organic, carbon-rich substrate in culture media. In this work, the influence of different concentrations of glycerol in the culture medium, the composition of fatty acids and proteins in Spirulina sp. LEB 18 biomass and their effect on its growth were investigated. The fatty acid profile of the biomass was altered, showing a 20% increase in the unsaturated concentration and a 60% reduction in the saturated concentration in the culture supplemented with 0.05 mol L⁻¹ of glycerol compared to those in the control. The addition of the substrate stimulated an increase in its cellular concentration (3.00 g L⁻¹, 0.05 mol L⁻¹), productivity (0.72 g L⁻¹ d⁻¹, 0.05 mol L⁻¹) and its protein production (69.78% w w⁻¹, 0.05 mol L⁻¹).     

Properties of a membrane-bound triglyceride lipase of rapeseed (Brassica napus L.) cotyledons

The properties of the alkaline lipase activity (EC 3.1.1.3) that was recovered almost completely from a microsomal membrane fraction of 4-d-old rapeseed (Brassica napus L.) cotyledons were studied employing a titrimetric test procedure. The apparent K_M was 6.5 mmol l^-1, with emulgated sunflower oil as the substrate. The products of triglyceride hydrolysis in vitro were glycerol, free fatty acids, and minor amounts of mono- and diglycerides. Maximum lipase activity depended on the preincubation of the lipolytic membrane fraction in 0.15 mol l^-1 NaCl and on the presence of at least 0.1 mol l^-1 NaCl in the test mixture. Desoxycholate and up to 0.1 mol l^-1 CaCl₂ also activated the enzyme while EDTA and detergents such as trito x-100, digitonin, tween 85, and sodium dodecylsulfate were inhibitory. The rapeseed lipase displayed a conspicuous substrate selectivity among different plant triglycerides; the activity was inversely correlated with the oleic acid content of the oils. Water-soluble triacetin and the phospholipid lecithin were not hydrolyzed. Increasing amounts of free fatty acids reduced lipase activity; erucic acid, a major component of rapeseed oil, exhibited the strongest effect, suggesting a possible role in the regulation of lipase activity in vivo. The data demonstrate that the lipolytic membrane fraction houses a triglyceride lipase with properties similar to other plant and animal lipases. It can both qualitatively and quantitatively account for the fat degradation in rapeseed cotyledons. The evidence that provides further reason to acknowledge the membranous appendices of the spherosomes as the intracellular site of lipolysis is discussed.     

The peculiarities of succinic acid production from rapeseed oil by Yarrowia lipolytica yeast

The process of succinic acid (SA) production represents the combination of microbial synthesis of α-ketoglutaric acid from rapeseed oil by yeast Yarrowia lipolytica VKM Y-2412 and subsequent decarboxylation of α-ketoglutaric acid by hydrogen peroxide to SA that leads to the production of 69.0 g l^?1 of SA and 1.36 g l^?1 of acetic acid. SA was isolated from the culture broth filtrate in a crystalline form. The SA recovery from the culture filtrate has certain difficulties due to the presence of residual triglycerides of rapeseed oil. The effect of different methods of the culture filtrate treatment and various sorption materials on the coagulation of triglycerides was studied, and as a result, the precipitation of residual triglycerides by acetone was chosen. The subsequent isolation procedures involved the decomposition of H₂O₂ in the filtrate followed by filtrate bleaching and acidification with a mineral acid, evaporation of filtrate, and SA extraction with ethanol from the residue. The purity of crystalline SA isolated from the culture broth filtrate achieved 97.6–100 %. The product yield varied from 62.6 to 71.6 % depending on the acidity of the supernatant.     

190 The peculiarities of regulation of Yarrowia lipolytica yeast and citric acid overproduction

The growth of Yarrowia lipolytica yeast as well the biosynthesis of citric acid on rapeseed oil were studied. It was indicated that the initial step of assimilation of rapeseed oil in the yeast Y. lipolytica is their hydrolysis by extracellular lipases with the formation of glycerol and fatty acids, which appear in the medium in the phase of active growth. The concentrations of these metabolites change insignificantly upon further cultivation. Lipase and the key enzymes of glycerol metabolism (glycerol kinase) and the glyoxylate cycle responsible for the metabolism of fatty acids (isocitrate lyase and malate synthase) are induced just at the beginning of the growth phase and remain active in the course of further cultivation. These results, taken together, suggest that glycerol and fatty acids according in the medium do not suppress the metabolism of each other. The fact that glycerol and fatty acids can be consumed simultaneously is of special importance for the development of the efficient regime of oil feeding, Y. lipolytica produced citric acid (175?g/L) with a yield of 150%. It should be noted that the simultaneous utilization of two different substrates is not typical of micro-organisms, which first assimilate one of the two available substrates (commonly, a carbohydrate), whereas the assimilation of the other substrate starts only after the first substrate is fully consumed from the medium. Indeed, upon the cultivation of Y. lipolytica on the mixture of glucose and oleic acid, the latter substrate began to be utilized only when the concentration of glucose decreased. The glycolytic enzyme pyruvate dehydrogenase was induced from the first hours of cultivation and remained at high levels until the exhaustion of glucose in the medium. At the same time, the activities of isocitrate lyase and malate synthase were very low during the metabolism of glucose, but were rapidly induced (approximately in 10 times) after the exhaustion of glucose in the medium. When Y. lipolytica was grown on the mixture of glucose and hexadecane, the dynamics of growth and substrate consumption was typical of the diauxie phenomenon: the utilization of hexadecane began only in several hours after the time when glucose was completely exhausted in the cultivation medium. In this case, the exhaustion of glucose arrested growth and the culture resumed growth only after a lag period. The assay of enzymes showed that the glycolytic enzyme pyruvate dehydrogenase was active during the phase of growth on glucose, whereas the enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase were active during the phase of growth on hexadecane. In recent years in the literature, there are data that the different sugars produce signals which modify the conformation of certain proteins that, in turn, directly or through a regulatory cascade affect the expression of the genes subject to catabolite repression. These genes are not all controlled by a single set of regulatory proteins (Cho et al. 2009), but there are different circuits of repression for different groups of genes (Gancedo 1990). We will discuss the possible metabolic regulation in the case of Y. lipolytica.     

1,3-Propanediol oxidoreductase production with Klebsiella pneumoniae DSM2026

We report a Klebsiella pneumoniae DSM2026 fermentation procedure for the efficient production of a key enzyme of 1,3-propanediol formation: 1,3-propanediol oxidoreductase (E.C. 1.1.1.202). The fermentation process is composed of an aerobic batch phase on glucose and glycerol and an anaerobic phase on glycerol. The role of the aerobic phase is to produce sufficiently high cell mass (12.9–14.6 g/l dry weight) and to activate the aerobic branch of the Klebsiella glycerol pathway, whereas in the anaerobic phase there is a rapid initiation of 1,3-propanediol oxidoreductase formation. A fast change from an aerobic to an anaerobic environment led to a redox imbalance, which resulted in the abrupt activation of the anaerobic branch of glycerol utilization, with the occurrence of a high 1,3-propanediol-oxidoreductase activity. A mathematical model with substrate inhibition showed that the adequate glycerol concentration for enzyme production was 14–16 g/l. The combination of the optimal substrate concentration together with the subsequent use of glucose and glycerol resulted in 90.6 ± 11.6 U enzyme activity referred to 1 l of fermentation broth and 10.3 ± 0.9 U/(1 h) productivity.     

Enhanced production of erythritol by Yarrowia lipolytica on glycerol in repeated batch cultures

Erythritol is an important natural sweetener, industrially produced only by fermentation on glucose media. Glycerol is an important renewable feedstock as it is the major by-product of the biodiesel production process; here we present an alternative way to convert this low-cost substrate into value-added products, such as erythritol. Repeated batch cultures (RBC) were performed to improve the productivity of erythritol from pure and crude glycerol. An acetate negative mutant of Yarrowia lipolytica Wratislavia K1 was found to be applicable for the production of high amounts of erythritol in RBC. When 20 % of fresh replaced medium was added, the strain Wratislavia K1 was able to produce 220 g l ^?1 erythritol, which corresponded to a 0.43 g g^?1 yield and a productivity of 0.54 g l^?1 h^?1. Additionally, the activity of the culture remained stable for more than 1,000 h, i.e., 11 cycles of the repeated batch bioreactors.     

Adaptation of acidogenic sludge to increasing glycerol concentrations for biohydrogen production

Hydrogen is a promising alternative as an energetic carrier and its production by dark fermentation from wastewater has been recently proposed, with special attention to crude glycerol as potential substrate. In this study, two different feeding strategies were evaluated for replacing the glucose substrate by glycerol substrate: a one-step strategy (glucose was replaced abruptly by glycerol) and a step-by-step strategy (progressive decrease of glucose concentration and increase of glycerol concentration from 0 to 5 g L^?1), in a continuous stirred tank reactor (12 h of hydraulic retention time (HRT), pH 5.5, 35 °C). While the one-step strategy led to biomass washout and unsuccessful H₂ production, the step-by-step strategy was efficient for biomass adaptation, reaching acceptable hydrogen yields (0.4?±?0.1 mol_H2?mol^?1 _{glycerol consumed}) around 33 % of the theoretical yield independently of the glycerol concentration. Microbial community structure was investigated by single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) fingerprinting techniques, targeting either the total community (16S ribosomal RNA (rRNA) gene) or the functional Clostridium population involved in H₂ production (hydA gene), as well as by 454 pyrosequencing of the total community. Multivariate analysis of fingerprinting and pyrosequencing results revealed the influence of the feeding strategy on the bacterial community structure and suggested the progressive structural adaptation of the community to increasing glycerol concentrations, through the emergence and selection of specific species, highly correlated to environmental parameters. Particularly, this work highlighted an interesting shift of dominant community members (putatively responsible of hydrogen production in the continuous stirred tank reactor (CSTR)) according to the gradient of glycerol proportion in the feed, from the family Veillonellaceae to the genera Prevotella and Clostridium sp., putatively responsible of hydrogen production in the CSTR.     

Sophorolipid production by <Emphasis Type="Italic">Candida bombicola</Emphasis> on oils with a special fatty acid composition and their consequences on cell viability

Sophorolipids production by the yeast Candia bombicola is most favourable when glucose is used as a carbon source in combination with a hydrophobic carbon source such as a common vegetable oil. Most vegetable oils are comprised of C16–C18 fatty acids, an ideal range for sophorolipid production. The use of oils with either shorter or longer fatty acids, such has coconut oil or meadowfoam oil, respectively, was evaluated. Such oils did not contribute to enhanced sophorolipid production when compared to cultures run on glucose as the sole carbon source. Moreover, a toxic effect of medium-chain fatty acids towards stationary C. bombicola cells was demonstrated.     

Microbial synthesis of polyhydroxybutyrate from glycerol: Gluconeogenesis, molecular weight and material properties of biopolyester

Glycerol is considered as an ideal feedstock for producing bioplastics via bacterial fermentation due to its ubiquity, low price, and high degree of reduction substrate. In this work, we study the yield and cause of limitation in poly(3‐hydroxybutyrate) (PHB) production from glycerol. Compared to glucose‐based PHB production, PHB produced by Cupriavidus necator grown on glycerol has a low productivity (0.92 g PHB/L/h) with a comparably low maximum specific growth rate of 0.11 h^?1. We found that C. necator can synthesize glucose from glycerol and that the lithotrophical utilization of glycerol (non‐fermentative substrate) or gluconeogenesis is an essential metabolic pathway for biosynthesis of cellular components. Here, we show that gluconeogenesis affects the reduction of cell mass, the productivity of biopolymer product, and the molecular chain size of intracellular PHB synthesized from glycerol by C. necator. We use NMR spectroscopy to show that the isolated PHB is capped by glycerol. We then characterized the physical properties of the isolated glycerol‐based PHB with differential scanning calorimetry and tensile tests. We found that although the final molecular weight of the glycerol‐based PHB is lower than those of glucose‐based and commercial PHB, the thermal and mechanical properties of the biopolymers are similar. Biotechnol. Bioeng. 2012; 109: 2808–2818. © 2012 Wiley Periodicals, Inc.     

Environmental conditions during glycerol bioconversion affect 3-hydroxypropionic acid bioproduction by Limosilactobacillus reuteri DSM 17938

3-Hydroxypropionic acid (3-HP) is a platform molecule whose biological production was carried out by the bacterium Limosilactobacillus reuteri according to a two-step process: first, a growth phase in batch mode on glucose, then a glycerol bioconversion into 3-HP in fed-batch mode. With the objective of improving 3-HP bioproduction, this study aimed at defining the operating conditions during the bioconversion phase that increases the bioproduction performance. A central composite rotatable design allowed testing various pH levels and specific glycerol feeding rates. By establishing response surfaces, optimal conditions have been identified that were different depending on the considered output variable (final 3-HP quantity, 3-HP production yield and production rate). Of them, 3-HP final quantity and 3-HP production yield were maximized at pH 6.0 and at specific glycerol feeding rates of 60 and 55 mg_gly g_CDW⁻¹ h⁻¹, respectively. The specific 3-HP production rate was the highest at the upper limit of the specific substrate feeding rate (80 mg_gly g_CDW⁻¹ h⁻¹) but was not affected by the pH. An additional experiment was carried out at pH 6.0 and a specific glycerol feeding rate of 80 mg_gly g_CDW⁻¹ h⁻¹ to validate the previous observations. In conclusion, the results showed a significant improvement of 3-HP concentration by 13%, of specific production rate by 34% and of 3-HP volumetric productivity by 39%, as compared to the initial values.     

Simple defined autoinduction medium for high-level recombinant protein production using T7-based Escherichia coli expression systems

Protein production under the control of lac operon regulatory elements using autoinduction is based on diauxic growth of Escherichia coli on lactose after consumption of more preferred carbon substrates. A novel simple and cost-effective defined autoinduction medium using a mixture of glucose, glycerol, and lactose as carbon substrate and NH₄⁺ as sole nitrogen source without any supplementation of amino acids and vitamins was developed for T7-based E. coli expression systems. This medium was successfully employed in 96-well microtiter plates, test tubes, shake flasks, and 15-L bioreactor cultivations for production of different types of proteins achieving an average yield of 500 mg L⁻¹ product. Cell-specific protein concentrations and solubility were similar as during conventional isopropyl β-d-1-thiogalactopyranoside induction using Luria-Bertani broth. However, the final yield of target proteins was about four times higher, as a higher final biomass was achieved using this novel defined autoinduction broth.