Free fatty acids reduce metabolic stress and favor a stable production of heterologous proteins in Pichia pastoris

ABSTRACT The growth of yeasts in culture media can be affected by many factors. For example, methanol can be metabolized by other pathways to produce ethanol, which acts as an inhibitor of the heterologous protein production pathway; oxygen concentration can generate aerobic or anaerobic environments and affects the fermentation rate; and temperature affects the central carbon metabolism and stress response protein folding. The main goal of this study was determine the implication of free fatty acids on the production of heterologous proteins in different culture conditions in cultures of Pichia pastoris. We evaluated cell viability using propidium iodide by flow cytometry and thiobarbituric acid reactive substances to measure cell membrane damage. The results indicate that the use of low temperatures and low methanol concentrations favors the decrease in lipid peroxidation in the transition phase from glycerol to methanol. In addition, a temperature of 14 ºC + 1%M provided the most stable viability. By contrast, the temperature of 18 ºC + 1.5%M favored the production of a higher antibody fragment concentration. In summary, these results demonstrate that the decrease in lipid peroxidation is related to an increased production of free fatty acids.

Statistical optimization of kojic acid production by a UV-induced mutant strain of Aspergillus terreus

ABSTRACT The ability of four Aspergillus strains for biosynthesis of kojic acid was evaluated among which Aspergillus terreus represented the highest level (2.21 g/L) of kojic acid production. Improvement kojic acid production ability of A. terreus by random mutagenesis using different exposure time to ultraviolet light (5-40 min) was then performed to obtain a suitable mutant of kojic acid production (designated as C5-10, 7.63 g/L). Thereafter, design of experiment protocol was employed to find medium components (glucose, yeast extract, KH2PO4 (NH4)2SO4, and pH) influences on kojic acid production by the C5-10 mutant. A 25-1 fractional factorial design augmented to central composite design showed that glucose, yeast extract, and KH2PO4 were the most considerable factors within the tested levels (p < 0.05). The optimum medium composition for the kojic acid production by the C5-10 mutant was found to be glucose, 98.4 g/L; yeast extract, 1.0 g/L; and KH2PO4, 10.3 mM which was theoretically able to produce 120.2 g/L of kojic acid based on the obtained response surface model for medium optimization. Using these medium compositions an experimental maximum Kojic acid production (109.0 ± 10 g/L) was acquired which verified the efficiency of the applied method.

Carotenoid production in sugarcane juice and synthetic media supplemented with nutrients by Rhodotorula rubra l02

ABSTRACT In order for the use of biological carotenoids to become feasible, it is necessary to have adequate low cost sources and improved methods of cultivation. The aim of this study was to evaluate the effect of supplementation with nitrogen, phosphorus, zinc, and magnesium, on the biomass and carotenoid volumetric production by yeast Rhodotorula rubra L02 using a complex medium (sugarcane juice) and synthetic media (sucrose and maltose) as substrates. The experimental design used for each substrate was randomized in blocks with 16 treatments and 3 repetitions. The treatments were compound for 15 different combinations of nutrients (N; Mg; Zn; P, N + Mg; N + Zn; N + P; Mg + Zn; Mg + P; Zn + P; N + P + Zn; N + P + Mg; N + Zn + Mg; P + Zn + Mg; N + Zn + Mg + P) alone and combined, and a control. The results were submitted to analysis of variance and Tukey test at 5% significance level. Among the treatments evaluated, the highest production of dry biomass, with both maltose and sucrose, was observed for Mg (1.60 g/L and 1.94 g/L, respectively). Additionally, another treatment that stood out in terms of biomass production was the control treatment with maltose (1.54 g/L). After the incubation time, killer activity was not observed since there was no formation of inhibition halo around the L02 yeast.

Screening of medium constituents for clavulanic acid production by Streptomyces clavuligerus

ABSTRACT Clavulanic acid is a β-lactam compound with potent inhibitory activity against β-lactamases. Studies have shown that certain amino acids play essential roles in CA biosynthesis. However, quantitative evaluations of the effects of these amino acids are still needed in order to improve CA production. Here, we report a study of the nutritional requirements of Streptomyces clavuligerus for CA production. Firstly, the influence of the primary nitrogen source and the salts composition was investigated. Subsequently, soybean protein isolate was supplemented with arginine (0.0-3.20 g L-1), threonine (0.0-1.44 g L-1), ornithine (0.0-4.08 g L-1), and glutamate (0.0-8.16 g L-1), according to a two-level central composite rotatable design. A medium containing ferrous sulfate yielded CA production of 437 mg L-1, while a formulation without this salt produced only 41 mg L-1 of CA. This substantial difference suggested that Fe2+ is important for CA biosynthesis. The experimental design showed that glutamate and ornithine negatively influenced CA production while arginine and threonine had no influence. The soybean protein isolate provided sufficient C5 precursor for CA biosynthesis, so that supplementation was unnecessary. Screening of medium components, together with experimental design tools, could be a valuable way of enhancing CA titers and reducing the process costs.

Exopolysaccharide production from Bacillus velezensis KY471306 using statistical experimental design

Abstract Exopolysaccharide (EPS) biopolymers produced by microorganisms play a crucial role in the environment such as health and bio-nanotechnology sectors, gelling agents in food and cosmetic industries in addition to bio-flocculants in the environmental sector as they are degradable, nontoxic. This study focuses on the improvement of EPS production through manipulation of different culture and environmental conditions using response surface methodology (RSM). Plackett-Burman design indicated that; molasses, yeast extract and incubation temperature are the most effective parameters. Box-Behnken RSM indicated that; the optimum concentration for each parameter was 12% (w/v) for molasses, 6 g/L yeast extract and 30 °C for incubation temperature. The most potent bacterial isolate was identified as Bacillus velezensis KY498625. After production, EPS was extracted, purified using DEAE-cellulose, identified using Fourier transform infrared (FTIR), gel permeation chromatography (GPC) and gas chromatography-mass spectroscopy (GC-MS). The result indicated that; it has molecular weight 1.14 × 105 D consisting of glucose, mannose and galactose.

Genetic characterization, evaluation of growth and production of biomass of strains from wild edible mushrooms of Lyophyllum of Central Mexico

Abstract The present study conducted a genetic characterization and determined growth rate and biomass production in solid and liquid media, using strains obtained from wild edible sporomes of Lyophyllum that grow in high mountains. Vegetative isolation was used to obtain a total of four strains, which were divided into two clades within the section Difformia: Lyophyllum sp. and Lyophyllum aff. shimeji. Growth rate and biomass production were influenced by both the culture media and the strains. In a potato dextrose agar medium, the strains presented a higher growth rate, while in a malt extract-peptone and yeast agar medium, the growth rate was lower, but with a higher biomass production that was equal to that in the malt extract-peptone and yeast liquid medium.

Statistical tools application on dextranase production from Pochonia chlamydosporia (VC4) and its application on dextran removal from sugarcane juice

ABSTRACT The aim of this study was to optimize the dextranase production by fungus Pochonia chlamydosporia (VC4) and evaluate its activity in dextran reduction in sugarcane juice. The effects, over the P. chlamydosporia dextranase production, of different components from the culture medium were analyzed by Plackett-Burman design and central composite design. The response surface was utilized to determine the levels that, among the variables that influence dextranase production, provide higher production of these enzymes. The enzymatic effect on the removal of dextran present in sugarcane juice was also evaluated. It was observed that only NaNO3 and pH showed significant effect (p<0.05) over dextranase production and was determined that the levels which provided higher enzyme production were, respectively, 5 g/L and 5.5. The dextranases produced by fungus P. chlamydosporia reduced by 75% the dextran content of the sugarcane juice once treated for 12 hours, when compared to the control treatment.

Pleurotus spp. cultivation on Brachiaria sp. straw treatment with alkaline water

Abstract The aim of this research was to evaluate the efficiency of aqueous alkali-treated Brachiaria straw for the cultivation of appropriate species of oyster mushroom. The substrate used in the cultivation of various Pleurotus spp. was soaked for 20 min by using two different procedures: (i) 0.5-2.0% Ca(OH)2 in 100 L water, and (ii) 50-250 L water. As a result, 1% Ca(OH)2 dissolved in 100 L water and 3.5 kg of Brachiaria straw presented the best production. The most suitable species for the application of the present method were P. pulmonarius and P. sapidus. The success of this technique is directly related to the concentration of Ca(OH)2 and water, the species, and the origin and quality of raw material used as the substrate in the production of oyster mushroom.

Phenotype-driven strategies for screening Candida parapsilosis complex for molecular identification

Abstract In this study, phenotypic methods presented >80% agreement with the molecular identification of 59 Candida parapsilosis complex. Growth at 15% NaCl or pH 7.0 significantly reduced cfu-counts of Candida orthopsilosis, suggesting these conditions may support the development of phenotypic methods for the differentiation of the cryptic species of C. parapsilosis complex.

First report of the production of a potent biosurfactant with α, ß-trehalose by Fusarium fujikuroi under optimized conditions of submerged fermentation

Abstract Biosurfactants have many advantages over synthetic surfactants but have higher production costs. Identifying microorganisms with high production capacities for these molecules and optimizing their growth conditions can reduce cost. The present work aimed to isolate and identify a fungus with high biosurfactant production capacity, optimize its growth conditions in a low cost culture medium, and characterize the chemical structure of the biosurfactant molecule. The fungal strain UFSM-BAS-01 was isolated from soil contaminated with hydrocarbons and identified as Fusarium fujikuroi. To optimize biosurfactant production, a Plackett-Burman design and a central composite rotational design were used. The variables evaluated were pH, incubation period, temperature, agitation and amount of inoculum in a liquid medium containing glucose. The partial structure of the biosurfactant molecule was identified by nuclear magnetic resonance spectrometry. F. fujikuroi reduced surface tension from 72 to 20 mN m−1 under the optimized conditions of pH 5.0, 37 °C and 7 days of incubation with 190 rpm agitation. The partial identification of the structure of the biosurfactant demonstrated the presence of an α,β-trehalose. The present study is the first report of the biosynthesis of this compound by F. fujikuroi, suggesting that the biosurfactant produced belongs to the class of trehalolipids.

Bioconversion of waste cooking oil glycerol from cabbage extract to lactic acid by Rhizopus microsporus

Abstract Glycerol from spent oil was processed by transesterification for biodiesel production. Although glycerol contains many types of impurities, it can be used as a C-source for lactic acid production by fungi, such as Rhizopus microsporus. In this study, we found that wild type R. microsporus (LTH23) produced more lactic acid than the mutant strains on cabbage glycerol media (CG media). More lactic acid was produced on CG media than on cabbage extract media (C media) by about two-fold in batch fermentation conditions. In addition, we found that lactic acid production in a fed-batch process was also slightly higher than in a batch process. To study the combined effects of pH, urea, and glycerol waste concentration on lactic acid production, a response surface methodology was used. The optimum pH, urea, and glycerol waste concentrations were pH 6.5, 3.75 g/L, and 17 g/L, respectively. The maximum lactic acid production predicted by this equation model was 4.03 g/L.

Cost-effective production of bacterial cellulose using acidic food industry by-products

Abstract To reduce the cost of obtaining bacterial cellulose, acidic by-products of the alcohol and dairy industries were used without any pretreatment or addition of other nitrogen sources. Studies have shown that the greatest accumulation of bacterial cellulose (6.19 g/L) occurs on wheat thin stillage for 3 days of cultivation under dynamic conditions, which is almost 3 times higher than on standard Hestrin and Schramm medium (2.14 g/L). The use of whey as a nutrient medium makes it possible to obtain 5.45 g/L bacterial cellulose under similar conditions of cultivation. It is established that the pH of the medium during the growth of Gluconacetobacter sucrofermentans B-11267 depends on the feedstock used and its initial value. By culturing the bacterium on thin stillage and whey, there is a decrease in the acidity of the waste. It is shown that the infrared spectra of bacterial cellulose obtained in a variety of environments have a similar character, but we found differences in the micromorphology and crystallinity of the resulting biopolymer.

Evaluation of a selective chromogenic medium for detecting vancomycin-resistant enterococci

ABSTRACT Rapid identification of vancomycin-resistant enterococci (VRE) can assist in choosing the appropriate treatment and preventing VRE spread. The performance of chromIDTM VRE agar was evaluated using 184 clinical isolates of Enterococcus spp. and reference strains. The test had a sensitivity of 95.52% but a low specificity of 30%.

Pectin lyase overproduction by Penicillium griseoroseum mutants resistant to catabolite repression

Abstract Expression of pectinolytic genes is regulated by catabolic repression limiting the production of pectin lyase (PL) if the natural inducer, pectin, is missing from the growth medium. Here, we report the isolation of Penicillium griseoroseum mutants resistant to 2-deoxy-d-glucose (DG) that show resistance to catabolite repression and overproduce PL. Three spontaneous and nine UV-induced mutants were obtained. Some mutants produced sectors (segments morphologically different) that were also studied. The mutants were analyzed for pectinases production on pectinase-agar plates and five mutants and two sectors showing larger clearing zones than the wild type were selected for quantitative assay. Although PL production higher than the wild type has been found, phenotype instability was observed for most of the mutants and, after transfers to nonselective medium, the DG resistance was no longer present. Only mutants M03 and M04 were stable maintaining the DG-resistance phenotype. When growing for 120 h in liquid medium containing glucose with or without pectin, both mutants showed higher PL production. In the presence of glucose as sole carbon source, the mutant M03 produced 7.8-fold more PL than the wild type. Due its phenotypic stability and PL overproduction, the mutant M03 presents potential for industrial applications.

Optimizing culture conditions for production of intra and extracellular inulinase and invertase from Aspergillus niger ATCC 20611 by response surface methodology (RSM)

Abstract The aim of this study was obtain a model that maximizes growth and production of inulinase and invertase by Aspergillus niger ATCC 20611, employing response surface methodology (RSM). The RSM with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. Results showed that the experimental data could be appropriately fitted into a second-order polynomial model with a coefficient of determination (R2) more than 0.90 for all responses. This model adequately explained the data variation and represented the actual relationships between the parameters and responses. The pH and temperature value of the cultivation medium were the most significant variables and the effects of inoculum size and agitation speed were slightly lower. The intra-extracellular inulinase, invertase production and biomass content increased 10-32 fold in the optimized medium condition (pH 6.5, temperature 30 °C, 6% (v/v), inoculum size and 150 rpm agitation speed) by RSM compared with medium optimized through the one-factor-at-a-time method. The process development and intensification for simultaneous production of intra-extracellular inulinase (exo and endo inulinase) and invertase from A. niger could be used for industrial applications.

Evaluation of short-chain-length polyhydroxyalkanoate accumulation in Bacillus aryabhattai

Abstract This study was focused on the polyhydroxybutyrate (PHB) accumulation property of Bacillus aryabhattai isolated from environment. Twenty-four polyhydroxyalkanoate (PHA) producers were screened out from sixty-two environmental bacterial isolates based on Sudan Black B colony staining. Based on their PHA accumulation property, six promising isolates were further screened out. The most productive isolate PHB10 was identified as B. aryabhattai PHB10. The polymer production maxima were 3.264 g/L, 2.181 g/L, 1.47 g/L, 1.742 g/L and 1.786 g/L in glucose, fructose, maltose, starch and glycerol respectively. The bacterial culture reached its stationary and declining phases at 18 h and 21 h respectively and indicated growth-associated PHB production. Nuclear Magnetic Resonance (NMR) spectra confirmed the material as PHB. The material has thermal stability between 30 and 140 °C, melting point at 170 °C and maximum thermal degradation at 287 °C. The molecular weight and poly dispersion index of the polymer were found as 199.7 kDa and 2.67 respectively. The bacterium B. aryabhattai accumulating PHB up to 75% of cell dry mass utilizing various carbon sources is a potential candidate for large scale production of bacterial polyhydroxybutyrate.

Cultivation of Pichia pastoris carrying the scFv anti LDL (-) antibody fragment. Effect of preculture carbon source

Abstract Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (-) under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol) used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8 h.

Enhanced production of erythritol and mannitol by Yarrowia lipolytica in media containing surfactants

Abstract Various chemical compounds, including surfactants, when introduced to culture media may increase the permeability of cellular membranes and thereby affect the quantity of metabolites excreted by cells. The aim of the present study was to evaluate the impact of detergents including Triton X-100, Span 20 and Tween 80 on erythritol production from glycerol by Yarrowia lipolytica Wratislavia K1 in a shake-flask experiment, batch and fed-batch cultures. When Span 20 was added to a fed-batch culture with glycerol as a carbon source (300 g L-1), erythritol production increased by 15% compared to the culture without the surfactant where it reached 142 g L-1 after 5 days, which corresponded to 0.47 g g-1 yield and productivity of 1.1 g L-1 h-1. Therefore, it was concluded that Span 20 considerably enhanced the production of this polyol from glycerol.

Sugarcane straw as a feedstock for xylitol production by Candida guilliermondii FTI 20037

Abstract Sugarcane straw has become an available lignocellulosic biomass since the progressive introduction of the non-burning harvest in Brazil. Besides keeping this biomass in the field, it can be used as a feedstock in thermochemical or biochemical conversion processes. This makes feasible its incorporation in a biorefinery, whose economic profitability could be supported by integrated production of low-value biofuels and high-value chemicals, e.g., xylitol, which has important industrial and clinical applications. Herein, biotechnological production of xylitol is presented as a possible route for the valorization of sugarcane straw and its incorporation in a biorefinery. Nutritional supplementation of the sugarcane straw hemicellulosic hydrolyzate as a function of initial oxygen availability was studied in batch fermentation of Candida guilliermondii FTI 20037. The nutritional supplementation conditions evaluated were: no supplementation; supplementation with (NH4)2SO4, and full supplementation with (NH4)2SO4, rice bran extract and CaCl2·2H2O. Experiments were performed at pH 5.5, 30 °C, 200 rpm, for 48 h in 125 mL Erlenmeyer flasks containing either 25 or 50 mL of medium in order to vary initial oxygen availability. Without supplementation, complete consumption of glucose and partial consumption of xylose were observed. In this condition the maximum xylitol yield (0.67 g g-1) was obtained under reduced initial oxygen availability. Nutritional supplementation increased xylose consumption and xylitol production by up to 200% and 240%, respectively. The maximum xylitol volumetric productivity (0.34 g L-1 h-1) was reached at full supplementation and increased initial oxygen availability. The results demonstrated a combined effect of nutritional supplementation and initial oxygen availability on xylitol production from sugarcane straw hemicellulosic hydrolyzate.

Surface response methodology for the optimization of lipase production under submerged fermentation by filamentous fungi

Abstract A Plackett–Burman Factorial Design of 16 experiments was conducted to assess the influence of nine factors on the production of lipases by filamentous fungi. The factors investigated were bran type (used as the main carbon source), nitrogen source, nitrogen source concentration, inducer, inducer concentration, fungal strain (Aspergillus niger or Aspergillus flavus were selected as good lipase producers via submerged fermentation), pH and agitation. The concentration of the yeast extract and soybean oil and the pH had a significant effect (p < 0.05) on lipase production and were consecutively studied through a Full Factorial Design 23, with the concentration of yeast extract and pH being significant (p < 0.05). These variables were optimized using a central composite design, obtaining maximum lipolytic activities with the use of 45 g/L of yeast extract and pH 7.15. The statistical model showed a 94.12% correlation with the experimental data.