Fermentation Procedure Research Articles

Some Important Criteria for Presentation and Analysis of Data Obtained from Fermentation Processes

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High-performance hydrolysis of wheat straw using cellulase and thermomechanical pretreatment

Wheat straw was ground and pretreated with a thermomechanical process before exposure to a mixture of cellulases (Celluclast). The thermomechanical process known as DIC (Détente Instantanée Contrôlée), which is similar to the steam explosion process, was used to pretreat the lignocellulosic biomass through a flash expansion. The results demonstrate that crushing the wheat straw increased the hydrolysis yields. The initial hydrolysis rates and the final yields of hydrolysis increased relative to those of the untreated straw as a result of the DIC pretreatment. After optimization, the hydrolysis process resulted in a glucose yield of 31g/100g of straw, which corresponds to approximately 90% of the theoretical cellulose value. A fermentation procedure was performed using the microorganism Saccharomyces cerevisiae, and the results confirmed the production of bioethanol (11.5g/100g of pretreated straw). The adsorption isotherms of cellulases on wheat straw and the enzyme kinetics of hydrolysis of the wheat straw were also studied using different concentrations of Celluclast. The kinetic parameters (maximal velocity, Vemax, and half-saturation constant, Ke) were determined from the initial velocities. Kad (adsorption coefficient) results show that the DIC pretreatment of the straw decreased Kad six-fold compared to that of the untreated straw (15.54 vs 2.52g of enzymes/g of biomass, respectively), confirming that the DIC pretreatment produced a substrate that is more accessible to cellulases.

Potential of Thermophilic Fungus Rhizomucor pusillus NRRL 28626 in Biotransformation of Antihelmintic Drug Albendazole

In the present investigation, thermophilic fungus Rhizomucor pusillus was used to study biotransformation of antihelmintic drug albendazole to produce its active metabolite, albendazole sulfoxide and novel metabolites of commercial interest. A two-stage fermentation procedure was followed for biotransformation of albendazole. The transformation was identified and structures were confirmed by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analysis. Four metabolites albendazole sulfoxide, the active metabolite, albendazole sulfone, N-methyl metabolite of albendazole sulfoxide, and a novel metabolite were produced. The study demonstrates the biotransformation ability of thermophilic fungus R. pusillus NRRL28626 in the production of, the active metabolite of albendazole which has industrial and economic importance, other metabolites and a novel metabolite in an ecofriendly way.

Study on the Microbial Hydrolysis of Maize Straw and the Water Reducing Properties of the Hydrolysate

A two-step fermentation procedure for maize straw hydrolysis by Phanerochaete chrysosporium, Trichoderma and Aspergillus nigerwere investigated in this study. 2 mL of P. chrysosporium (107spores/mL) wasadded to 50 mL medium for the first fermentation stage. The optimal culture conditions were 28°C of culture temperature, 4.0 of pH, 8 days of culture time, 2 mL of Trichoderma suspensions (107spores/mL) and 1 mL of A. niger suspensions (107spores/mL) of the inoculums size on the second fermentation stage. Under the optimal conditions, the crude degradation rate achieved 48.2%. Via sulfonation, oxidate-lignosulfonate alkali lignin (OLAL) was achieved from maize straw hydrolysate(MSH).OLAL and MSH can be partial substitution for common water reducers and OLAL was more efficient compared to that of MSH.

Fungal transformation of pimaradienoic acid and its schistosomicidal activity against Schistosoma mansoni

In the present work, the microbial transformation of pimaradienoic acid (PA, 1) (Figure 1) was performed using submerged shaken liquid culture of Aspergillus ochraceus (1.8×106 spores/mL). The microorganism was grown by a two-stage fermentation procedure [1]. PA was added as a dimethylsulfoxide solution (0.1g/L) and incubated for 3 days. The culture was filtered and the aqueous layer was extracted with ethyl acetate to furnish the extract codified as AoPA. Chemical and NMR studies of AoPA allowed us to isolate and to identify two PA derivatives (Figure 1: Compounds 2 and 3).

Coproduction of Acetaldehyde and Hydrogen during Glucose Fermentation by Escherichia coli

Escherichia coli K-12 strain MG1655 was engineered to coproduce acetaldehyde and hydrogen during glucose fermentation by the use of exogenous acetyl-coenzyme A (acetyl-CoA) reductase (for the conversion of acetyl-CoA to acetaldehyde) and the native formate hydrogen lyase. A putative acetaldehyde dehydrogenase/acetyl-CoA reductase from Salmonella enterica (SeEutE) was cloned, produced at high levels, and purified by nickel affinity chromatography. In vitro assays showed that this enzyme had both acetaldehyde dehydrogenase activity (68.07 ± 1.63 μmol min(-1) mg(-1)) and the desired acetyl-CoA reductase activity (49.23 ± 2.88 μmol min(-1) mg(-1)). The eutE gene was engineered into an E. coli mutant lacking native glucose fermentation pathways (ΔadhE, ΔackA-pta, ΔldhA, and ΔfrdC). The engineered strain (ZH88) produced 4.91 ± 0.29 mM acetaldehyde while consuming 11.05 mM glucose but also produced 6.44 ± 0.26 mM ethanol. Studies showed that ethanol was produced by an unknown alcohol dehydrogenase(s) that converted the acetaldehyde produced by SeEutE to ethanol. Allyl alcohol was used to select for mutants with reduced alcohol dehydrogenase activity. Three allyl alcohol-resistant mutants were isolated; all produced more acetaldehyde and less ethanol than ZH88. It was also found that modifying the growth medium by adding 1 g of yeast extract/liter and lowering the pH to 6.0 further increased the coproduction of acetaldehyde and hydrogen. Under optimal conditions, strain ZH136 converted glucose to acetaldehyde and hydrogen in a 1:1 ratio with a specific acetaldehyde production rate of 0.68 ± 0.20 g h(-1) g(-1) dry cell weight and at 86% of the maximum theoretical yield. This specific production rate is the highest reported thus far and is promising for industrial application. The possibility of a more efficient "no-distill" ethanol fermentation procedure based on the coproduction of acetaldehyde and hydrogen is discussed.

Ethanol production from sugar beet molasses by S. cerevisiae entrapped in an alginate–maize stem ground tissue matrix

A new alginate–maize stem ground tissue matrix was developed as a Saccharomyces cerevisiae carrier for ethanol fermentation from sugar beet molasses. There were several fermentation procedures in the present study: using free cells and alginate-entrapped cells with and without maize stem ground tissue supplementation (F; F + C; AB; AB + C), and using a new combined alginate–maize stem ground tissue carrier (ABC). It was found that addition of maize stem ground tissue meal (C), with honeycomb configuration, provided high surface areas for cell attachment and biofilm growth, and also increased alginate matrix porosity, enabling better mass transfer characteristic, better physical strength and stability of beads. The highest values of process parameters were obtained in the case of new carrier (ABC): the ethanol concentration of 60.36 g/l, percentage of the theoretical ethanol yield of 96.56%, ethanol yield of 0.493 g/g and the volumetric ethanol productivity of 2.51 g/l h. The medium supplementation with maize stem ground tissue significantly decreased acetaldehyde and acetic acid content, did not affect fusel alcohol and ethylacetate content of the distillate.

The Influence of Adding Buckwheat Sprouts on the Fermentation Characteristics of Yakju

The purpose of this study was to investigate the fermentation characteristics of Yakju using fresh sprouts from common buckwheat, a Daisan cultivar, and a tartary buckwheat Daikwan 3-3 cultivar to develop a functional Yakju, which is a traditional Korean liquor. As fermentation time increased, alcohol concentration and total sugar content (expressed as Brix degrees) increased, whereas reducing sugar content decreased. In particular, alcohol formation capability was maximized from the fourth to the seventh days of the second mashing stage during the fermentation procedure, which corresponded to the abrupt rise in mashing body temperature. The pH increased slightly when the titratable acidity was kept from increasing as fermentation proceeded. Quercetin and rutin were not present in the control group but their presence in Yakju with added buckwheat sprouts continuously increased with an increase in the fermentation period. Quercetin and rutin contents were higher in the Yakju with added Daikwan3-3 buckwheat sprouts than Yakju with added Daisan buckwheat sprouts. In conclusion, adding buckwheat sprouts improved Yakju quality during fermentation. Particularly, Yakju with added Daikwan3-3 buckwheat sprouts had superior fermentation characteristics and quality.

Probiotics Bacteria from Egyptian Infants cause Cholesterol Removal in Media and Survive in Yoghurt

One of the most significant groups of probiotic organisms are the lactic acid bacteria, commonly used in fermented dairy products. In this study, cultures were isolated from two infants. After screening for the classic properties of probiotic organisms, four promising isolates were identified as two strains of Lactobacillus acidophilus (P106, P110), strain of Lactobacillus plantarum (P164) and Lactobacillus. pentosus (P191)which were tested for capability to remove cholesterol and to deconjugate sodium taurocholate from the culture medium. Results showed that a considerable variation existed among cultures in their growth viability in the presence of bile salt, deconjugation of sodium tauro-cholate and assimilation of cholesterol from the medium. All tested strains removed less cholesterol from the broth (ranged from 4.02-24.32%) compared to those grown in broth supplemented with 0.2% bile salts (from 29.02 to 45.3). Lactobacillus acidophilus P106 appeared to be more active in bile salt hydrolase compared to the other strains, and therefore, is regarded as a suitable candidate probiotic and adjunct culture.These strains were employed to make yo-ghurt and, in order to achieve a short production time; a two-stage fermentation procedure was used with Streptococ-cus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus providing the rapid acidification. Storage trials at 4o C showed that the viability of the probiotic cultures was retained over 15 days.

A kinetic study on extraction and transformation phenomena of phenolic compounds during red wine fermentation

SummaryThe aim of this work was both to model the kinetics of the extraction and transformation of phenolic compounds during fermentation and to propose an index to compare fermentation procedures. Six lots of Sangiovese grapes from five vineyards were used for red wine fermentation tests on lab and industrial scale during 2005 and 2006 vintages. Total phenol index, colour parameters, anthocyanin and pigmented polymer contents were measured during fermentations as variables related to phenolics. Microvinifications performed during vintage 2005 were used to model the evolution of total phenol index at 280 nm, malvidine content, colour parameter a* and pigmented polymer content during fermentation. Vinifications performed during vintage 2006 were used to validate the models. A specific adimensional number, called equivalent index, was set up to specify the operating conditions of red wine fermentation being a function of the extraction and transformation phenomena of phenolic compounds.

Reaction engineering studies for the production of 2-hydroxyisobutyric acid with recombinant Cupriavidus necator H 16

Recombinant Cupriavidus necator H 16 with a novel metabolic pathway using a cobalamin-dependent mutase was exploited to produce 2-hydroxyisobutyric acid (2-HIBA) from renewable resources through microbial fermentation. 2-HIBA production capacities of different strains of C. necator H 16 deficient in the PHB synthase gene and genetically engineered to enable the production of 2-HIBA from the intracellular PHB precursor (R)-3-hydroxybutyryl-CoA were evaluated in 48 parallel milliliter-scale stirred tank bioreactors (V = 11 mL). The effects of media composition, limitations, pH, and feed rate were studied with respect to the overall process performances of the different recombinant strains. 2-HIBA production was at a maximum at nitrogen limiting conditions and if the pH was controlled between 6.8 and 7.2 under fed-batch operating conditions (intermittent fructose addition). The final concentration of 2-HIBA was 7.4 g L(-1) on a milliliter scale. Best reaction conditions identified on the milliliter scale were transferred to a laboratory-scale fed-batch process in a stirred tank bioreactor (V = 2 L). Two different process modes for the production of 2-HIBA, a single-phase and a dual-phase fermentation procedure, were evaluated and compared on a liter scale. The final concentration of 2-HIBA was 6.4 g L(-1) on a liter scale after 2 days of cultivation.

Improvement of the Functional Qualities of Sea Tangle Extract through Fermentation by Aspergillus oryzae

This study was conducted to evaluate the potential of a microbial fermentation procedure to improve the functional qualities of seaweeds. Aspergillus oryzae, which has been used in traditional Korean fermented foods, was inoculated and cultivated in an aqueous extract of sea tangle (Laminaria japonica). Fermentation of the sea tangle extract by A. oryzae for 4 days resulted in a 3-fold increase in -aminobutyric acid (GABA) content. GABA is known to be a bioactive compound. Fungal fermentation of the extract also enhanced its antioxidant activity and increased its total content of phenolic compounds. It was assumed that these changes stemmed from the biodegradation of active compounds of the sea tangle packaged within its rigid structural matrix or occurred as result of fungal fermentation. These results suggested that the application of microbial fermentation to the processing of seaweeds will help in the development of processed foods to meet consumer demands.

Properties of field-sprouted sorghum and its performance in ethanol production

The objective of this research was to investigate physicochemical and biochemical characteristics of field-sprouted grain sorghum and its fermentation performance in ethanol production. Five field-sprouted grain sorghum varieties, which received abnormally high rainfall during harvest, were used in this study. Enzyme activities, microstructure, flour pasting properties, kernel hardness, kernel weight, kernel size, flour size and particle distribution of field-sprouted grain sorghum were analyzed. The effect of germination (i.e., sprouting) on conversion of grain sorghum to ethanol was determined by using a laboratory dry-grind ethanol fermentation procedure. Sprouted sorghum had increased α-amylase activity; degraded starch granules and endosperm cell walls; decreased kernel hardness, kernel weight, kernel size, and particle size; and decreased pasting temperature and peak and final viscosities compared with non-sprouted grain sorghum. The major finding is that the time required for sprouted sorghum to complete fermentation was only about half that of non-sprouted sorghum. Also, ethanol yield from sprouted sorghum was higher (416–423 L/ton) than that from non-sprouted sorghum (409 L/ton) on a 14% moisture basis.

Optimization of cellulases production by Trichoderma citrinoviride on marc of Artemisia annua and its application for bioconversion process

The production of cellulases by Trichoderma citrinoviride fermented on marc of Artemisia annua, and bioconversion of the same marc by produced cellulase system was studied. The effects of pretreatments, substrate concentration, particle size, initial pH, temperature and concentration of the medium components on production of FPase, endoglucanase and β-glucosidase were monitored and comparatively evaluated. Among the three pretreatment processes, alkali hydrolysis with autoclaving was found to be most suitable for production of all the three enzymes. Optimum production of FPase, endoglucanase and β-glucosidase was obtained at 96 h, 96 h and 72 h of fermentation period, respectively. Substrate concentration of 1% with particle size between 200 μm and 475 μm gave the higher yields. Higher production of all the three enzymes was obtained with initial pH value of 5.5, temperature of 28 °C and 75% of mineral salt solution. Partially purified enzyme system obtained by optimized fermentation procedure, was applied for saccharification. Forty six percent of saccharification was noticed after 48 h of incubation on alkali hydrolyzed and autoclaved substrate which was 3.26 fold more than that of unpretreated substrate.

High-Gravity Brewing and Distilling—Past Experiences and Future Prospects

High-gravity brewing employs wort (15–20°P original gravity) at a higher than normal concentration, and to obtain sales-gravity beer, dilution with water (usually carbon filtered and deoxygenated) is required at a later stage in processing. Using this method, increased production demands can be met without significant expansion of brewing, fermenting, and storage facilities. Scotch whisky production, particularly grain whisky, also is increasingly employing high-gravity techniques (18–21°P wort) during a continuous fermentation procedure. The principal advantage to this procedure is the more efficient use of existing processes. The disadvantages of high-gravity brewing include decreased beer foam stability, a variety of stress effects on yeast, and problems with beer flavor matching compared with sales-gravity brewed beers. The stress effects of high-gravity wort also include enlargement of the yeast vacuole and changes in the topography of the yeast cell surface. Hydrodynamic stress effects on yeast are also a problem, particularly when employing centrifugation at the end of fermentation to crop yeast for recycling into a subsequent fermentation. Finally, difficulties encountered in both brewing and distilling include the inability of yeast to completely utilize the largest fermentable wort sugar—maltotriose. This is particularly the case during grain whisky production by continuous fermentation under high-gravity wort conditions.

Secretory expression of functional barley limit dextrinase by Pichia pastoris using high cell-density fermentation

Heterologous production of large multidomain proteins from higher plants is often cumbersome. Barley limit dextrinase (LD), a 98 kDa multidomain starch and α-limit dextrin debranching enzyme, plays a major role in starch mobilization during seed germination and is possibly involved in starch biosynthesis by trimming of intermediate branched α-glucan structures. Highly active barley LD is obtained by secretory expression during high cell-density fermentation of Pichia pastoris. The LD encoding gene fragment without signal peptide was subcloned in-frame with the Saccharomyces cerevisiae α-factor secretion signal of the P. pastoris vector pPIC9K under control of the alcohol oxidase 1 promoter. Optimization of a fed-batch fermentation procedure enabled efficient production of LD in a 5-L bioreactor, which combined with affinity chromatography on β-cyclodextrin–Sepharose followed by Hiload Superdex 200 gel filtration yielded 34 mg homogenous LD (84% recovery). The identity of the recombinant LD was verified by N-terminal sequencing and by mass spectrometric peptide mapping. A molecular mass of 98 kDa was estimated by SDS–PAGE in excellent agreement with the theoretical value of 97419 Da. Kinetic constants of LD catalyzed pullulan hydrolysis were found to K m,app = 0.16 ± 0.02 mg/mL and k cat,app = 79 ± 10 s −1 by fitting the uncompetitive substrate inhibition Michaelis–Menten equation, which reflects significant substrate inhibition and/or transglycosylation. The resulting catalytic coefficient, k cat,app/ K m,app = 488 ± 23 mL/(mg s) is 3.5-fold higher than for barley malt LD. Surface plasmon resonance analysis showed α-, β-, and γ-cyclodextrin binding to LD with K d of 27.2, 0.70, and 34.7 μM, respectively.

Immobilized Enzymes Availability for Glycerol – 1,3 Propanediol Bioconversion

The increasing abundance of glycerol, its renewability, and attractive pricing make it an appealing platform chemical. Glycerol can be utilized in various ways of biological transformation to industrially valuable products. There are some important derivatives among these such as 3-hydroxypropionaldehyde ((3-HPA) commercial name: reuterin), dihydroxyacetone (DHA), and 1,3-propanediol (PDO). PDO has the largest potential need. Several bacteria are able to ferment glycerol to PDO under anaerobic conditions. A new recombinant technology was worked out with the leadership of DuPont and Genecor in the last decade, in which a recombinant Escherichia coli converts glucose directly to PDO. Microbiological production of a molecule has always lower yield than an enzymatic method and usually produces a lot of by-products that can be avoided using enzymatic bioconversion. For years we have been working on a new enzymatic process in order to produce PDO and DHA simultaneously from glycerol. For this enzymatic way three key enzymes are needed: Glycerol-dehydratase (GDHt), 1,3-propanediol-oxydoreductase (PDOR), glycerol-dehydrogenase (GDH). These enzymes must be produced by microbial fermentation. We would like to present the results of developing the fermentation media in economic aspect. Our pleriminary results (not publicated) indicated that the 2xYT medium is perfect for our Clostridium strain. However in economic aspect the concentrations of yeast extract and bactotryptone are too high to scale-up this fermentation procedure. The first purpose of this study was to optimize the fermentation media. Optimized media concentrations are yeast extract, 6 g∙l-1 and bactotryptone, 2 g∙l-1 (instead of 10 and 16 g∙l-1 respectively). The other aim of our work was to develop immobilized enzymes suitable for glycerol to propanediol bioconversion and develop a method to measure the immobilized enzymes activity. We tried a covalent method applying chitosan matrix activated with glutaraldehyde. Chitosan – a natural polyaminosaccharide obtained from chitin – was chosen as support material to bind the three key enzymes from the crude enzyme solution. We could detect that PDOR and GDH enzyme bind to chitosan beads. Immobilized enzymes activity was higher than of the soluble enzymes.

EFFECT OF THE USE OF DIFFERENT LACTIC STARTERS ON THE MICROBIOLOGICAL AND PHYSICOCHEMICAL CHARACTERISTICS OF NATURALLY BLACK TABLE OLIVES OF GEMLIK CULTIVAR

ABSTRACT The effect of different lactic acid bacteria cultures on the physicochemical and microbiological characteristics of brined black olives of Gemlik cultivar at low fermentation temperature was studied. Fermentation was carried out according to the traditional Gemlik method with modifications like low salt concentration and lactic starter addition. The brines with 7% salt concentration were inoculated with lactic acid bacteria (Lactobacillus brevis, Leuconostoc cremoris and L. paramesenteroides), which were previously isolated from olive fruits at low temperatures and a commercial strain of Lb. plantarum. Fermentation procedures were carried out at controlled temperatures (between 10–12C). Lactic acid bacteria survival was accompanied by yeast development, no Pseudomonas and Enterobacter species were detected in all treatments during fermentation. The highest total titratable acidity, lowest pH and least yeast growth were determined at the brines and fermentation products, which were inoculated with L. cremoris. PRACTICAL APPLICATIONS The use of suitable starter cultures is necessary to improve the microbiological control of the naturally black table olive process, help to standardize the fermentation, increase the lactic acid yield and accordingly provide the production of olives with high quality. The requirements mentioned for starter cultures include a rapid and predominant growth, homofermentative metabolism, tolerance to salt, acid and polyphenols, and few growth factor requirements. Especially at the regions where olives were picked later when environmental temperatures are lower, the use of a starter culture that has the ability to grow at low temperatures may be necessary. Use of such starter cultures may help to increase acidification, to control some types of spoilage and to shorten the fermentation process.

Expression and purification of soluble HIV-1 envelope glycoprotein gp160 mutant from Saccharomyces cerevisiae

Here we report the expression of HIV-1 gp160 and its mutated proteins in Saccharomyces cerevisiae. Two strong hydrophobic regions, aa 511-537 and aa 679-703, were predicted by GCG Wisconsin Package software and removed to investigate the solubility of the mutated gp160 (gp160Delta12). The results showed that gp160Delta12 assumes high solubility as to be present in supernatant of cell lysate exclusively. The mutant exists as trimeric form in solutions via some inter-molecule disulfide bonds, which can be associated to monomer with the reduced reaction of DTT. The fermentation procedure was optimized to get high cell density yield and expression level as approximately 10 mg/L. After purification with electro elution, gp160Delta12 was checked as glycosylation form by Endo-H deglycosylating catalysis. The ELISA performed with a panel of human sera suggests that the purified gp160Delta12 shares some determinants with gp120 and gp41, but exposes some distinct epitopes that react with early HIV-infected antibody. Thus, we may provide a novel antigen for immunodetection assay, vaccine candidate, and other relative research purposes.

Production of 6-pentyl-α-pyrone with Trichoderma atroviride and its mutant in a novel extractive liquid-surface immobilization (Ext-LSI) system

A novel extractive fermentation procedure, tentatively named an extractive liquid-surface immobilization (Ext-LSI) system, for the production of water-insoluble secondary metabolites with fungi was developed. The system using a unique polymeric micro-material, a ballooned polyacrylonitrile microshpere (MS), was applied to the production of 6-pentyl-α-pyrone (6PP), a fungicidal secondary metabolite, with a newly isolated Trichoderma atroviride AG2755-5 and its nitrosoguanidine (NTG)-mutant. The resulted mutant AG2755-5NM398 was immobilized on the surface of a liquid medium by using the MS. Following the formation of a fungus-MS mat, low toxic hydrophobic solvent, dimethyl silicone oil, was added onto the fungus-MS mat. The optimum carbon and nitrogen sources were fructose and malt extract, respectively. The higher the initial medium pH, the more the 6PP-accumulation was observed. The best MS and extractive organic solvent were MFL-80SDE (non-coated type) and KF-96L-1CS (dimethyl silicone oil). Thus, produced 6PP was spontaneously extracted from cells to the organic phase to reach 7.1 g L −1 of the accumulation in the organic phase.