Fermentation Process For Production Research Articles

Rewiring carbon catabolite repression for microbial cell factory

Carbon catabolite repression (CCR) is a key regulatory system found in most microorganisms that ensures preferential utilization of energy-efficient carbon sources. CCR helps microorganisms obtain a proper balance between their metabolic capacity and the maximum sugar uptake capability. It also constrains the deregulated utilization of a preferred cognate substrate, enabling microorganisms to survive and dominate in natural environments. On the other side of the same coin lies the tenacious bottleneck in microbial production of bioproducts that employs a combination of carbon sources in varied proportion, such as lignocellulose-derived sugar mixtures. Preferential sugar uptake combined with the transcriptional and/or enzymatic exclusion of less preferred sugars turns out one of the major barriers in increasing the yield and productivity of fermentation process. Accumulation of the unused substrate also complicates the downstream processes used to extract the desired product. To overcome this difficulty and to develop tailor-made strains for specific metabolic engineering goals, quantitative and systemic understanding of the molecular interaction map behind CCR is a prerequisite. Here we comparatively review the universal and strain-specific features of CCR circuitry and discuss the recent efforts in developing synthetic cell factories devoid of CCR particularly for lignocellulose- based biorefinery.

Fermentative production of isobutene.

Isobutene (2-methylpropene) is one of those chemicals for which bio-based production might replace the petrochemical production in the future. Currently, more than 10 million metric tons of isobutene are produced on a yearly basis. Even though bio-based production might also be achieved through chemocatalytic or thermochemical methods, this review focuses on fermentative routes from sugars. Although biological isobutene formation is known since the 1970s, extensive metabolic engineering is required to achieve economically viable yields and productivities. Two recent metabolic engineering developments may enable anaerobic production close to the theoretical stoichiometry of 1isobutene + 2CO2 + 2H2O per mol of glucose. One relies on the conversion of 3-hydroxyisovalerate to isobutene as a side activity of mevalonate diphosphate decarboxylase and the other on isobutanol dehydration as a side activity of engineered oleate hydratase. The latter resembles the fermentative production of isobutanol followed by isobutanol recovery and chemocatalytic dehydration. The advantage of a completely biological route is that not isobutanol, but instead gaseous isobutene is recovered from the fermenter together with CO2. The low aqueous solubility of isobutene might also minimize product toxicity to the microorganisms. Although developments are at their infancy, the potential of a large scale fermentative isobutene production process is assessed. The production costs estimate is 0.9 € kg−1, which is reasonably competitive. About 70% of the production costs will be due to the costs of lignocellulose hydrolysate, which seems to be a preferred feedstock.

Membrane Processes in Biorefineries: From Feedstock Preparation to Downstream Processing

1. IntroductionThe third and latest wave of biotechnology is the so called „white biotechnology“ aiming to replace the C2/C3 chemistry based on oil and gas by biological processes. The backbone of biotechnology is the conversion by fermentation which is widely established in the production of antibiotics, enzymes, bioethanol and organic acids. In the past, fermentation processes had to partly compete with chemical conversion but the foreseeable shortage of raw materials for chemical conversion has revived the interest in fermentation in recent years. Since the 1970’s, cross-flow membrane processes have established themselves in the downstream processing of fermentation products for recovery and purification. It is expected that this role will extend into the new concept of biorefineries. The first part of this presentation will provide an overview of the existing potential and future opportunities of membrane processes within the concept of biorefineries in general and the second part provides an application study for a wheat-based biorefinery in details. 2. Membrane processes in biorefineriesThe aim of biorefineries is the full utilisation of feedstock to simultaneously produce e.g. food, biofuels and biochemicals. The integrated production of biomaterials can be based on e.g. sugar, starch and cellulose-based feedstock and as such extend current sugar, starch and pulp factories. Membrane processes have been identified as a key separation technology due to their high selectivity and low energy consumption. The initial step in the concept of biorefineries is conversion of the feedstock, e.g. cellulosic or starch-based materials, into sugars. The sugar stream might be polished by micro-filtration (MF) or ultrafiltration (UF) and concentrated by reverse osmosis (RO) before fermentation. During the fermentation step, the fermentation products can be continuously removed by using either MF/UF or pervaporation (PV) in a side stream or submerged in the fermenter to avoid product inhibitions. In the downstream process after the fermenter, membrane processes can be used for the polishing and concentration of the fermentation products, e.g. biofuels/biochemicals. 3. Application study: Membrane processes in a wheat-based biorefinery The presentation will provide an overview of membrane applications in a wheat-biorefinery focused on four applications with reference to the four key production steps: starch extraction, starch conversion to sweetener, fermentation and downstream processing. The first step in wheat-based biorefineries is extraction of the starch from the wheat. For this the wheat flour is mixed with water and then separated by a 3-phase decanter resulting in an A-starch fraction, a gluten and B-starch fraction, and a fraction consisting of solubles and pentosanes. In order to optimise the water consumption it is possible to apply UF for concentrating the solubles and pentosanes and recovering water for recycling in the process. The overall water balance for the starch extraction can be improved by using this concept. An important step in the subsequent conversion of starch to sweeteners is the removal of the mud fraction after liquification and saccarfication. A combination of UF with a decanter can be used as an alternative to rotary vacuum filters achieving higher purities and a mud fraction which is not contaminated with filter aid kieselguhr and can therefore be added directly in the bioethanol production. Hence, the closed process avoids potentially hazardous filter aids and results in a value-added by-product. Continuous fermentation is an important feature in the concept of biorefineries. MF/UF, either submerged or as side-stream, can be applied to recover the active principles directly from the fermentation broth. In this way, the production can be moved from batch to continuous thus preventing product inhibitions. This concept is valid for bioethanol as such but also other biochemicals such as organic acids or bioplastics. Evaporation is often used in the downstream processing as a stand-alone unit or in combination with RO for concentration of the fermentation products. The resulting evaporator condensate from the concentration can be polished by RO resulting in a permeate stream which can be recycled in the process thus improving the water balance of the process. Pilot and/or production results will be presented for all four focus applications. 4. Conclusions and outlookThis paper demonstrates the great potential of membrane processes in the concept of biorefineries and thus in solving the energy and environmental problems of the future. (Less)

Effects of pretreatment of natural bacterial source and raw material on fermentative biohydrogen production

Effects of pretreatment of natural bacterial source and raw material on biohydrogen production in fermentative biohydrogen production process were investigated systematically. Biohydrogen production from stale corn slurry was found to be feasible and effective by A1 (water soak) pretreated compost and B1 (gelatinization) pretreated stale corn. The results were further confirmed by the verification test with working volume of 8 L, in which the maximum hydrogen yield of 262 mL H 2/g-substrate, hydrogen production rate of 39 mL/g h −1 and the corresponding hydrogen content of 50% was observed at fixed substrate concentration of 10 g/L, working pH 5.0–5.5 and 36 ± 1 °C. The effluent was mostly composed of acetate and butyrate. Subsequently, two new hydrogen producing strains were isolated from the effluent sludge in the running bioreactor, and they were preliminarily identified as Clostridium and Enterobacter, respectively, according to the routine screening examinations.

Design of Simulation System for Batch Process

Process monitoring and fault diagnosis of batch process is a research focus in the industrial control field. In this paper, penicillin fermentation is taken as the research background, a visual batch process simulation system is designed based on mathematical models of an actual production process. By introducing different fault signals to the penicillin fermentation simulation process, the designed system can be used to simulate the real penicillin fermentation production process clearly. In the end, an ideal experimental simulation data for batch process fault diagnosis is provided.

Establishment of an alternative phosphoketolase-dependent pathway for fructose catabolism in Ralstonia eutropha H16

The β-proteobacterium Ralstonia eutropha H16 utilizes fructose and gluconate as carbon sources for heterotrophic growth exclusively via the Entner-Doudoroff pathway with its key enzyme 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase. By deletion of the responsible gene eda, we constructed a KDPG aldolase-negative strain, which is disabled to supply pyruvate for energy metabolism from fructose or gluconate as sole carbon sources. To restore growth on fructose, an alternative pathway, similar to the fructose-6-phosphate shunt of heterofermentative bifidobacteria, was established. For this, the xfp gene from Bifidobacterium animalis, coding for a bifunctional xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp; Meile et al. in J Bacteriol 183:2929-2936, 2001), was expressed in R. eutropha H16 PHB(-)4 Δeda. This Xfp catalyzes the phosphorolytic cleavage of fructose 6-phosphate to erythrose 4-phosphate and acetylphosphate as well as of xylulose 5-phosphate to glyceralaldehyde 3-phosphate and acetylphosphate. The recombinant strain showed phosphoketolase (PKT) activity on either substrate, and was able to use fructose as sole carbon source for growth, because PKT is the only enzyme that is missing in R. eutropha H16 to establish the artificial fructose-6-phosphate shunt. The Xfp-expressing strain R. eutropha H16 PHB(-)4 Δeda (pBBR1MCS-3::xfp) should be applicable for a novel variant of a plasmid addiction system to stably maintain episomally encoded genetic information during fermentative production processes. Plasmid addiction systems are often used to ensure plasmid stability in many biotechnology relevant microorganisms and processes without the need to apply external selection pressure, like the addition of antibiotics. By episomal expression of xfp in a R. eutropha H16 mutant lacking KDPG aldolase activity and cultivation in mineral salt medium with fructose as sole carbon source, the growth of this bacterium was addicted to the constructed xfp harboring plasmid. This novel selection principle extends the applicability of R. eutropha H16 as production platform in biotechnological processes.

Prospects for microbial biodiesel production

As the demand for biofuels for transportation is increasing, it is necessary to develop technologies that will allow for low-cost production of biodiesel. Conventional biodiesel is mainly produced from vegetable oil by chemical transesterification. This production, however, has relatively low land-yield and is competing for agricultural land that can be used for food production. Therefore, there is an increasing interest in developing microbial fermentation processes for production of biodiesel as this will allow for the use of a wide range of raw-materials, including sugar cane, corn, and biomass. Production of biodiesel by microbial fermentation can be divided into two different approaches, (1) indirect biodiesel production from oleaginous microbes by in vitro transesterification, and (2) direct biodiesel production from redesigned cell factories. This work reviews both microbial approaches for renewable biodiesel production and evaluates the existing challenges in these two strategies.

Efficient and direct glutathione production from raw starch using engineered Saccharomyces cerevisiae

Glutathione is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae. We demonstrated that expression of amylase genes in glutathione-producing S. cerevisiae enables direct use of starch as a carbon source, thus eliminating the Crabtree effect that is caused by excess glucose. Consequently, cell growth and glutathione productivity were significantly improved. This approach is potentially applicable to a variety of fermentative processes for production of value-added chemicals under aerobic conditions.

Effect of process integration on the exergy balance of a two-stage process for fermentative hydrogen production

In this paper, a process for the fermentative production of hydrogen is analysed with respect to its exergy efficiency. Parametric studies show the influence of used feedstock, applied process parameters as well as process and heat integration measures on exergy efficiency. It is shown that heat integration and recirculation of fermentor effluents reduce process irreversibilites and the amount of exergy leaving the process in waste streams. Nevertheless, depending on the used feedstock, a large amount of exergy leaves the process via by-products. Internal use of by-products and waste materials for providing process heat could increase chemical exergy efficiency up to 74%.

Development of a solid-state fermentation process for production of an alpha amylase with potentially interesting properties

Ca-independency with potential activity and stability at low pH are among the most interesting characteristics of alpha-amylase in starch industry. In this attempt the synergetic effect of low pH on activity of crude Ca-independent alpha-amylase isolated from a native Bacillus sp. KR-8104 in solid-state fermentation (SSF) was studied using wheat bran (WB) as a substrate. The effects of different parameters including moisturizing agents, solid substrate to moisture ratio, particle size, incubation temperature and period, inoculum (v/w) and supplementation with 1% (w/w) different carbon and nitrogen sources on enzyme production were investigated. Maximum enzyme production of 140U/g dry fermented substrate was obtained from wheat bran moistened with tap water at a ratio of 1:1.5 and supplemented with 1% (w/w) NH(4)NO(3) and 1% (w/w) lactose after 48h incubation at 37 degrees C. Even though the production of alpha-amylase was lower at 40 and 45 degrees C, the viable cell count was higher. In addition response surface methodology (RSM) was applied to find optimum conditions of temperature and pH on crude amylase activity. Using central composite design (CCD) a quadratic mathematical model equation was derived for the prediction of enzyme activity. The results showed that the model was in good agreement with experimental results, with R(2)=0.90 (p<0.0001) and the low pH has a synergetic effect on enzyme activity at higher temperature.

Continuous 2-keto-l-gulonic acid fermentation from l-sorbose by Ketogulonigenium vulgare DSM 4025

A single-stage continuous fermentation process for the production of 2-keto-L-gulonic acid (2KGA) from L-sorbose using Ketogulonigenium vulgare DSM 4025 was developed. The chemostat culture with the dilution rate that was calculated based on the relationship between the 2KGA production rate and the 2KGA concentration was feasible for production with high concentration of 2KGA. In this system, 112.2 g/L of 2KGA on the average was continuously produced from 114 g/L of L-sorbose. A steady state of the fermentation was maintained for the duration of more than 110 h. The dilution rate was kept in the range of 0.035 and 0.043 h(-1), and the 2KGA productivity was 3.90 to 4.80 g/L/h. The average molar conversion yield of 2KGA from L-sorbose was 91.3%. Under the optimal conditions, L-sorbose concentration was kept at 0 g/L. Meanwhile, the dissolved oxygen level was changing in response to the dilution rate and 2KGA concentration. In the dissolved oxygen (DO) range of 16% to 58%, it was revealed that the relationship between DO and D possessed high degree of positive correlation under the L-sorbose limiting condition (complete consumption of L-sorbose). Increasing D closer to the critical value for washing out point of the continuous fermentation, DO value tended to be gradually increased up to 58%. In conclusion, an efficient and reproducible continuous fermentation process for 2KGA production by K. vulgare DSM 4025 could be developed using a medium containing baker's yeast without using a second helper microorganism.

Development of a fermentation process for production of calcium- l-lactate

This study is an effort to develop an efficient process for low-cost production of calcium- l-lactate (CaL 2). CaL 2 showed higher inhibitory effect on lactate production than ammonium- l-lactate (NH 4) at lactate concentration lower than 100 g/L, but it showed lower inhibitory effect at higher lactate concentration. The strain Lactobacillus rhamnosus (NBRC 3863) was resistant to glucose inhibition and final CaL 2 concentration increased with initial glucose concentration increasing up to 190 g/L. High-concentration CaL 2 induced, however, flocculation of fermentation broth, causing incomplete fermentation. It was confirmed that stirring speed markedly influenced flocculation, an increase of the stirring speed leading to an earlier flocculation. Formation of colloidal CaL 2 during fermentation was considered to be the cause of flocculation in this study. Addition of YE raised fermentation efficiency and made complete fermentation feasible. In the fermentation with 25 g/L YE, glucose was completely consumed and final CaL 2 concentration of 220 g/L was obtained, when flocculation did not yet occur. In this study, CaL 2 crystals and activated carbon powder were added as nucleation sites to get earlier crystallization of colloids and to decrease the inhibitory effect of CaL 2, and so to increase productivity. Of the two nucleation sites, activated carbon powder showed higher performance in CaL 2 production. Finally, the feasibility of crystallization-based recovery without concentration step was discussed.

Fumaric acid production by fermentation

The potential of fumaric acid as a raw material in the polymer industry and the increment of cost of petroleum-based fumaric acid raises interest in fermentation processes for production of this compound from renewable resources. Although the chemical process yields 112% w/w fumaric acid from maleic anhydride and the fermentation process yields only 85% w/w from glucose, the latter raw material is three times cheaper. Besides, the fermentation fixes CO2. Production of fumaric acid by Rhizopus species and the involved metabolic pathways are reviewed. Submerged fermentation systems coupled with product recovery techniques seem to have achieved economically attractive yields and productivities. Future prospects for improvement of fumaric acid production include metabolic engineering approaches to achieve low pH fermentations.

Monoterpenoid biosynthesis inSaccharomyces cerevisiae

Plant monoterpenoids belong to a large family of plant secondary metabolites with valuable applications in cosmetics and medicine. Their usual low levels and difficult purification justify the need for alternative fermentative processes for large-scale production. Geranyl diphosphate is the universal precursor of monoterpenoids. In yeast it occurs exclusively as an intermediate of farnesyl diphosphate synthesis. In the present study we investigated the potential use of Saccharomyces cerevisiae as an alternative engineering tool. The expression of geraniol synthase of Ocimum basilicum in yeast allowed a strong and specific excretion of geraniol to the growth medium, in contrast to mutants defective in farnesyl diphosphate synthase which excreted geraniol and linalool in similar amounts. A further increase of geraniol synthesis was obtained using yeast mutants defective in farnesyl diphosphate synthase. We also showed that geraniol synthase expression affects the general ergosterol pathway, but in a manner dependent on the genetic background of the strain.

Amylase production by Penicillium fellutanumisolated from mangrove rhizosphere soil

The effects of pH, temperature, incubation time, salinity, sources of carbon and nitrogen were tested in submerged fermentation process in production of α-amylase by Penicillium fellutanum isolated from coastal mangrove soil. The production medium without addition of seawater and with provision of maltose as carbon source, peptone as nitrogen source, incubated for 96 h, maintained with pH of 6.5 at 30oC, was found optimal for production of α-amylase by P. fellutanum. Key words: α-amylase, Penicillium fellutanum, mangroves, rhizosphere soil, Rhizophora.

Amylase production by Penicillium fellutanum isolated from mangrove rhizosphere soil

The effects of pH, temperature, incubation time, salinity, sources of carbon and nitrogen were tested in submerged fermentation process in production of �-amylase by Penicillium fellutanum isolated from coastal mangrove soil. The production medium without addition of seawater and with provision of maltose as carbon source, peptone as nitrogen source, incubated for 96 h, maintained with pH of 6.5 at 30 o C, was found optimal for production of �-amylase by P. fellutanum.

Bioseparations.

Here we review key applications of separation technology in applied biology. We first sketch out the field as a whole, but then narrow our scope to the processing of fermentation products, particularly to high-value biologicals such as proteins and nucleotides. We go on to provide a qualitative overview describing the importance and general nature of this large field, major trends, and the strategies that have proven most fruitful in evolving effective separation and purification processes. We then give a detailed description of individual separations equipment and the principles governing their operation. We concentrate throughout on making the available literature accessible to the reader; we provide what is hoped to be a representative set of basic references. However, these references, in turn, include some that suggest promising new developments as well as a number of more specialized reviews. We hope that our overall result provides the reader with access to the most relevant literature.

Negative H2S character and flocculation as yeast strain markers for inoculum recovery

Routine identification of yeast behavior is essential to measure the control of the alcohol production process and to maintain product quality standards. This work utilized the non-hydrogen sulfide production and flocculation traits as characteristic strain markers for the evaluation of cell recycling during the alcoholic fermentation process for production of sugarcane alcohol. This study evaluated the behavior of a recombinant yeast bank made by protoplast fusion, for strain screening purposes; strain fermentative kinetics in comparison to commercial baker yeast; viability and recovery of the selected strain on differential media, after five consecutive fermentation batches; and the recovery of the selected strain from fermentation with mixed strain cultures. The strain selected for the H2S negative character kept its viability during successive recyclings, with contamination levels not detected by the method of analysis. It also presented a kinetic behavior similar to that of baker yeast, either in single or mixed culture fermentations, opening new possibilities for further work on quality control of cell recycling in the alcoholic fermentation process.

Use of catabolite repression mutants for fermentation of sugar mixtures to ethanol

Use of agricultural biomass, other than corn-starch, to produce fuel ethanol requires a microorganism that can ferment the mixture of sugars derived from hemicellulose. Escherichia coli metabolizes a wide range of substrates and has been engineered to produce ethanol in high yield from sugar mixtures. E. coli metabolizes glucose in preference to other sugars and, as a result, utilization of the pentoses in hemicellulose-derived sugar mixtures is delayed and may be incomplete. Residual sugar lowers the ethanol yield and is problematic for downstream processing of fermentation products. Therefore, a catabolite repression mutant that simultaneously utilizes glucose and pentoses would be useful for fermentation of complex substrate mixtures. We constructed ethanologenic E. coli strains with a glucose phosphotransferase (ptsG) mutation and used the mutants to ferment glucose, arabinose, and xylose, singly and in mixtures, to ethanol. Yields were 87-94% of theoretical for both the wild type and mutants, but the mutants had an altered pattern of mixed sugar utilization. Phosphotransferase mutants metabolized the pentoses simultaneously with glucose, rather than sequentially. Based upon fermentations of sugar mixtures, a catabolite-repression mutant of ethanologenic E. coli is expected to provide more efficient fermentation of hemicellulose hydrolysates by allowing direct utilization of pentoses.

Role of Data Mining in Drug Development

Increasing attention is being paid to reducing the time taken from discovery to defining a final fermentation production process. Multivariate statistical data analysis and chemometric techniques can play an important role in contributing to the achievement of this aim by providing biologists and fermentation scientists with sophisticated data visualisation and data exploration tools. This paper presents a study of the application of a range of feature extraction techniques to data from shake flask experiments. investigatingrecombinant protein expression. carried out during the discovery phase of drug development.