Enhanced production of L-(+)-lactic acid in chemostat by Lactobacillus casei DSM 20011 using ion-exchange resins and cross-flow filtration in a fully automated pilot plant controlled via NIR

Due to the lack of suitable in-process sensors, on-line monitoring of fermentation processes is restricted almost exclusively to the measurement of physical parameters only indirectly related to key process variables, i.e., substrate, product, and biomass concentration. This obstacle can be overcome by near infrared (NIR) spectroscopy, which allows not only real-time process monitoring, but also automated process control, provided that NIR-generated information is fed to a suitable computerized bioreactor control system. Once the relevant calibrations have been obtained, substrate, biomass and product concentration can be evaluated on-line and used by the bioreactor control system to manage the fermentation. In this work, an NIR-based control system allowed the full automation of a small-scale pilot plant for lactic acid production and provided an excellent tool for process optimization. The growth-inhibiting effect of lactic acid present in the culture broth is enhanced when the growth-limiting substrate, glucose, is also present at relatively high concentrations. Both combined factors can result in a severe reduction of the performance of the lactate production process. A dedicated software enabling on-line NIR data acquisition and reduction, and automated process management through feed addition, culture removal and/or product recovery by microfiltration was developed in order to allow the implementation of continuous fermentation processes with recycling of culture medium and cell recycling. Both operation modes were tested at different dilution rates and the respective cultivation parameters observed were compared with those obtained in a conventional continuous fermentation. Steady states were obtained in both modes with high performance on lactate production. The highest lactate volumetric productivity, 138 g L(-1) h(-1), was obtained in continuous fermentation with cell recycling.     

Fermentation Kinetics and Continuous Process of L-Asparaginase Production

For the purpose of obtaining L-asparaginase in quantities from Erwinia aroideae, cell growth and enzyme formation were investigated in both batch and continuous fermentation. Using yeast extract as a growth-limiting substrate, the relationship between specific growth rate and substrate concentration was found to fit the Monod equation. The optimum temperature for enzyme production was 24 C, although cell growth was higher at 28 C. The enzyme yield reached its maximum of 4 IU/ml during the negative acceleration growth phase which occurs just prior to stationary growth. Compared to batch fermentations, the continuous fermentation process gave a lower enzyme yield except when the fermentation was conducted at a dilution rate of 0.1 hr(-1). The graphical method frequently used for prediction of continuous fermentation does not apply to L-asparaginase production by E. aroideae. The optimum temperature for enzyme production in continuous process was 24 C, which was the same as in batch process. Increasing the temperature from 24 to 28 C resulted in a 20% loss of enzyme yield.     

Current industrial practice in solid state fermentations for secondary metabolite production: the Biocon India experience

Solid substrate fermentation at Biocon was originally envisaged for the production of enzymes, used in the food processing industry. The original process developed at Biocon was a hygienically designed automated tray culture process. Plants using this process still continue to run effectively at Biocon, and produce a variety of products meeting and exceeding FCC/JECFA specifications for food products. Biocon recently designed, developed and patented a new bioreactor, the PlaFractor™ (pronounced play-fractor) for carrying out fermentations that use solid matrices—a term covering both nutritive support matrices as well as non-nutritive matrices impregnated with medium.Using the PlaFractor™ process it is now possible to extend the use of solid matrix fermentation for the production of enzymes, biocontrol agents and pharmaceutical products, that require elaborate containment—under precisely defined conditions. The production takes place in computer controlled bioreactors, using complex fermentation control algorithms. All the operations of solid matrix fermentation, i.e. sterilization, cooling, inoculation, fermentation and process control, product recovery and post-fermentation sterilization, are all done in one single equipment, which was not hitherto possible. All the advantages of traditional solid state fermentation, over submerged fermentation, like low energy consumption, low water requirement, high mass transfer coefficient, no foaming, and high product concentrations are retained. In addition, techniques that are important to submerged fermentation, like fed-batch fermentation, process parameter profiling, air and media sterilization, operation under aseptic environments, and ease of handling, can now be easily applied to solid state fermentation, because of the way this bioreactor is designed.A production plant, built around this bioreactor has already been operating for more than a year.     

Oxygen enriched air supply in Escherichia coli processes: production of biomass and recombinant human growth hormone

In order to investigate the impact of high oxygen and carbon dioxide concentrations, Escherichia coli was grown in batch cultivations where the air supply was enriched with either oxygen or carbon dioxide. The effect of elevated concentrations of oxygen and carbon dioxide on stochiometric and kinetic constants was studied this way. The maximum growth rate was significantly reduced, the production of acetic acid and the biomass yield coefficient on glucose increased in cultures with carbon dioxide enriched air, compared to reference cultivations and cultivations with oxygen enriched air. The application of oxygen enriched air was studied in high cell density cultivations of Escherichia coli. Two production processes were chosen to investigate the impact of oxygen enrichment. Biomass concentration, specific growth rate, yield coefficient, respiration, mixed acid fermentation products and the product yield and quality for the recombinant product were investigated. First, a process for the production of biomass was investigated. Exponential growth could proceed for a longer time and higher growth rates could be maintained with oxygen enriched air supply. However, a higher specific oxygen consumption rate per glucose was measured after the start of the oxygen enrichment, indicating higher maintenance and consequently the growth rate and yield coefficient decreased drastically in the end of the process. Second, a process for the production of recombinant human growth hormone (rhGH) was investigated. Although the glucose feed rate and all medium components were doubled, the amount of produced biomass could only be increased by 77% when oxygen enriched air (40% oxygen) supply was applied. This was due to a decreased yield coefficient of biomass per glucose. The total amount of produced product was decreased by almost 50% compared to the control, although less proteolytically degraded variants were produced.     

Enhanced lignin peroxidase synthesis by Phanerochaete Chrysosporium in solid state bioprocessing of a lignocellulosic substrate

Summary A solid state fermentation (SSF) process for the production of lignin peroxidase was optimized to enhance enzyme production by Phanerochaete chrysosporium. Optimization of the corncob SSF medium caused a significant reduction in fermentation time to give maximum lignin peroxidase yield. Supplementation of the SSF medium by low concentrations of peptone, yeast extract and Tween-80 enhanced lignin peroxidase production. Maximum yield of lignin peroxidase was 13.7 U/gds (units per gram dry substrate) noted after 5 days of SSF with 70% moisture and 20% (v/w) inoculum.     

Indirect method for quantification of cellular biomass in a solidscontaining medium used as pre-culture for cellulase production

A process that combines the advantages of solid state fermentation (SSF) and submerged fermentation (SmF) could increase the efficiency of cellulase production required in the cellulosic ethanol industry. Due to the difficulty of measuring cellular biomass in the presence of solids, we developed a novel methodology for indirect quantification of biomass during production of the preculture for a combined fermentation process. Cultivation of Aspergillus niger was initiated as SSF using sugar cane bagasse as a solid substrate. Experiments were conducted in the absence of bagasse to determine growth kinetic parameters. Changes in glucose and biomass concentrations were measured. and the data were used for simulation employing a simple unstructured model. Parameters were estimated by applying a combination of Simulated Annealing (SA) and Levenberg-Marquardt (LM) algorithms to search for minimization of the error between model estimates and experimental data. Growth kinetics followed the Contois model, with a maximum specific growth rate (μ_max) of 0.042/h, a yield coefficient for biomass formation (Y_x/s) of 0.30 g/g and a death constant (k_D) of 0.005/h.These parameters were used to simulate cellular growth in the solids-containing medium. The proposed model accurately described the experimental data and succeeded in simulating the cell concentration profile. The selected pre-culture conditions (24 h as SSF followed by 48 h as SmF) were applied for cellulase production using the combined fermentation process and resulted in an endoglucanase activity (1,052 ± 34 U/L) greater than that obtained using the conventional SmF procedure (824 ± 44 U/L). Besides the standardization of pre-culture conditions, this methodology could be very useful in systems where direct measurement of cell mass is not possible.     

Application of dynamic calorimetry for monitoring fermentation processes

The rate of heat evolution (kcal/liter-hr) in mycelial fermentations for novobiocin and cellulase production with media containing noncellular solids was measured by an in situ dynamic calorimetric procedure. Thermal data so obtained have proved significant both in monitoring cell concentration during the trophophase (growth phase) and in serving as a physiological variable in the fermentation process. The validity of this technique has been demonstrated by closing the overall material and energy balances. The maintenance energy in a batch fermentation can also be calculated by integrating heat evolution data. This integration method is applicable to a fermentation lacking a precise cell growth curve. The maintenance coefficient, obtained for the novobiocin fermentation by Streptomyces niveus, is equal to 0.028 g glucose equivalent/g cell-hr. The production of novobiocin in the idio-phase (production phase) also correlates well with the amount of energy catabolixed for maintenance and this results in an observed conversion yield of glucose to novobiocin of 11.8 mg of novobiocin produced per gram of glucose catabolized. A new physiological variable, kilocalories of heat evolved per millimole of oxygen consumed, has been proposed to monitor the state of cells during the fermentation. This method may provide a simple way to monitor on-line shifts in the efficiency of cell respiration and changes in growth yields during a microbial process.     

Dynamic optimization of hybridoma growth in a fed-batch bioreactor

This study addressed the problem of maximizing cell mass and monoclonal antibody production from a fed-batch hybridoma cell culture. We hypothesized that inaccuracies in the process model limited the mathematical optimization. On the basis of shaker flask data, we established a simple phenomenological model with cell mass and lactate production as the controlled variables. We then formulated an optimal control algorithm, which calculated the process-model mismatch at each sampling time, updated the model parameters, and re-optimized the substrate concentrations dynamically throughout the time course of the batch. Manipulated variables were feed rates of glucose and glutamine. Dynamic parameter adjustment was done using a fuzzy logic technique, while a heuristic random optimizer (HRO) optimized the feed rates. The parameters selected for updating were specific growth rate and the yield coefficient of lactate from glucose. These were chosen by a sensitivity analysis. The cell mass produced using dynamic optimization was compared to the cell mass produced for an unoptimized case, and for a one-time optimization at the beginning of the batch. Substantial improvements in reactor productivity resulted from dynamic re-optimization and parameter adjustment. We demonstrated first that a single offline optimization of substrate concentration at the start of the batch significantly increased the yield of cell mass by 27% over an unoptimized fermentation. Periodic optimization online increased yield of cell mass per batch by 44% over the single offline optimization. Concomitantly, the yield of monoclonal antibody increased by 31% over the off-line optimization case. For batch and fed-batch processes, this appears to be a suitable arrangement to account for inaccuracies in process models. This suggests that implementation of advanced yet inexpensive techniques can improve performance of fed-batch reactors employed in hybridoma cell culture.     

Towards the design of an optimal strategy for the production of ergosterol from Saccharomyces cerevisiae yeasts

The total yield of ergosterol produced by the fermentation of the yeast Saccharomyces cerevisiae depends on the final amount of yeast biomass and the ergosterol content in the cells. At the same time ergosterol purity—defined as percentage of ergosterol in the total sterols in the yeast—is equally important for efficient downstream processing. This study investigated the development of both the ergosterol content and ergosterol purity in different physiological (metabolic) states of the microorganism S. cerevisiae with the aim of reaching maximal ergosterol productivity. To expose the yeast culture to different physiological states during fermentation an on‐line inference of the current physiological state of the culture was used. The results achieved made it possible to design a new production strategy, which consists of two preferable metabolic states, oxidative‐fermentative growth on glucose followed by oxidative growth on glucose and ethanol simultaneously. Experimental application of this strategy achieved a value of the total efficiency of ergosterol production (defined as product of ergosterol yield coefficient and volumetric productivity), 103.84 × 10^?6 g L^?1h^?1, more than three times higher than with standard baker's yeast fed‐batch cultivations, which attained in average 32.14 × 10^?6 g L^?1h^?1. At the same time the final content of ergosterol in dry biomass was 2.43%, with a purity 86%. These results make the product obtained by the proposed control strategy suitable for effective down‐stream processing. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:838–848, 2017     

温度对丙酮酸生物合成动力学、能荷和氧化-还原度的影响

在光滑球拟酵母(Torulopsis glabrata620)生产丙酮酸的过程中,温度对丙酮酸生物合成有着重要的影响。考察了不同发酵温度下基质消耗、细胞生长、丙酮酸合成及能荷水平和氧化-还原度等方面的差异。在恒温发酵中,维持较高的发酵温度可以增强糖耗,促进菌体生长,加速丙酮酸积累,但前期胞内能荷水平较高,菌体消耗较多葡萄糖合成菌体,后续产酸能力不足,导致丙酮酸得率降低;维持较低的发酵温度可以在发酵后期提供稳定的产酸能力,但菌体代谢缓慢,后期胞内NADH/NAD 水平较高,丙酮酸生产强度降低。因此仅仅采取单一的温度控制策略很难达到丙酮酸高产量、高产率和高生产强度的统一。     

Effect of air supplement on the performance of continuous ethanol fermentation system

For the purpose of improving ethanol productivity, the effect of air supplement on the performance of continuous ethanol fermentation system was studied. The effect of oxygen supplement on yeast concentration, cell yield, cell viability, extracellular ethanol concentration, ethanol yield, maintenance coefficient, specific rates of glucose assimilation, ethanol production, and ethanol productivity have been evaluated, using a high alcohol tolerant Saccharomyces cerevisiae STV89 strain and employing a continuous fermentor equipped with an accurate air metering system in the flow rate range 0-11 mL air/L/h. It was found that, when a small amount of oxygen up to about 80mu mol oxygen/L/h was supplied, the ethanol productivity was significantly enhanced as compared to the productivity of the culture without any air supplement. It was also found that the oxygen supplement improved cell viability considerably as well as the ethanol tolerance level of yeast. As the air supply rate was increased, from 0 to 11 mL air/L/h while maintaining a constant dilution rate at about 0.06 h(-1), the cell concentration increased from 2.3 to 8.2 g/L and the ethanol productivity increased from 1.7 to 4.1 g ethanol/L/h, although the specific ethanol production rate decreased slightly from 0.75 to 0.5 g ethanol/g cell/h. The ethanol yield was slightly improved also with an increase in air supply rate, from about 0.37 to 0.45 ethanol/g glucose. The maintenance coefficient increased by only a small amount with the air supplement. This kind of air supplement technique may very well prove to be of practical importance to a development of a highly productive ethanol fermentation process system especially as a combined system with a high density cell culture technique.     

Production of tannase from Aspergillus ruber under solid-state fermentation using jamun (Syzygium cumini) leaves

Tannase producing fungal strains were isolated from different locations including garbages, forests and orchards, etc. The strain giving maximum enzyme yield was identified to be Aspergillus ruber. Enzyme production was studied under solid state fermentation using different tannin rich substrates like ber leaves (Zyzyphus mauritiana), jamun leaves (Syzygium cumini), amla leaves (Phyllanthus emblica) and jawar leaves (Sorghum vulgaris). Jamun leaves were found to be the best substrate for enzyme production under solid-state fermentation (SSF). In SSF with jamun leaves, the maximum production of tannase was found to be at 30 °C after 96 h of incubation. Tap water was found to be the best moistening agent, with pH 5.5 in ratio of 1:2 (w/v) with substrate. Addition of carbon and nitrogen sources to the medium did not increase tannase production. Under optimum conditions as standardized here, the enzyme production was 69 U/g dry substrate. This is the first report on production of tannase by A. ruber, giving higher yield under SSF with agro-waste as the substrate.     

Kinetic study of the conversion of different substrates to lactic acid using Lactobacillus bulgaricus

Lactic acid fermentation includes several reactions in association with the microorganism growth. A kinetic study was performed of the conversion of multiple substrates to lactic acid using Lactobacillus bulgaricus. Batch experiments were performed to study the effect of different substrates (lactose, glucose, and galactose) on the overall bioreaction rate. During the first hours of fermentation, glucose and galactose accumulated in the medium and the rate of hydrolysis of lactose to glucose and galactose was faster than the convesion of these substrates. Once the microorganism built the necessary enzymes for the substrate conversion to lactic acid, the conversion rate was higher for glucose than for galactose. The inoculum preparation was performed in such a way that healthy young cells were obtained. By using this inoculum, shorter fermentation times with very little lag phase were observed. The consumption patterns of the different substrates converted to lactic acid were studied to determine which substrate controls the overall reaction for lactic acid production. A mathematical model (unstructured Monod type) was developed to describe microorganism growth and lactic acid production. A good fit with a simple equation was obtained. It was found experimentally that the approximate ratio of cell to substrate was 1 to 10, the growth yield coefficient (Y(XS)) was 0.10 g cell/g substrate, the product yield (Y(PS)) was 0.90 g lactic acid/g substrate, and the alpha parameter in the Luedeking-Piret equation was 9. The Monod kinetic parameters were obtained. The saturation constant (K(S)) was 3.36 g/L, and the specific growth rate (microm ) was 1.14 l/h.     

Propionic acid fermentation by Propionibacterium acidipropionici: effect of growth substrate

Summary Batch propionic acid fermentations by Propionibacterium acidipropionici with lactose, glucose, and lactate as the carbon source were studied. In addition to propionic acid, acetic acid, succinic acid and CO₂ were also formed from lactose or glucose. However, succinic acid was not produced in a significant amount when lactate was the growth substrate. Compared to fermentations with lactose or glucose at the same pH, lactate gave a higher propionic acid yield, lower cell yield, and lower specific growth rate. The specific fermentation or propionic acid production rate from lactate was, however, higher than that from lactose. Since about equimolar acid products would be formed from lactate, the reactor pH remained relatively unchanged throughout the fermentation and would be easier to control when lactate was the growth substrate. Therefore, lactate would be a preferred substrate over lactose and glucose for propionic acid production using continuous, immobilized cell bioreactors. Correspondence to: S. T. Yang     

Value-added food: single cell protein

The alarming rate of population growth has increased the demand for food production in third-world countries leading to a yawning gap in demand and supply. This has led to an increase in the number of hungry and chronically malnourished people. This situation has created a demand for the formulation of innovative and alternative proteinaceous food sources. Single cell protein (SCP) production is a major step in this direction. SCP is the protein extracted from cultivated microbial biomass. It can be used for protein supplementation of a staple diet by replacing costly conventional sources like soymeal and fishmeal to alleviate the problem of protein scarcity. Moreover, bioconversion of agricultural and industrial wastes to protein-rich food and fodder stocks has an additional benefit of making the final product cheaper. This would also offset the negative cost value of wastes used as substrate to yield SCP. Further, it would make food production less dependent upon land and relieve the pressure on agriculture. This article reviews diversified aspects of SCP as an alternative protein-supplementing source. Various potential strains and substrates that could be utilized for SCP production are described. Nutritive value and removal of nucleic acids and toxins from SCP as a protein-supplementing source are discussed. New processes need to be exploited to improve yield. In that direction the solid state fermentation (SSF) method and its advantages for SCP production are highlighted.     

Lactic acid fermentation of crude sorghum extract

Crude extract from sweet sorghum supplemented with vetch juice was utilized as the carbohydrate source for fermentative production of lactic acid. Fermentation of media containing 7%(w/v) total sugar was complex completed in 60–80 hr by Lactobacillus plantarum, product yield averaging 85%. Maximum acid production rates were dependent on pH, initial substrate distribution, and concentration, the rates varying from 2 to 5 g(liter·hr.) The lactic acid yield was lowered to 67% under limited medium supplementation. The fermented ammoniated product contained over eight times as much equivalent crude protein (N × 6.25) as the original medium. Unstructured kinetic models were developed for cell growth, lactic acid formation, and substrate consumption in batch fermentation. With the provision of experimentally determined kinetic parameters, the proposed models accurately the fermentation process.     

等离子体作用结合氧限制模型选育利福霉素SV高产菌株

利福霉素SV毒性低、疗效高、抗菌谱广,主要由地中海拟无枝酸菌发酵生产,其发酵过程属于耗氧发酵,供氧直接影响产物形成.为减少发酵过程氧限制影响,进一步提高利福霉素发酵产量,通过构建定向氧限制模型,将常温常压等离子体诱变和无水亚硫酸钠氧限制筛选模型相结合,建立了利福霉素生产菌株24孔板快速培养的高通量筛选方法,高效选育出能...     

Multiplicity of steady states in a bioreactor during the production of 1,3-propanediol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis>

In recent decades, the production of compounds from microorganisms has increased significantly. Glycerol as a source of substrate appears to have great potential, due to its large supply because of the increase in biodiesel production. This paper will discuss the multiplicity of steady states for the production of 1,3-propanediol from glycerol by Clostridium butyricum, employing a model that takes into account inhibition by fermentation products. The theoretical study of bifurcation enabled us to make a qualitative adjustment to the various experimental steady states, using the theoretical steady states obtained from the AUTO2007 program. The theoretical model parameters were varied to fit qualitatively the values of the experimental steady states. In addition, this work is a qualitative study, using experimental steady states that can be used as an initial study for more advanced work on optimizing the production of 1,3-propanediol.     

Immobilization of co-culture of Saccharomyces cerevisiae and Scheffersomyces stipitis in sodium alginate for bioethanol production using hydrolysate of apple pomace under separate hydrolysis and fermentation

Apple pomace as a substrate for bioethanol production is interesting due to its abundance and sustainable availability in varied states like Himachal Pradesh (H.P.), Jammu and Kashmir, Uttarakhand and Arunachal Pradesh, India. In the current study, apple pomace which is the main fruit industrial waste of H.P. was evaluated as feedstock for bioethanol production by the process of enzymatic saccharification using multiple carbohydrases. Microwave pretreatment of the apple pomace resulted in the efficient removal of lignin and crystalline structure of cellulose fibre. The enzymatic saccharification of the pretreated biomass was done by optimizing parameters for maximal saccharification leads to production of 27.50?mg/g of reduce, ng sugar. An enhanced ethanol yield of 44.46?g/l and fermentation efficiency of 58% by immobilized co-culture of Saccharomyces cerevisiae MTCC 3089 and Scheffersomyces stipitis NCIM 3498 under SHF as compared to fermentation performed with free yeast cells, i.e. 34.46?g/l of ethanol and 45% of fermentation efficiency.     

Microbial and physiological characterization of weakly amylolytic but fast-growing lactic acid bacteria: a functional role in supporting microbial diversity in pozol,a Mexican fermented maize beverage

Pozol is an acid beverage obtained from the natural fermentation of nixtamal (heat- and alkali-treated maize) dough. The concentration of mono- and disaccharides from maize is reduced during nixtamalization, so that starch is the main carbohydrate available for lactic acid fermentation. In order to provide some basis to understand the role of amylolytic lactic acid bacteria (ALAB) in this fermented food, their diversity and physiological characteristics were determined. Forty amylolytic strains were characterized by phenotypic and molecular taxonomic methods. Four different biotypes were distinguished via ribotyping; Streptococcus bovis strains were found to be predominant. Streptococcus macedonicus, Lactococcus lactis, and Enterococcus sulfureus strains were also identified. S. bovis strain 25124 showed extremely low amylase yield relative to biomass (139 U g [cell dry weight](-1)) and specific rate of amylase production (130.7 U g [cell dry weight](-1) h(-1)). In contrast, it showed a high specific growth rate (0.94 h(-1)) and an efficient energy conversion yield to bacterial cell biomass (0.31 g of biomass g of substrate(-1)). These would confer on the strain a competitive advantage and are the possible reasons for its dominance. Transient accumulation of maltooligosaccharides during fermentation could presumably serve as energy sources for nonamylolytic species in pozol fermentation. This would explain the observed diversity and the dominance of nonamylolytic lactic acid bacteria at the end of fermentation. These results are the first step to understanding the importance of ALAB during pozol fermentation.