Use of an exchange filtration technique to obtain synchronous sporulation in an extended batch fermentation

A fermentor system with an external filtration loop has been developed to control the growth and sporulation of yeast in a single vessel. Excess growth medium, instead of being removed by centrifugation, is removed by filtration and replaced with acetate sporulation medium. The technique did give 80% sporulation after 20 hr, greatly improving the rate and degree of synchrony of sporulation and it also eliminated the contamination hazard of the conventional harvest technique, centrifugation, and resuspension of vegetative cells in sporulation medium. Furthermore it permits proper control of the environmental conditions throughout the growth, exchange, and sporulation phase. In this technique 100% recycle of biomass is achieved without any packing of the cells on the filter. This technique has wide application in the study of industrial fermentation that involves microbial differentiation such as the production of ergot alkaloids, bacitracin, and cephalosporin.     

Citric acid production by Candida lipolytica Y 1095 in cell recycle and fed-batch fermentors

The effect of dissolved oxygen on citric acid production and oxygen uptake by Candida lipolytica Y 1095 was evaluated in cell recycle and fed-batch fermentation systems. The maximum observed volumetric productivity, which occurred at a dilution rate of 0.06 h(-1), a dissolved oxygen concentration of 80%, and a biomass concentration of 5% w/v, in the cell recycle system, was 1.32 g citric acid/L . h. At these same conditions, the citric acid yield was 0.65 g/g and the specific citric acid productivity was 24.9 mg citric acid/g cell . h. In the cell recycle system, citric acid yields ranged from 0.45 to 0.72 g/g. Both the volumetric and specific citric acid productivities were dependent on the dilution rate and the concentration of dissolved oxygen in the fermentor. Similar productivities (1.29 g citric acid/L . h) were obtained in the fed-batch system operated at a cycle time of 36 h, a dissolved oxygen concentration of 80%, and 60 g total biomass. Citric acid yields in the fed-batch fermentor were consistently lower than those obtained in the cell recycle system and ranged from 0.40 to 0.59 g/g. Although citric acid yields in the fed-batch fermentor were lower than those obtained in the cell recycle system, higher citric:isocitric acid ratios were obtained in the fed-batch fermentor. As in the cell recycle system, both the volumetric and specific citric acid productivities in the fed-batch fermentor were dependent on the cycle time and dissolved oxygen concentration. (c) 1995 John Wiley & Sons, Inc.     

The rotorfermentor. II. Application to ethanol fermentation.

The kinetics of microbial growth and product formation are described as applied to the high cell concentration scheme of the rotorfermentor. A bench scale pilot plant was designed and built in order to demonstrate the operational feasibility of the rotorfermentor. The fermentation of glucose to ethanol by Saccharomyces cerevisiae ATCC 4126 was used. When the rotorfermentor was used with a glucose feed concentration of 104 g/liter almost 100% glucose utilization was obtained and the ethanol productivity rate was 27.3 g ethanol/liter hr which was found to be about 10 times greater than the ethanol productivity obtained from an ordinary continuous stirred tank (CST) fermentor. The ethanol experimental results obtained from the rotorfermentor and an ordinary CST fermentor were used as a basis to assess the economic feasibility of the rotorfermentor. The economics of an industrial scale ordinary CST fermentor with and without cell recycle is compared with a rotorfermentor unit for the same ethanol production throughput. For the process conditions considered in this case, calculations showed that the rotorfermentor may replace both a CST fermentor and cell centrifuge resulting in lower capital equipment costs and lower power consumption requirements.     

Preliminary study of a suspended growth predenitrification system

A laboratory study has been conducted to obtained preliminary process information of a suspended growth Predenitrification (SGPDN)system. System performance was evaluated, in terms of chemical oxygen demand (COD) removal, NH(3)-N removal, system biomass yield and inventory, and effluent qualities, at different solids retention times (SRTs) and recycle ratios. Chemical oxygen demand removal in an SGPDN system occurs mainly in the anoxic reactor, which accounts for 94% of total COD removal. The overall COD removal rate is independent of recycle ratio (ranging from 2-5) used in this study; however, effluent COD increase with increasing recycle ratio. The observed anoxic and aerobic COD removal rates decrease with increasing SRT. The NH(3)-N removal in an SGPDN system is induced by two mechanisms: assimilatory NH(3)-N requirement for biomass production in the anoxic reactor and nitrification in the aerobic reactor. The observed anoxic NH(3)-N removal rate relates directly to the anoxic COD removal rate and agrees fairly well with the assimilatory NH(3)-N requirement theoretically predicted. The overall NH(3)-N removal rate is independent of SRTs and recycle ratios used in this study. Biomass yield in an SGPDN system occurs mainly in the anoxic reactor. However, uniform distribution of biomass throughout the entire system is obtained because of the high recycle rate used. The observed biomass yield (Y(O)) decreases with increasing STR. Tertiary treatment efficiency can be achieved in an SGPDN system. More than 90% reduction in feed COD., feed NH(3)-N, and NO(2) + NO(3)-N is obtained at all SRTs and recycle ratios used in this study. Higher MLVSS loading rates can be applied to a final clarifier without impairing its separation efficiency because of the excellent settleability of the Predenitrification activated sludge.     

Production of cellulases by Trichoderma reesei QM 9414 in fed-batch and continuous-flow culture with cell recycle

The scope in improving enzyme productivities from the cellulose fermentation process is examined in laboratory-scale fermentors. The maximum productivity (30 IU/liter hr) is attained in a continuous-culture process with cell recycle using modified medium containing 0.5% cellulose. Optimum dilution rate and recycle ratio are determined as 0.025 hr-1 and 1.2, respectively, for the process. The system is analyzed and steady-state equations for predicting enzyme protein concentrations in the fermentor are developed. In fed-batch cultures, slow addition of cellulose at high concentrations can improve enzyme productivity by as much as 33% over a batch process. The scope and results of using modified medium for cellulase production are also presented.     

Citric acid production from glucose. II. Growth and excretion kinetics in a trickleflow fermentor

The growth and citric acid production kinetics of Saccharomycopsis lipolytica on glucose is investigated in a trickle-flow fermentor. Liquid hold-up and oxygen-transfer coefficient in the reactor column filled with cylindrical wood chips have been determined and found in agreement with chemical engineering correlations. Citric acid production starts at the end of the growth phase and proceeds at a constant specific rate of 0.025 hr^?1for about 80 hr. The fermentor can then be regenerated by addition of ammonia, which induces new growth and excretion phases. Comparing the metabolic behavior of free and immobilized cells, two main kinetic differences are observed. First, the growth phase is linear with the bound cells instead of exponential in the stirred fermentor. Second, in the trickle-bed fermentor acid productivity and oxygen acid yield are reduced by 30%. Oxygen diffusional limitations, mainly in the biomass film, and alterations in bound cell metabolism are shown to be responsible of the kinetic modifications. Simple modelizations of oxygen diffusion effects are also presented to support the interpretation of the experimental data.     

Rapid ethanol fermentation of cellulose hydrolysate. I. Batch versus continuous systems

Rapid fermentation of bagasse hydrolysate to ethanol under anaerobic conditions by a strain of Saccharomyces cerevisiae has been studied in batch and continuous cultures at pH 4.0 and 30°C temperature with cell recycle. By using a 23.6 g/liter cell concentration, a concentation of 9.7% (w/v)ethanol was developed in a period of 6 hr. The rate of fermentation was found to increase with supplementation of yeast vitamins in the hydrolysate. In continuous culture employing cell recycle and a 0.127 v/v/m air flow rate, a cell mass concentration of 48.5 g/liter has been achieved. The maximum fermentor productivity of ethanol obtained under these conditions was 32.0 g/liter/hr, which is nearly 7.5 times higher than the normal continuous process without cell recycle and air sparging. The ethanol productivity was found to decrease linearly with ethanol concentration. Conversion of glucose in the hydrolysate to ethanol was achieved with a yield of 95 to 97% of theoretical.     

Single-cell protein production from spent sulfite liquor utilizing cell-recycle and computer monitoring

Spent sulfite liquor (SSL), a waste product of the paper pulping industry, is produced at a rate of 1 ton (dry basis) per ton of pulp. The sugar content of SSL is about 30 g/L. To reduce the biological oxygen demand (BOD) of SSL before disposal, torula yeast (Candida utilis) is produced by a continuous culture process, the productivity of which is limited by sugar concentration and cell growth rate. To increase productivity, a recycle system has been designed and tested. Cells were sedimented continuously with a flocculating agent (bentonite) before being recycled to the fermentor. A bentonite concentration of 0.02 g/g cell was required. A computer monitoring system based on material balancing techniques was developed to monitor and control the recycle system. With this computer system, productivity was raised to 6.1 g/L h, with cell concentration up to 65 g/L in the recycle stream and 24 g/L in the fermentor. This represents a productivity increase of 150% over continuous culture with no recycle.     

Comparison of batch and semicontinuous cultures for production of protein from mesquite wood by Brevibacterium sp. JM98A

The production of protein by a Brevibacterium sp. JM98A usingmesquite wood as the substrate was compared in batch and semicontinuous cultures. A 14 liter glass fermentor with automatic pH, temperature, and foam control was used for the study. A pH range of 6.6 to 7.2 was optimum for the growth of JM98A. The batch and semicontinuous cultures were compared on the basis of viable cell counts, protein production, CMC-Ase (β-1,4-glucanase) activity, and filter paper cellulase (β-1,4-glucan cellobiohydrolyase) activity. Total hexose, cellulose, and reducing sugar consumption were measured. The semicontinuous process yielded 2.97 times as much protein in 72 hr as the batch cultures. Most of the biomass resulted from the utilization of soluble sugars rather than from the degradation of cellulose during the semicontinuous process.     

Production of single cell protein from rice polishings using Candida utilis

Microbial protein was produced from defatted rice polishings using Candida utilis in shake-flasks and a 14-l fermentor to optimize fermentation conditions before producing biomass in a 50-l fermentor. The organism supported maximum values of 0.224 h⁻¹, 0.94, 1.35, 1.75, 2.12 g l⁻¹ h⁻¹, 0.62 g cells g⁻¹ substrate utilized and 0.38 g g⁻¹ for specific growth rate, true protein productivity, crude protein productivity, cell mass productivity, substrate consumption rate, cell yield, crude protein yield, respectively in 50-l fermentor studies using optimized cultural conditions. Maximum values compared favourably or were superior to published data in literature. The biomass protein in the 50-l fermentor contained 22.3, 27.8, 19.2, 9.5, 38.12, 8.5 and 0.27% true protein, crude protein, crude fibre, ash, carbon, cellulose and RNA content, respectively. The dried biomass showed a gross metabolizable energy value of 2678 kcal kg⁻¹ and contained all essential and non-essential amino acids. Yeast biomass as animal feed may replace expensive feed ingredients currently being used in poultry feed and may improve the economics of feed produced in countries like Pakistan. This revised version was published online in August 2006 with corrections to the Cover Date.     

Continuous ethanol production by a flocculating strain of Kluyveromyces marxianus: bioreactor performance

A flocculating strain of Kluyveromyces marxianus was used for alcoholic fermentation in a continuous bioreactor working with zero residual concentration in effluent. Specific kinetic parameters were improved by increasing dilution rate, which is similar to results obtained with ultrafiltration systems. Specific biomass accumulation rate had always a value greater than 92.5% of specific biomass growth rate and was independent of the dilution rate. Productivity is shown to be 12.5 times greater than in conventional continuous operation and is directly proportional to dilution rate. Maximum biomass concentration also presents a linear relationship with dilution rate. The largest obtained biomass concentration is 8 times greater than in a conventional continuous fermentor.     

Very slow growth of Escherichia coli.

A recycling fermentor (a chemostat with 100% biomass feedback) was used to study glucose-limited behavior of Escherichia coli B. The expectation from mass transfer analysis that growth would asymptotically approach a limit mass determined by the glucose provision rate (GPR) and the culture's maintenance requirement was not met. Instead, growth proceeded at progressively lower rates through three distinct phases. After the fermentor was seeded, but before glucose became limiting, growth followed the usual, exponential path (phase 1). About 12 h postseeding, residual glucose in the fermentor fell below 1 microgram . ml-1 and the growth rate (dx/dt) became constant and a linear function of GPR (phase 2). The specific growth rate, mu, therefore fell continuously throughout the phase. Biomass yield and glucose assimilation (13%) were near the level for exponential growth, however, and independent of GPR over a broad range. At a critical specific growth rate (0.04 h-1 for this strain), phase 2 ended abruptly and phase 3 commenced. In phase 3, the growth rate was again constant, although lower than in phase 2, so that mu continued to fall, but growth rates and yields were praboloid functions of GPR. They were never zero, however, at any positive value of GPR. By inference, the fraction of metabolic energy used for maintenance functions is constant for a given GPR, although different for phases 2 and 3, and independent of biomass. In both phases 2 and 3, orcinol, diphenylamine, and Lowry reactive materials were secreted at near-constant rates such that over 50% as much biosynthetic mass was secreted as was retained by the cells.     

Continuous culture in combined backmix-plug-flow-tubular-loop fermentor configurations

Several combinations of backmix, tubular-loop, and plug-flow fermentors with and without culture recycle were studied by computer simulations. The steady-state concentrations of cell mass in a continuous culture were calculated as a function of dilution rate using Monod growth kinetics. It was found theoretically and verified for one case experimentally that the maximum dilution rate, over which microbial cells were washed out from the fermentor, could be elevated well beyond the maximum specific growth rate if a particular fermentor combination was used. A combination of two backmix fermentors has been analyzed previously by Sinclair and Brown. Application of this type of fermentor combination as a seed tank for performing continuous culture of microbes in a plug-flow reactor was shown with special reference to fermentation production using the kinetics proposed by Luedeking and Piret, van Dedem and Moo-Young, and Brown and Vass.     

A simple capillary viscosity meter for the on-line measurement and control of the biomass concentration in high-density cultures

A capillary viscosity meter was used for the on-line determination of the biomass concentration in a fermentation broth. At high cell densities the viscosity of the broth increases, which can be measured as the pressure drop over a capillary. Calibration aspects of this viscosity meter are presented, and the use of the device for the control of the biomass concentration in a membrane recycle fermentor is demonstrated.     

脱色希瓦氏菌(Shewanella decolorationis) S12还原不同电子受体的厌氧发酵罐培养方法

脱色希瓦氏菌Shewanella decolorationisS12在厌氧环境下能够使用多种电子受体进行厌氧呼吸。为了取得足够的细胞量用于膜蛋白质组学等科学研究的需要,本研究选取无机小分子(硝酸钠)、金属离子(柠檬酸铁)和有机大分子(偶氮染料苋菜红)作为电子受体,在使用确定成分的无机盐培养基条件下,使用不同浓度的电子供体和碳源对S12进行厌氧条件下静置和发酵罐的优化培养,采用连续补充电子受体的培养方式,确认了电子供体和碳源的合适浓度,建立了S12厌氧发酵罐培养方法。相比传统的静置厌氧培养,厌氧发酵罐培养方法在保证了严格厌氧条件下高效率还原电子受体的同时,还极大的提高了细胞生长密度。连续补充电子受体的厌氧发酵罐培养的S12最大细胞密度最大分别可达到静置厌氧培养细胞密度的325,304,369倍,而生长时间也比静置厌氧培养分别缩短了26.5%,17.6%,7.5%。这为需要大量细胞和蛋白的细菌厌氧呼吸生长实验建立了可行方法,对于进行兼性厌氧呼吸的微生物的大规模厌氧培养具有借鉴意义。     

Production of ethanol by Clostridium thermosaccharolyticum: I. Effect of cell recycle and environmental parameters

The direct microbial conversion (DMC) process for the production of ethanol from lignocellulosic biomass is limited by low volumetric ethanol production rates due to the low cell densities of Clostridium thermosaccharolyticum which is a key organism for ethanol production in this process. Hence, this study focuses on the use of a continuous- culture cell recycle system to improve the volumetric ethanol productivity and yield of the fermentation of xylose by C. thermosaccharolyticum. Early experiments with the continuous-culture cell recycle system showed a two-fold improvement in volumetric ethanol productivity. However, the ethanol yield at the higher dilution rates suffered because of the large amount of lactate produced. The manipulation of two environmental parameters-iron concentration in the nutrient medium and the N(2) purge rate of the fermentor headspace-allowed a dramatic reduction in the lactate production and a simultaneous improvement in the ethanol titer and yield. Under the improved conditions of increased iron concentration (12.5 mg/L FeSO(4) . 7H(2)O) and decreased N(2) purge rate (0.1 L/min), a continuous culture of C. thermosaccharolyticum operating at a dilution rate of 0.24 h(-1) and 50% cell recycle produced 8.6 g/L ethanol and less than 1 g/L each of acetate and lactate. The volumetric ethanol productivity was 2.2 g/L/h, which is 8 times larger than obtained for a continuous culture operated with no cell recycle and the same specific growth rate.     

Effect of feed zone in fed-batch fermentations of Saccharomyces cerevisiae

In production-scale, fed-batch fermentations, feed is often added to a single point at the top of the fermentor, which, combined with poor mixing, results in formation of a "feed zone" rich in nutrients. Frequent exposure of the culture to high concentrations of nutrients in the feed zone for sufficient duration can produce unexpected effects on its performance. The effect of the feed zone was evaluated by conducting aerobic fed-batch fermentations of Saccharomyces cerevisiae with both complex and defined media. The broth was recirculated between a recycle loop and a bench-scale fermentor, and feed was intermittently added into the recycle loop to simulate the circulation of cells through the feed zone. Experiments were carried out for a range of residence times in the recycle loop from 0.5 to 12 min. Biomass yields from the complex-media fermentations were not affected by exposure to high nutrient levels in the recycle loop for residence times up to 12 min. Ethanol consumption was reduced by as much as 50% for residence time in the loop up to 3 min. Very long exposure of yeast cells to excess nutrient levels (12 min) gave acetic acid formation. In a defined medium, the simulated feed zone effect increased biomass yield by up to 10%, but had no effect on ethanol levels. This study indicates that the feed zone effect on biomass yield in yeast fermentation, using complex substrates, will be negligible under fully aerobic conditions.     

The physiology of lactate production by Lactobacillus delbreuckii in a chemostat with cell recycle

The physiology of lactate production by Lactobacillus delbreuckii NRRL B-445 in a continuous fermenter with partial cell recycle has been studied and compared with that observed in a conventional chemostat. Partial cell recycle was achieved using a hollow-fiber ultrafiltration cartridge. The biomass growth yield was reduced in the recycle fermenter while culture viability and the cellular content of polysaccharide, protein, carbon, and nitrogen remained constant, suggesting an enlarged specific rate of glucose consumption for nonanabolic (e.g., maintenance) functions. The volumetric productivity of lactate was enhanced in the recycle fermenter due to the complete utilization of glucose. The yield of lactate from biomass and the molar product ratio, lactate: ethanol plus acetate, decreased with increasing recycle ratio. Enhanced formation of ethanol and acetate occurred in the recycle fermenter although lactate remained the major product. The change in product profile was due to glucose limitation. The specific activity of lactate dehydrogenase remained constant during recycle fermentation. These physiological observations have implications for the future application of cell recycle to production processes.     

Cell retention culture with an internal filter module: continuous ethanol fermentation

A new internal filter feedback system with a stainless steel filter was introduced and its application for continuous ethanol fermentation was investigated. The filter performance was highly influenced by agitation speed and yeast concentration. Retention coefficient with a filter of 2 mum pore size was found more than 97.5%, and the filter was suitable for yeast separation. Maximum yeast concentration was 157 g/L and the best operable cell concentration was between 90 and 150 g/L. Which was similar to that obtained in the external membrane cell recycle culture. The cell concentration in the fermentor was maintained by manipulation of dilution rate and bleed ratio with the growth rate. The internal filter feedback system was successfully operated for more than 10 days. This study shows that the internal filter feedback system with a stainless steel filter can be used high-density cell culture and ethanol fermentation. Furthermore, it can be scaled up more easily than the external cell recycle system. (c) 1993 John Wiley & Sons, Inc.     

Control of nitrate concentration in fermentations of Corynebacterium glutamicum

A nitrate control system has been devised for the maintenance of stable nitrate concentrations throughout fed-batch fermentations of Corynebacterium glutamicum. The feedback control system was based on the use of a nitrate-ion-selective electrode to directly monitor the nitrate levels in the fermentor and an automatic controller to activate a nitrate feed pump. The electrode which was used for controlling the nitrate level was stable through-out the fermentation period. The apparent maximum specific growth rate, biomass production, protein production, biomass yields on glucose and nitrate, and amino acid production were all optimal at approximately 50mM nitrate.