Cellulose fermentation by continuous cultures of Ruminococcus albus and Methanobrevibacter smithii

Summary The hydrolysis and fermentation of cellulose (Avicel) by continuous cultures of Ruminococcus albus strain 7 and Methanobrevibacter smithii strain PS were studied. Cellulose destruction ranged from ca. 22% to 71% for 0.25 to 2.27 days solids retention time, respectively. The cellulose hydrolysis rate constant (k) was 1.3 days^–1. Concentrations of soluble reducing sugars were low, showing that cellulose hydrolysis was the rate-limiting step of cellulose fermentation. The estimated methane-based molar growth yield for M. smithii was 2.8 g mol^–1. Its maximum specific growth rate was ca. 4 days^–1. The dissolved H₂ half-saturation constant (K _s ) for methanogenesis was ca. 1 M. The final products of the co-culture were primarily acetate, CH₄ and CO₂ and low levels of ethanol and H₂. The co-culture produced more H₂ (used for reduction of CO₂ to CH₄) and acetate than a monoculture of R. albus. These differences coulb be accounted for by the lower production of ethanol, confirming to the theory of interspecies H₂ transfer. Offprint requests to: M. J. Wolin     

Production of 4-decanolide by semicontinuous fermentation of Tyromyces sambuceus

Summary Tyromyces sambuceus was cultivated in a stirred tank reactor under semicontinuous conditions for 70 days. Over longer periods of time in submersion, values of 220 and 330 mg·L^–1 of 4-decanolide could be maintained; after dispersion of the headspace mycelia, a single peak product concentration of 880 mg·L^–1 was attained. The productivity was subject to large variations, peak values were 18 and 11 mg·L^–1·h^–1. Depending on the culture conditions, specific growth rates of up to 0.05 h^–1 were reached.     

Improved performance of intensive semicontinuous cultures of Scenedesmus by biomass recirculation

The microscopic green alga, Scenedesmus obliquus, was used in a semicontinuous culture system for the tertiary treatment of urban wastewater, with the simultaneous production of usable biomass. Partial biomass recycling was used to increase the productivity of the system by overcoming the limits imposed by the low maximal growth rate of the alga. The biomass to be recycled was collected by simple gravity settling of the removed culture.The culture system was operated at different dilution rates and its productivity measured at each rate. An evaluation of the crude nutrient composition of the algae produced at each dilution rate was also carried out.The system was found to operate stably at dilution rates of up to 0.8 day(-1) which represents a 20% net increase over the maximum dilution rate allowed under the same conditions in a system without recirculation. The composition of the biomass produced varied little over a range of dilution rates, which may be of relevance to its projected end-use.The study indicated that such a system can exploit available light to the full and should be of particular value for the treatment of low-strength wastes such as we employed.     

Glycerol production by semicontinuous fed-batch fermentation with immobilized cells of Saccharomyces cerevisiae

Summary Semicontinuous fed-batch glycerol fermentations with Saccharomyces cerevisiae cells immobilized in sintered glass Raschig rings were carried out in fixed-bed loop reactors with a working volume of either 0.8 l or 8 l. The influence of biomass, temperature and CO₂ gassing on the glycerol yield was examined. The highest glycerol yield of 85 g l^–1 was achieved at 30° C and average CO₂ gassing rate of 0.4 v/v m with a theoretical glycerol yield of 67%. Fed-batch fermentations with free cells indicated an inhibition mechanism of the glycerol produced, affecting the fermentation capacity of the yeast strain used.Offprint requests to: H.-J. Rehm     

Approximation of continuous growth of Cephalotaxus harringtonia plant cell cultures using fed-batch operation

In Cephalotaxus harringtonia plant cell cultures, periods of batch growth that are limited by hexose uptake are too short to make an accurate estimate of the Monod saturation constant. Continuous cultures are infeasible on a laboratory scale, and semicontinuous cultures require too frequent sampling. Fed-batch operation, consisting of intermittent removal from a culture that is fed continuously, was investigated as a possible solution to these problems. For a constant feed rate, computer simulations showed that a steady state can be achieved which is useful for studying growth at different specific growth rates. In terms of the dilution rate it was confirmed that the operation is essentially equivalent to continuous culture when the samples represent a small fraction of the total culture volume. Experiments with glucose or fructose as the carbon source were carried out in shake flasks fed by a multichannel syringe pump. Results indicate that Monod kinetics based on medium glucose levels cannot adequately describe growth under these conditions. Monod's expression for specific growth rate using internal glucose concentration gives an improved correlation.     

Citric acid production from sucrose by recombinant Yarrowia lipolytica using semicontinuous fermentation

The genetically modified yeast strain Yarrowia lipolytica H222‐S4(p67ICL1)T5 is able to utilize sucrose as a carbon source and to produce citric and isocitric acids in a more advantageous ratio as compared to its wild‐type equivalent. In this study, the effect of pH of the fermentation broth (pH 6.0 and 7.0) and proteose‐peptone addition on citric acid production by the recombinant yeast strain were investigated. It was found that the highest citric acid production occurred at pH 7.0 without any addition of proteose‐peptone. Furthermore, two process strategies (fed‐batch and repeated fed‐batch) were tested for their applicability for use in citric acid production from sucrose by Y. lipolytica. Repeated fed‐batch cultivation was found to be the most effective process strategy: in 3 days of cycle duration, approximately 80 g/L citric acid was produced, the yield was at least 0.57 g/g and the productivity was as much as 1.1 g/Lh. The selectivity of the bioprocess for citric acid was always higher than 90% from the very beginning of the fermentation due to the genetic modification, reaching values of up to 96.4% after 5 days of cycle duration.     

Kinetic studies of the lactic acid fermentation in batch and continuous cultures

The fermentation kinetics of the homofermentative organism Lactobacillus delbrueckii in a glucose-yeast extract medium is studied in both batch and continuous culture under conditions of controlled pH. From a graphical analysis of the batch data, a mathematical model of the process is derived which relates bacterial growth, glucose utilization, and lactic acid formation. The parameters in the model represent the activity of the organism and are a function of pH, having a maximum value at about 5.90. In a continuous stirred tank fermentor (CSTF), the effect of pH, feed concentration, and residence time is observed. The feed medium is a constant ratio of two parts glucose to one part yeast extract plus added mineral salts. An approximate prediction of the steady-state behavior of the CSTF can be made using a method based on the kinetic model derived for the batch case. In making step changes from one steady state to another, the transient response is observed. Using the kinetic model to simulate the transient period, the calculated behavior qualitatively predicts the observed response.     

Biosensor online control of citric acid production from glucose by Yarrowia lipolytica using semicontinuous fermentation

Our study aimed at the development of an effective method for citric acid production from glucose by use of the yeast Yarrowia lipolytica. The new method included an automated bioprocess control using a glucose biosensor. Several fermentation methodologies including batch, fed‐batch, repeated batch and repeated fed‐batch cultivation were tested. The best results were achieved during repeated fed‐batch cultivation: Within 3 days of cycle duration, approximately 100 g/L citric acid were produced. The yields reached values between 0.51 and 0.65 g/g and the selectivity of the bioprocess for citric acid was as high as 94%. Due to the elongation of the production phase of the bioprocess with growth‐decoupled citric acid production, and by operating the fermentation in cycles, an increase in citric acid production of 32% was achieved compared with simple batch fermentation.     

Acetonobutylic fermentation: improvement of performances by coupling continuous fermentation and ultrafiltration

The technology of coupling ultrafiltration and fermentation has been tested with the acetonobutylic fermentation in continuous mode. The device developed was sterilizable by steam and permitted drastic cleaning of the ultrafiltration (UF) membrane without interrupting the continuous fermentation. It has been shown to be an easily operated and reliable experimental tool for studying high-cell-density cultures and inhibition phenomena. With total recycle of biomass, a dry weight concentration of 125 g/L was attained, which greatly enhanced the volumetric solvent productivity of acetonobutylic fermentation in averaging 4. 5 g/L h for significant periods of time (>70 h) and maintaining solvent concentration and yield at acceptable levels.     

Factors controlling eicosapentaenoic acid production in semicontinuous cultures of marine microalgae

Three marine microalgal species with a high content of eicosapentaenoic acid (EPA), Phaeodactylum tricornutum, Isochrysis galbana and Porphyridium cruentum, were cultured semicontinuously in order to study the effect of renewal rate on EPA productivity. The percentage of EPA in total fatty acids increased with increasing renewal rates in nitrogen limited cultures, but while for Phaeodactylum tricornutum and Isochrysis galbana a plateau around 20–25% of total fatty acids was reached with renewal rates that were not nitrogen-limiting, in Porphyridium cruentum EPA percentage increased continuously with increasing renewal rate even for those cultures that were nitrogen sufficient. Maximal EPA productivities of4.6 mg L^-1 day^-1 for Isochrysis galbana and 5.2 mg L^-1 day^-1 for Phaeodactylum tricornutum were achieved with renewal rates of 20% and 30% respectively. On the other hand for Porphyridium cruentum maximal EPA productivity, 5.3 mg L^-1 day^-1, was obtained with the maximal renewal rate tested. Results indicate that different culture strategies should be adopted for the production of a particular polyunsaturated fatty acid depending on the microalgal species being used. This revised version was published online in August 2006 with corrections to the Cover Date.     

Theory of semicontinuous and continuous cultivation applied to the yeastTorula utilis

Были выведены математические зависимости между скоростью разведения и скоростью роста в условиях полунепрерывной культивации микроорганизмов. Было доказано, что вполне непрерывный способ культивации представляет особыи—крайний—случай полунепрерывных процессов и позволяет обработку максимального количества субстрата на единицу времени и объема ферментора. При изучении оптимальных условий размножения дрожжей Torula utilis было экспериментально доказано, что непрерывный процесс представляет одновременно наиболее удобный способ осуществления ферментации, позволяющий полное использование субстрата и мощности оборудования. Было доказано, что в случаях, когда дрожжи используют один источник углерода или же различные истичники углерода одновременно и с одинаковой скоростью, одноступенчатый способ ферментации более выгоден, чем многоступенчатый процесс.     

Propionic acid fermentation: improvement of performances by coupling continuous fermentation and ultrafiltration

The present paper presents a study of propionic acid production from whey by using Propionibacterium acidipropionici in batch and continuous fermentation with cell recycle. The experimental investigation is carried through with a biomass concentration (DW) of 112kg/m³. The highest propionic acid productivity is 2.14 kg/(m³ h). Biomass concentration is 9 times as high, propionic acid productivity 6 times as high as compared to batch results.     

Optimized nikkomycin production by fed-batch and continuous fermentation

We performed fed-batch and continuous fermentations to extend the time of maximal nikkomycin production by Streptomyces tendae Tü 901/S 2566. This was achieved by the fed-batch culture technique. Furthermore, high productivity was obtained at slow growth rates in a continuous fermentation process. Different dilution rates with and without carbon limitation were done and the results were compared. Correspondence to : T. Schüz     

Microbial biomass production by continuous fermentation of bark hydrolysate

Summary The fungus Aspergillus niger, an unidentified filamentous fungus (strain no. PDDCC8239) and the yeast Candida tropicalis were grown in continuous culture in stirred tank reactors at dilution retes varying between 0.02–0.1 h^-1 on a bark extract medium made by dilute acid hydrolysis of Pinus radiata bark. Maximum yields were 5.1, 18.7, and 61.5 mg biomass·g^-1 bark for the unidentified fungus, A. niger and C. tropicalis respectively. Culturing in a tower fermenter under otherwise identical environmental conditions increased the yield of A. niger to 27.3 mg biomass·g^-1 bark. The yield of C. tropicalis represents a productivity of 0.26g biomass·l^-1·h^-1 which exceeds other reported values of bark fermentations. Analysis of the medium and spent broth revealed significant breakdown of tannin material by C. tropicalis during growth. This ability may be of value in the treatment of tannin-based industrial effluents.     

Application of multistage continuous fermentation for production of fuel alcohol by very-high-gravity fermentation technology

A fermentation system to test the merging of very-high-gravity (VHG) and multistage continuous culture fermentation (MCCF) technologies was constructed and evaluated for fuel ethanol production. Simulated mashes ranging from 15% to 32% w/v glucose were fermented by Saccharomyces cerevisiae and the dilution rates were adjusted for each glucose concentration to provide an effluent containing less than 0.3% w/v glucose (greater than 99% consumption of glucose). The MCCF can be operated with glucose concentrations up to 32% w/v, which indicates that the system can successfully operate under VHG conditions. With 32% w/v glucose in the medium reservoir, a maximum of 16.73% v/v ethanol was produced in the MCCF. The introduction of VHG fermentation into continuous culture technology allows an improvement in ethanol productivity while producing ethanol continuously. In comparing the viability of yeast by methylene blue and plate count procedures, the results in this work indicate that the methylene blue procedure may overestimate the proportion of dead cells in the population. Ethanol productivity (Yps) increased from the first to the last fermentor in the sequence at all glucose concentrations used. This indicated that ethanol is more effectively produced in later fermentors in the MCCF, and that the notion of a constant Yps is not a valid assumption for use in mathematical modeling of MCCFs. Journal of Industrial Microbiology & Biotechnology (2001) 27, 87–93. Received 20 January 2001/ Accepted in revised form 28 April 2001     

Exopolysaccharide, pigment and protein production by the marine microalga Chroomonas sp. in semicontinuous cultures

The marine microalga Chroomonas sp. isolated from Venezuela was grown in semicontinuous culture in order to study the effect of renewal rate and nutrient concentration on alloxanthin, chlorophyll a, carotenoid, carbohydrate, exopolysaccharide, protein and cell productivity. Maximal cell productivity of 8.43 ± 1.8 and 8.81 ± 2.3 × 10⁹ cell l^–1 day^–1 were achieved with renewal rates of 30 and 40%. Maximal protein and chlorophyll productivity of 64.64 ± 2.3 and 2.72 ± 0.3 mg l^–1 day^–1 were obtained with renewal rate of 20 and 30%. Biochemical composition of Chroomonas sp. was influenced by renewal rate. Nutrient concentration seems not to affect cell, protein, chlorophyll and carotenoid productivity. However, carbohydrate and exopolysaccharide productivity of 7.56 ± 0.4 and 9.57 ± 1.2 mg l^–1 day^–1 were increased at 12 mM NaNO₃(P < 0.05). Also, alloxanthin and chlorophyll a production analysed by HPLC, were higher between 8 and 12 mM NaNO₃ at a renewal rate of 30%. Results demonstrated that a renewal rate of 30% and nutrient concentration at 8 mM NaNO₃ optimize the cell, protein, carbohydrate, chlorophyll a, and exopolysaccharide productivity in semicontinuous cultures of Chroomonas. This microalga, as biological source of commercially valuable compounds, shows high capacity for changing its productivity and biochemical composition in semicontinuous system on the basis of nutrient concentration and the renewal rate.     

Extremum seeking scheme for continuous fermentation processes described by an unstructured fermentation model

This paper examines the problem of maximising the productivity of a class of fermentation processes described by an unstructured fermentation process model. For a given dilution rate, an extremum seeking adaptive control has been used to maximise the productivity of a fermentation process. The concept behinds the extremum seeking method is to iteratively adjust the feed substrate rate in order to steer the process to yield a maximum productivity. The main advantage of the extremum seeking adaptive control is it does not require any structural information of the modeling uncertainty.     

Modelling of continuous acetonobutylic fermentation

A model of continuous acetonobutylic fermentation is proposed. This model correctly portrays the predominantly solvents formation observed at acidic extracellular pH and predominantly acids production at more neutral pH, as well as observed effects of dilution rate and feed substrate on products' concentrations. A fair agreement between experimental and theoretical predictions is achieved for a broad range of operating variables.     

Assessment of fed-batch, semicontinuous, and continuous epothilone D production processes

Epothilone D is a member of a class of potent antineoplastic natural products produced by myxobacteria. Previously, we have described a fed-batch epothilone D production process in which an adsorber resin is incorporated into the bioreactor setup to capture and stabilize the product in situ, preventing its degradation within the bioreactor. The capture of epothilone D by these relatively large resin beads enables the development of continuous and semicontinuous culturing systems incorporating bead retention mechanisms to completely retain the product within the bioreactor, increasing the epothilone D product titer by almost 3-fold in both cases over a baseline fed-batch system. These product retention strategies, described here for production of the epothilones, are generally applicable to any system using adsorber resins as a method to capture product during a microbial cultivation.