Repeated Batch Culture Research Articles

Extractive bioconversion for lactic acid production using solid sorbent and organic solvent

Extraction of lactic acid from fermentation broths have been studied. The extractant selected for this research was paraffin oil which, in addition to having satisfactory physical properties, is non-toxic and does not affect the fermentability of the broth. Several amine compounds and a polymeric resin (Bonopore) have been tested to increase the extraction efficiency of the paraffin oil. Addition of these compounds gave higher KD values. However, all of the amine compounds tested are highly toxic to the free cells of L. delbrueckii (NRRL B-445). Bonopore, the polymer giving good absorption pattern and no deleterious effects was tested with paraffin oil in repeated batch culture of L. delbrueckii.

The Production of Secondary Metabolites by Plant Cells Cultivated in Bioreactors1

Plant cell cultures are a potential source of pharmaceutically important plant metabolites. In the past few years a lot of success has been achieved in the field of the cultivation of plant cells on a large scale. Special bioreactor systems, such as airlift or drum-type fermentors have been devised for the mass cultivation of plant cells. Classical stirred-tank bioreactors (up to 75000 l volume) have also been used for the cultivation of plant cells and the production of important plant metabolites. Plant cells proliferate much slower than microbial cells. In consequence, the time taken to grow a plant cell suspension from the shake-flask level (300 ml) to the production scale (20,000 l) takes about 3 to 6 weeks. Nevertheless, the cost analyses available show that the production of valuable chemicals by a suitable plant cell culture process could be commercially viable. Plant cells have been grown in batch and repeated batch culture, and single- stage as well as two-stage processes have been developed for the production of secondary metabolites by fermentor-cultured plant cells. There are, however, only few cell culture processes that have attracted industrial attention as yet. Biological rather than technological problems are the main obstacles to a more common use of fermentor-cultured plant cells in industry.

Studies on Production of Lipids in Fungi. XXII.

Nine fungi of Mortierella genus were cultivated batchwise in a 30L-fermenter in high con-centration glucose medium (100-200g/L). As a result, four strains attained a γ-linolenic acid (GLA) productivity exceeding 2g/L. The strain content of GLA in the fatty acids of the lipids obtained was from 6.6-7.8%. Using M. ramanniana var. angulispora IFO 8187, one of the four strains, the effects of various cultivation conditions on GLA productivity were investigated. Culti-vation at a higher content of GLA in the total lipids was generally noted in strains with lower lipid productivity, thus giving lower GLA yield. Consequently, cultivation to obtain high GLA yield is possible in a cultivation of high lipid productivity rather than high GLA content. Two types of repeated batch cultures with one and two fermenters, respectively, were made to obtain higher lipid productivity. A maximum lipid productivity of 13.9g/L/d was realized in the 2nd fermenter with a double stage repeated batch culture. GLA yield in the repeated batch culture increased with lipid productivity.

A feed-harvest culturing method to improve lignin peroxidase production by Phanerochaete chrysosporium INA-12 immobilized on polyurethane foam

Phanerochaete chrysosporium INA-12 immobilized with polyurethane foam produced high lignin peroxidase levels in 24-h repeated batch culture when the biomass was partially regenerated on day 5 (40.6 nkat·ml −1). The enzyme synthesis continued at a high level for eight batches, with only 7% activity lost every 24 h.

Adsorbents for extractive bioconversion applied to the acetone-butanol fermentation

Four different polymeric resins were tested as adsorbents in extractive bioconversion applied to the fermentative production of acetone and butanol by Clostridium acetobutylicum. The polymers were tested for their ability to adsorb butanol from pure solutions, and fermentation broths. Furthermore, the effect on the fermentability of the media was tested. The pH was increased to prevent adsorption of intermediates such as acetic and butyric acids. Bonopore, the polymer giving the best adsorption pattern with no undesirable effects, was tested in repeated batch cultures with C. acetobutylicum.

CH4 production from H2 and CO2 byMethanobacterium thermoautotrophicum cells fixed on hollow fibers

Methane was produced from H2 and CO2 byMethanobacteriumthermoautotrophicum cells fixed on the surface of hollow fibers. The mineral solution permeated through the inside of fibers was consumed by the cells, while the gaseous substrate flowing outside the fibers was directly metabolized to methane. Methane production was proportional to hollow fiber length i.e., contact area between cell layer and gas phase. In repeated batch cultures, the production rates of methane and cell mass were 33.1 L/L reactor/day and 1.75 g cells/L reactor/day, respectively with 90% conversion rate.

Stability of plasmid PBR322 in Escherichia coli immobilized on cotton cloth during use as resident inoculum.

E. coli cells harbouring plasmid pBR322 which confers ampicillin resistance were immobilized on cotton cloth. The resulting film was used as an inoculum in daily repeated batch culture in ampicillin-free medium. During two months, the film was able to produce cultures which, at the late log phase, showed little sensitivity to 10 mg/ml ampicillin. Thus such a bacterial film can effectively be used as an inoculum for the production of recombinant DNA products by means of pBR322 or its derivatives in the absence of ampicillin.

Enhancement of ?-linolenic acid production by Mucor ambiguus with nonionic surfactants

γ-Linolenic acid (GLA) production by Mucor ambiguus IFO 6742, immobilised in Biomass Support Particles (BSPs), has been investigated in a fluidized-bed fermenter in the presence of nonionic surfactants. In this system, repeated batch cultivation was achieved at higher yield and productivity than by conventional methods, since microbial lipids inlcuding GLA were significantly secreted into the culture broth and/or on the surface of the cell wall.

Simulation and optimization of fed-batch culture for ethanol production from molasses

Two simulation methods for ethanol production from molasses by a flocculating yeast, Saccharomyces cerevisiae AM12, were investigated and molasses feeding was optimized. The first method was based on a deterministic model with fixed kinetic parameters and the second was based on regression analysis. The amount of ethanol produced in a fed-batch culture with multiple additions of molasses was simulated by both of these two methods. Simulated results of a fed-batch culture were compared with those of a simple batch culture by a model of regression analysis. The intermittent addition of molasses gave better production than a single addition at the beginning; more frequent addition may further improve production. The experimental results suggested the same. The effect of the amount of the added molasses on ethanol production was investigated by simulation. Repeated batch culture with and without intermittent addition of molasses in each batch was also done.

Effects of discharged fraction in repeated batch culture for hyaluronate lyase production

The production of hyaluronate lyase (EC 4.2.21), HL, by Streptococcus equisimilis strain SHL-03 has been studied in repeated batch culture with various harvest volumes (discharged fraction, α = 0.875, 0.75, 0.625, and 0.50). The optimum discharged fraction to obtain the highest production was α = 0.75. The productivity of this system was 73% higher than that of a classical batch culture. The process could be repeated for 15 cyclase (245 h) with stable hyaluronate lyase production rate and yield. These results suggest that this system could provide a practical procedure to increase the productivity of an industrial plant without changes in facilities and with minimal investment.

Continuous production of lignin peroxidase by Phanerochaete chrysosporium

Phanerochaete chrysosporium spores were immobilized both in agarose and agar gel beads, and used for the production of lignin peroxidase in repeated batch cultures on carbon-limited medium both with 0.5 g l −1 glucose and without glucose. Veratryl alcohol was used as an activator of enzyme production. The biocatalyst was more stable in agarose gel with the maximum activity of 245 U l −1 obtained in a 70 h batch. The biocatalyst could be used for at least 12 batches on the glucose medium with a gradual decrease in lignin peroxidase activity after the sixth batch. Further, mycelium pellets grown on carbon-limited medium were employed both in vertical and horizontal column reactors for the continuous production of lignin peroxidase. The bioreactor produced lignin peroxidase for at least 20 days in the horizontal system at 49 h residence time, with a maximum activity of 95 U l −1.

Chemoautotrophic growth of Thiocystis violacea, Chromatium gracile and C. vinosum in the dark at various O2‐concentrations

AbstractThe influence of various O2‐concentrations on chemoautotrophic growth was determined in the range from microaerobic to aerobic conditions. Chromatium vinosum D, Thiocystis violacea 2311 and Chromatium gracile 25‐1 were grown chemoautotrophically using consecutively repeated batch cultures. Rapid growth of all three strains occurred only under microaerobic conditions. The fastest growth (μ = 0.058) was obtained with Chromatium gracile 25‐1 grown in the presence of 1 mg O2 · 1−1. For optimal chemoautotrophic growth sulfur‐containing cells had to be supplied continuously with extracellular electron donor. Multiplication of thiocystis violacea 2311 and Chromatium gracile 25‐1 was significantly inhibited after several cell generations, while Chromatium vinosum D could be grown unlimited. In case of the latter bacterium any given O2‐concentration determined a unique growth rate and a unique specific bchl α content. The analysis of absorption spectra of whole cells revealed that different pigment complexes were regulated separately by oxygen. Growth yields were not dependent on the O2‐concentration. A growth yield of 12 g dry cell mass per mol of thiosulfate oxidized was determined for Chromatium vinosum D growing in the micro‐ to semiaerobic O2‐concentration range. When compared to phototrophically grown cells, the catalase content of chemotrophically grown cells was 2 times lower while the maximal specific respiration rate of the cells was 7 times higher. The ecological significance of the kinetics of chemotrophic growth under various growth conditions is discussed.

Increase of hyaluronate lyase productivity by repeated batch fermentation

The production of hyaluronate lyase (E.C.4.2.2.1.), HL, byStreptococcusequisimilis strain SHL-03 has been studied under repeated batch culture conditions with fixed harvest volume (discharged fraction, α=0.875). The productivity of this system was 49.2% higher than that of a classical batch culture. The process could be repeated at least for 15 cycles (245 hours) with a productivity of 33.77 U/ml h and a yield of 12,800-U/g Glu.

Improved performance with multiple fermentors for repeated batch cultivation for non‐growth‐associated products

Based on batch cultivation data for the production of L-glutamic acid from glucose, a comparative evaluation was made for repeated batch cultivations using one and more fermentors. The problem was formulated as maximizing the productivity of metabolic product with the specified conversion with respect to the cell age and the volume fraction used as seed for the subsequent repeated batch cultivation. Simulations were carried out with the assumption of no lag in product formation for the cases where the total operation time was specified as 200 h with reproducible batch cultivation cycles. The product production was assumed to be solely a function of product concentration. The computation results show the advantage of using more than one fermentor from the viewpoints of productivity and conversion, which will apply in general to non-growth-associated product production with delay time. In particular, three fermentors are recommended for the production of L-glutamic acid chosen as an example in this article.

Properties of Trichoderma reesei cells immobilized by the irradiation technique

Aerobic cells of Trichoderma reesei have been immobilized by the radiation polymerization technique using fibrous substances and hydroxyethyl methacrylate. The enzyme [cellulase, 1,4-(1,3;1,4)-β- d-glucan 4-glucanohydrolase, EC 3.2.1.4] productivity and growth of the cells in the immobilized growing cells have been studied. The enzyme (filter paper) activity in the immobilized cells was comparable to that of the intact cells, showing that the cells immobilized with fibrous materials grow and become adhered to the surface of the fibrils. The filter paper activity of the immobilized cells was affected mainly by monomer concentration and the content of the fibrous materials, as well as the irradiation dose. It was demonstrated that in repeated batch culture of the immobilized cells the filter paper activity gave a constant value, and leakage of the cells was not observed .

Genetic construction of yeast strains for high ethanol production

To improve ethanol production capability, a flocculent yeast,Saccharomyces cerevisiae TJ1, was hybridized with other high-alcohol producing strains by protoplast fusion. A fusant strain of strain TJ1 hybridized with an Awamori-brewing strain, N1, produced 12.4 (w/v)% of ethanol even at 40°C. The fusant showed good flocculence and good sedimentation. In a repeated batch culture with cell reuse at 37°C, an average productivity of 13 g/l·h was attained.

Metabolite repression of fructose 1,6-diphosphatase in yeast

The growth of Yarrowia lipolytica on glycerol was studied in bioreactor repeated batch cultures and three distinct phases, namely biomass production phase, lipogenic phase and citric acid production phase were identified during growth cycle. In each phase, yeast cells were characterised by specific morphological and biochemical features. Though high activity of NAD+ dependent iso-citric dehydrogenase (NAD+–ICDH) was detected during biomass production phase, this activity was significantly decreased afterwards inducing lipogenesis. A further drop in NAD+–ICDH activity to minimal levels and a decrease in glycerol kinase activity were observed during the citric acid production phase. Surprisingly, citric acid production was accompanied by storage (neutral) lipid turnover, along with remarkable biosynthesis of glycolipids, sphingolipids and phospholipids. Oleic acid was the major fatty acid in all lipid fractions and phosphatidylcholine was the main phospholipid. This study allows concluding that Y. lipolytica successfully converts glycerol via phosphorylation pathway into valuable biotechnological products, such as single cell oil and citric acid.