Energy conservation in malolactic fermentation by Lactobacillus plantarum and Lactobacillus sake

A comparably poor growth medium containing 0.1% yeast extract as sole non-defined constituent was developed which allowed good reproducible growth of lactic acid bacteria. Of seven different strains of lactic acid bacteria tested, only Lactobacillus plantarum and Lactobacillus sake were found to catalyze stoichiometric conversion of l-malate to l-lactate and CO₂ concomitant with growth. The specific growth yield of malate fermentation to lactate at pH 5.0 was 2.0 g and 3.7 g per mol with L. plantarum and L. sake, respectively. Growth in batch cultures depended linearly on the malate concentration provided. Malate was decarboxylated nearly exclusively by the cytoplasmically localized malo-lactic enzyme. No other C₄-dicarboxylic acid-decarboxylating enzyme activity could be detected at significant activity in cell-free extracts. In pH-controlled continuous cultures, L. plantarum grew well with glucose as substrate, but not with malate. Addition of lactate to continuous cultures metabolizing glucose or malate decreased cell yields significantly. These results indicate that malo-lactic fermentation by these bacteria can be coupled with energy conservation, and that membrane energetization and ATP synthesis through this metabolic activity are due to malate uptake and/or lactate excretion rather than to an ion-translocating decarboxylase enzyme.     

Comparative evaluation of ethanol production by xylose-fermenting yeasts presented high xylose concentrations

Summary Three strains ofPichia stipitis and three ofCandida shehatae were compared withPachysolen tannophilus in their abilities to ferment xylose at concentrations as high as 200 g/L when subjected to both aerobic and microaerophilic conditions. Evaluations based on accumulated ethanol concentrations, ethanol productivities, xylose consumption, and ethanol and xylitol yields were determined from batch culture time courses. Of the strains considered,P.stipitis NRRL Y-7124 seemed most promising since it was able to utilize all but 7 g/L of 150 g/L xylose supplied aerobically to produce 52 g/L ethanol at a yield of 0.39 g per gram xylose (76% of theoretical yield) and at a rate comparable to the fastest shown byC.shehatae NRRL Y-12878. For all strains tested, fermentation results from aerobic cultures were more favorable than those from microaerophilic cultures.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

The effect of aeration on D-xylose fermentation byPachysolen tannophilus,Pichia stipitis,Kluyveromyces marxianus andCandida shehatae

Summary The fermentation of D-xylose byPachysolen tannophilus Y2460,Pichia stipitis Y7124,Kluyveromyces marxianus Y2415 andCandida shehatae Y12878 was investigated in aerobic, anaerobic and microaerophilic batch cultures. The aeration rate greatly influenced the fermentations; growth, rate of ethanol production and oxidation of ethanol are affected. Of the strains tested,Pichia stipitis appears superior; under anaerobic conditions it converts D-xylose (20 g/l) to ethanol with a yield of 0.40 g/l and it exhibits the highest ethanol specific productivity (3.5 g of ethanol per g dry cell per day) under microaerophilic conditions.     

Top and bottom yeasts together accelerate ethanol production in molasses fermentation

Summary Alcohol producing top and bottom yeasts were employed individually and together to assess their role in enhancing the rate of ethanol production, in cane molasses fermentation, at 30°C. The combination of top yeastS.cerevisiae NCIM 3281, and bottom yeastS.uvarum NCIM 3509, improved the enthanol production rate by 32.6% in batch fermentation and 25.2% in recycling yeasts cell fermentation as compared to their mean value of individual ethanol production activity.     

Utilization of grape must and concentrated rectified grape must to produce gluconic acid by Aspergillus niger,in batch fermentations

Summary Grape must and concentrated rectified grape must were used for the gluconic acid synthesis using Aspergillus niger batch cultures. The latter substrate was the better, with a production, at 72 h, of 67.43 g/l and a yield (calculated on converted glucose) of 0.96. Citric acid was also observed as a by-product. In order to decrease the residual fructose content, at the end of the gluconate production cycle, an experimental model of sequential fermentation A. niger — Rhizopus arrhizus was proposed for the synthesis of gluconic and fumaric acid. The use of Glucose-isomerase (EC 5.3.1.5) to convert fructose to glucose was also tested.     

Rapid derepression of in vitro nitrogenase activity in a Rhizobium strain which nodulates legumes and the nonlegume Parasponia

Using defined media and controlled gaseous conditions in vitro nitrogenase activity, as monitored by acetylene reduction, was detected after 16 hours of derepression. Specific activity of nitrogenase increased progressively over a period of 100 hours. The method used here utilises rapidly agitated cultures of Rhizobium strain ANU289, incubated at 28°C at cell densities of ca. 1×10⁹ cells ml^–1. The optimal medium for rapid derepression contained basic physiological salts with 3 mM glutamate and 50 mM sodium succinate being the only carbon and nitrogen additives. The gas phase was kept constant by a continuous flow of an air-nitrogen mixture with oxygen being maintained at 0.2%. The described culture system provides the opportunity to observe the regulation of nitrogenase activity in a near-chemostat situation.     

Production of D-xylanases by thermophilic fungi using different methods of culture

Summary Seven thermophilic strains of fungi were examined for their ability to produce D-xylanase in Liquid and solid-state fermentations. It was confirmed that the best producers of xylanase, among microorganisms used, wereH. Lanuginosa andS. thermophile in liquid fermentation, andT. aurantiacus andH. lanuginosa in solid-state fermentations. The higher productivity of xylanase, namely 18,72 IU/ml, was obtained in liquid culture ofH. lanuginosa. The pH and temperature optima of enzymes from liquid and solid-state cultures of fungi used were also presented.     

Shikimate pathway activity in shake and fermenter cultures of Cinchona succirubra

Cell suspension cultures of Cinchona succirubra were cultivated in shake cultures and for the first time in airlift fermenters. Under both conditions L-tryptophan exerts a stimulatory effect on alkaloid formation. In this context the regulatory pattern of some shikimate pathway enzymes was investigated in non-supplemented and tryptophan supplemented Cinchona cell cultures. A remarkable increase of tryptophan decarboxylase (TDC) activity was observed in Cinchona cells under the influence of tryptophan. Apparently, like in some other indole alkaloid producing cell cultures, a high TDC activity is a prerequisite for alkaloid formation. Growth pattern and some enzyme activities of C. succirubra fermenter cultures at controlled and non-regulated pH levels were followed. Optimum growth and alkaloid formation were recorded under non-regulated (normal) pH conditions.Abbreviations TDC tryptophan decarboxylase - try L-tyrosine - phe L-phenylalanine - DAHP 3-deoxy-D-arabino-heptulosonic acid-7-phosphate - trp L-tryptophan - E-4-P erythrose-4-phosphate - PEP phosphoenolpyruvate - MDH malate dehydrogenase - G-6-PDH glucose-6-phosphate dehydrogenase - 6-PG-DH 6-phosphogluconate dehydrogenase - Ch-mutase chorismate mutase - AS-synthase anthranilate synthase - n.d. not determined     

Ethanol fermentation of hexose and pentose wood sugars produced by hydrogen-fluoride solvolysis of aspen chips

Summary Hexose and pentose sugars, produced by hydrogen-fluoride solvolysis of aspen wood chips, were totally consumed in a coculture fermentation by Zymomonas mobilis and a mutant of Clostridium saccharolyticum. Z. mobilis converted the glucose to ethanol, while the mutant, which was improved in both ethanol production and tolerance, converted the xylose component to ethanol. A high conversion efficiency of wood sugars to ethanol was obtained, and the cells after the fermentation were successfully used for cell recycle.NRCC no. 23211     

Electron flow shift inClostridium acetobutylicum fermentation by electrochemically introduced reducing equivalent

Electrochemical energy as a source of reducing equivalent was applied to the cultures ofClostridum acetobutylicum to understand the effects of the pressure by reducing equivalent on anaerobic bacteria. The fermentation byC. acetobutylicum with methyl viologen and electrochemical energy produced more butanol (up to 26%) than the control culture, whilst less acetone (up to 25%) was produced. But no effect was observed on the growth of the culture. These results were indirectly supported byin vitro electrochemical reduction of NAD⁺ and artificial electron carriers.     

Bdellovibrio in methylotrophic bacteria

Summary During the production of single cell protein ofMethylomonas sp. using methanol as the sole carbon source in the pilot plant scale, we isolated aBdellovibrio strain from an abnormal fermentation broth. The abnormality of fermentation caused byBdellovibrio was much like phage infection. However, the plaques formed byBdellovibrio enlarged progressively when plated with host.     

Ethanol production by Zymomonas mobilis CP4 from sugar cane chips

Summary The fermentation of large sugar cane chips (1.0–1.5 in) to ethanol by Zymomonas mobilis CP4 (Z. mobilis) was studied in two glass fermentors operating with culture circulation for agitation (the EX-FERM type): a. A laboratory scale(2.5 liter) cylindrical vessel; b. A bench scale (8 liter) wide vessel. Z. mobilis cultures consumed 89–96% of the cane sucrose, converting it to ethanol by 90–97% of the theoretical yield in the laboratory scale fermentor and by 83–90% in the bench scale fermentor culture. Comparative Saccharomyces spp. cultures in laboratory fermentor consumed 96–98% of the cane sucrose, with ethanol conversion of only 75–79% of the theoretical yield.These preliminary results indicated that sucrose in agricultural size sugar cane chips was ethanol fermentable as compared to small size sugar cane chips or to sugar cane juice. Z. mobilis CP4 cultures converted sucrose more efficiently to ethanol than Saccharomyces spp. as shown in the laboratory scale fermentor studies.The ethanol yields in a wide bench scale fermentor cultures were slightly lower than in a laboratory fermentor.     

Batch and membrane-assisted cell recycling in ethanol production byCandida shehatae

Summary During xylose fermentation byCandida shehatae ATCC 22984 with batch cell recycling, the volumetric ethanol fermentation rate increased two-fold, and the xylitol production rate increased three-fold as the cell density increased to ten-fold. In continuous fermentation with membrane-assisted cell recycle, the fermentation rates increased almost linearly with increasing agitation rates up to 300 rpm. The maximum continuous ethanol production rates obtained with 90 and 200 g L^–1 xylose were respectively 2.4 and 4.4 g L^–1h^–1. The cell density was 65–70 g (dry wt) L^–1. Ethanol yields ranged from 0.26 to 0.41 g g^–1.     

Effect of NaCl on lipid content of plasma membranes isolated from roots and cell suspension cultures of the dicot halophyte Kosteletzkya virginica (L.) Presl.

Lipid concent was examined in plasma membrane fractions isolated by discontinuous sucrose density-gradient centrifugation from both salinized and unsalinized roots and cell suspension cultures of Kosteletzkva virginica (L.) Presl., seashore mallow, a halophytic dicot. The distribution of marker enzymes along the gradient indicated that plasma membranes of roots and cell cultures accumulated primarily at the 34%/45% interface. Total sterol and phospholipid content increased significantly in plants and cell suspensions grown on salinized nutrient media. In addition, K. virginica plasma membranes were constitutively rich in sterols, and a high sterol-to-phospholipid ratio was maintained or elevated under saline conditions. These results are discussed in relation to membrane composition as a mechanism involved in the cellularly based salt tolerance of K. virginica.     

Benomyl: A broad spectrum fungicide for use in plant cell and protoplast culture

The systemic fungicide methyl-1-(butylcarbamoyl)-2-benzimidazole carbamate (benomyl), is a broad spectrum fungicide. Benomyl at concentrations up to 50 mg/l does not inhibit the growth of suspension cultures ofNicotiana tabacum, Datura innoxia, Daucus carota, Glycine canescens, andSolanum tuberosum nor growth ofN. tabacum orN. plumbaginifolia protoplasts if benomyl is dissolved by autoclaving or boiling. Addition of benomyl dissolved in dimethyl sulfoxide results in a visible toxicity. Benomyl, at 6.25–50 mg/l preventsPenicillium spp. growth in both protoplast and cell cultures and can be used to remove fungal contaminates after one to three transfers without visibly retarding plant cell growth. Due to the broad spectrum of fungicidal activity, and nontoxicity at high concentrations when dissolved by boiling or autoclaving, benomyl can be used effectively to control or prevent fungal contamination in plant cell and protoplast cultures.     

Processing and fermentation of Jerusalem artichoke for ethanol production

Summary Processing and fermentation trials on Jerusalem artichoke (Helianthus tuberosus L.) tubers, and on pure inulin media were carried out. Acid and thermal treatments, pure and mixed cultures of yeast, and enzyme preparations were investigated. Best ethanol yields on either substrate were obtained with pH 2 thermal treatments, resulting in 131.6 liters ethanol per metric ton fresh tuber.     

Specific differences in tolerance to exogenous berberine among plant cell cultures

The tolerance of plant cells to exogenously administered berberine, an antimicrobial isoquinoline alkaloid, was studied using berberine-producing and nonproducing cell suspension cultures. Both Coptis japonica and Thalictrum flavum cells, which have an intrinsic ability to synthesize berberine, took up exogenous berberine from the culture medium by an energy-requiring active transport to accumulate it exclusively in vacuoles. By contrast, T. minus cells, which excrete indigenous berberine mostly into the medium, did not take up exogenously supplied berberine, indicating that the alkaloid transport in this species is unidirectional. No inhibition of cell growth by exogenous berberine was observed in the three berberine-producing cell cultures. On the other hand, a small amount of exogenous berberine strongly inhibited cell growth in the berberine-free cultures of Datura innoxia, Catharanthus roseus, and Paeonia albiflora. The berberine taken up actively by Datura cells could not be transported into vacuoles but was dispersed in the cytoplasm, causing a severe inhibition of cell growth.     

Dialysis cultures

Dialysis techniques are discussed as a means for effective removal of low-molecular-mass components from fermentation broth to reach high cell density. Reactor systems and process strategies, the relevant properties of membranes and examples for high-density fermentation with dialysis, and problems related to scale-up are addressed. The dialysis technique has turned out to be very efficient and reliable for obtaining high cell densities. As in dialysis processes the membranes are not perfused, membrane clogging is not a problem as it is for micro- and ultrafiltration. By applying a “nutrient-split” feeding strategy, the loss of nutrients can be avoided and the medium is used very efficiently. The potential of dialysis cultures is demonstrated on the laboratory scale in a membrane dialysis reactor with an integrated membrane and in reactor systems with an external dialysis loop. In dialysis cultures with different microorganisms (Staphylococci, Escherichia coli, extremophilic microorganisms, Lactobacilli) the cell densities achieved were up to 30 times higher than those of other fermentation methods. The technique enables high cell densities to be attained without time-consuming medium optimization. For animal cell cultures the concept of a fixed bed coupled with dialysis proved to be very effective. Received: 24 March 1998 / Received revision: 18 June 1998 / Accepted: 19 June 1998     

Development of a fermentation procedure to produce a tempe-related food using common beans as substrate

Summary A basic procedure was developed to produce a tempe-like product using the mouldRhizopus oligosporus and black common beans (Phaseolus vulgaris) as substrate. The initial pH of the substrate was 5.8, and fermentation was conducted at 37°C with a relative humidity of 70% for 72 hrs. Levels of soluble solids and soluble protein increased dramatically as a result of fermentation. Some changes were as well observed in fatty acid contents of fermented samples. It was concluded that the common bean used was an acceptable substrate for preparing this product.     

Pilot scale production and preservation of a new malolactic culture Leuconostoc oenos 44.40 For use in secondary wine fermentation

Pilot scale production of a starter culture Leuconostoc oenos 44.40 for the secondary fermentation of wine has been demonstrated. Cultures harvested in mid-log phase gave maximum viability after lyophilization. Lyophilized cultures retained their viability best when packaged under nitrogen and stored in dry cool conditions.