Rapid and efficient production of L-lactate from xylose using electrodialysis culture-associated product separation

L-Lactate was produced from xylose using electrodialysis culture (ED-C)-associated product separation. In a medium containing 50g xylose/l, the ED-C was completed in only 32h (i.e. less than half the time taken by the control culture, without electrodialysis). At 80g xylose/l, the control culture was unable to consume more than 50g xylose/l, whereas the ED-C showed increased xylose consumption and was completed by 45h. The maximum rate of lactate production in the ED-C was higher than that in the control culture. ED-C was also carried out (at 80g initial xylose/l) with a supply of fresh xylose-free medium. This ED-C was completed within 30h, which represents a reduction in fermentation time of 15h when compared to ED-C without addition of xylose-free medium. Thus, rapid production of L-lactate was achieved by using ED-C which supplied fresh xylose-free medium.     

Synergism of cellulase enzymes in mixed culture solid substrate fermentation

Sugar cane bagasse was subjected to a mixed culture, solid substrate fermentation with Trichoderma reesei QM9414 and Aspergillus terreus SUK-1 to produce cellulase and reducing sugars. The highest cellulase activity and reducing sugar amount were obtained in mixed culture. The percentage of substrate degradation achieved employing mixed culture was 26% compared to 50% using separate cultures of the two molds. This suggests that the synergism of enzymes in mixed culture solid substrate fermentation have lower synergism than in pure culture.     

Experiences with the use of a starter culture in the fermentation of maize for ‘kenkey’ production in Ghana

Controlled fermentation of maize was carried out using six strains of Lactobacillus fermentum and one strain of yeast, Saccharomyces cerevisiae, isolated from traditionally fermented maize dough as starter cultures for inoculum enrichement. The fermentations were monitored by pH, acidity, microbiological analysis and taste panel evaluation of two products, kenkey and koko, prepared from the fermented doughs. The strains of L. fermentum used as starter culture dominated the microflora during fermentation and in most inoculated doughs the required pH was attained by 24 h instead of 48 h of dough fermentation. Higher contents of lactic acid bacteria and yeasts were observed in inoculated doughs at the initial stages of fermentation but the spontaneously fermented doughs attained similar lactic acid bacteria and yeasts counts by 24 h of dough fermentation. The organoleptic quality of kenkey and koko prepared from doughs fermented with starter culture for 48 h was not significantly different from the traditional products. Kenkey prepared from doughs fermented for 24 h with starter culture were found to be unacceptable by the taste panel although similarly produced koko was acceptable.The authors are with the Food Research Institute, Council for Scientific and Industrial Research, P.O Box M 20. Accra, Ghana.     

Influence of culture modes on the microbial production of hyaluronic acid by <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis>

The principal objective of this study was to assess the effects of culture modes including batch culture, pulse fed-batch culture, constant feeding rate fed-batch culture, and exponential fed-batch culture on the production of hyaluronic acid (HA) by Streptococcus zooepidemicus. Batch cultures had the highest levels of HA productivity, whereas fed-batch cultures were more favorable with regard to cell growth, and exponential fed-batch cultures evidenced the highest cell concentrations. A two-step culture model was proposed to enhance HA production: an exponential fed-batch culture was conducted prior to 8 h and then sucrose supplementation was applied for 8 h to start the batch fermentation of S. zooepidemicus. HA production and productivity were increased by 36 and 37% in the proposed two-step culture process as compared with that observed in the batch culture, respectively. The proposed two-step culture model can be applied in the production of secondary metabolites, and particularly of the exopolysaccharides.     

Use of frozen bagged seed inoculum for secondary metabolite and bioconversion processes at the pilot scale

Frozen bagged seed inoculum was prepared, thawed and tested for seven cultures. Thawing techniques were developed and other key influences on thawing rate were quantified; seed bag thawing without a water bath rarely required more than 4 to 5 h and was as short as 0.5 to 1 h for lower fill volume bags. Testing included growth of bagged seed as a function of bag fill volume (0.5, 1.0, 2.0, and 3.5 L), comparison of culture age at time of bagging, growth of bagged versus laboratory-prepared seed, productivity of production cultures derived from bagged versus laboratory-prepared seed, growth of bagged seed as a function of volume percent glycerol added at time of bagging, and growth of bagged seed as a function of frozen storage time and temperature. For each culture tested, conditions were developed such that seed tanks inoculated with bagged seed showed only minimal delay in attaining the target oxygen uptake rate (OUR) relative to seed tanks inoculated with laboratory-prepared inoculum. Although the bag fill volume did influence culture growth in some cases, bag fill volumes required were reasonable (typically 2.0 to 3.5 L) compared with laboratory seed inoculum volumes of 2.0 L. In the most remarkable example, frozen bagged seed was prepared from a second-stage seed-tank cultivation of Glarca lozoyensis, then thawed and inoculated into first-stage seed medium. It grew to the desired OUR in a similar timeframe as laboratory-prepared inoculum inoculated into first-stage seed medium. Thus, the frozen bagged seed replaced an existing laboratory inoculum preparation period of 7 days without an appreciable delay in either of the two subsequent seed-tank growth stages. Furthermore, productivities were found to be comparable for bagged-seed-derived and laboratory-seed-derived production cultivations for four different fermentation processes.     

Ethanol production from xylose by Thermoanaerobacter ethanolicus in batch and continuous culture

The fermentation of xylose by Thermoanaerobacter ethanolicus ATCC 31938 was studied in pH-controlled batch and continuous cultures. In batch culture, a dependency of growth rate, product yield, and product distribution upon xylose concentration was observed. With 27 mM xylose media, an ethanol yield of 1.3 mol ethanol/mol xylose (78% of maximum theoretical yield) was typically obtained. With the same media, xylose-limited growth in continuous culture could be achieved with a volumetric productivity of 0.50 g ethanol/liter h and a yield of 0.42 g ethanol/g xylose (1.37 mol ethanol/mol xylose). With extended operation of the chemostat, variation in xylose uptake and a decline in ethanol yield was seen. Instability with respect to fermentation performance was attributed to a selection for mutant populations with different metabolic characteristics. Ethanol production in these T. ethanolicus systems was compared with xylose-to-ethanol conversions of other organisms. Relative to the other systems, T. ethanolicus offers the advantages of a high ethanol yield at low xylose concentrations in batch culture and of a rapid growth rate. Its disadvantages include a lower ethanol yield at higher xylose concentrations in batch culture and an instability of fermentation characteristics in continuous culture.     

Seed stage development for improved fermentation performance: Increased milbemycin production byStreptomyces hygroscopicus

Fermentation development for improved culture productivity can be achieved in a number of ways. Conventional approaches usually concentrate initially on optimisation of the final stage fermentation. However an understanding of the seed stage and its further development can lead to an improvement in final stage productivity. A significant increase in the production of milbemycin VM44866 byStreptomyces hygroscopicus was achieved by manipulation of several factors associated with the seed stage fermentation. Juvenile seeds and seed media containing reduced levels of carbohydrates overcame the detrimental effects of passaging and seed age associated with the standard (control) process. The effect of final stage inoculum level was seed medium-dependent and seed fermentation incubation temperature also affected subsequent milbemycin VM44866 production. These findings were extended to a second milbemycin-producing strain and these results have demonstrated the potential benefits of seed stage optimisation for improved final stage production.     

Development of starter culture for improved processing of Lafun,an African fermented cassava food product

Aims: To select appropriate micro‐organisms to be used as starter culture for reliable and reproducible fermentation of Lafun. Methods and Results: A total of 22 cultures consisting of yeast, lactic acid bacteria (LAB) and Bacillus cereus strains predominant in traditionally fermented cassava during Lafun processing were tested as potential starter cultures. In an initial screening, Saccharomyces cerevisiae 2Y48P22, Lactobacillus fermentum 2L48P21, Lactobacillus plantarum 1L48P35 and B. cereus 2B24P31 were found to be the most promising of the cultures and were subsequently tested in different combinations as mixed starter cultures to ferment submerged cassava roots. Saccharomyces cerevisiae, inoculated singly or combined with B. cereus, gave the softest cassava root after 48 h of fermentation according to determination of compression profile and stress at fracture. Overall, sensory quality testing showed that Lafun obtained from S. cerevisiae‐fermented cassava gave the most preferred stiff porridge. Saccharomyces cerevisiae 2Y48P22 showed pectinase production in a model system. Conclusions: The results suggest that S. cerevisiae 2Y48P22 is the most efficient organism for cassava softening during the fermentation. Therefore, it could be combined with LAB and used as starter for Lafun processing. Significance and Impact of the Study: Starter cultures are made available for controlled fermentation of Lafun.     

Metabolic and energetic aspects of the growth of Clostridium butyricum on glucose in chemostat culture

The influence of a number of environmental parameters on the fermentation of glucose, and on the energetics of growth of Clostridium butyricum in chemostat culture, have been studied. With cultures that were continuously sparged with nitrogen gas, glucose was fermented primarily to acetate and butyrate with a fixed stoichiometry. Thus, irrespective of the growth rate, input glucose concentration specific nutrient limitation and, within limits, the culture pH value, the acetate/butyrate molar ratio in the culture extracellular fluids was uniformly 0.74±0.07. Thus, the efficiency with which ATP was generated from glucose catabolism also was constant at 3.27±0.02 mol ATP/mol glucose fermented. However, the rate of glucose fermentation at a fixed growth rate, and hence the rate of ATP generation, varied markedly under some conditions leading to changes in the Y _glucose and Y _ATP values. In general, glucose-sufficient cultures expressed lower yield values than a correponding glucose-limited culture, and this was particularly marked with a potassium-limited culture. However, with a glucose-limited culture increasing the input glucose concentration above 40g glucose·l^-1 also led to a significant decrease in the yield values that could be partially reversed by increasing the sparging rate of the nitrogen gas. Finally glucose-limited cultures immediately expressed an increased rate of glucose fermentation when relieved of their growth limitation. Since the rate of cell synthesis did not increase instantaneously, again the yield values with respect to glucose consumed and ATP generated transiently decreased.Two conditions were found to effect a change in the fermentation pattern with a lowering of the acetate/butyrate molar ratio. First, a significant decrease in this ratio was observed when a glucose-limited culture was not sparged with nitrogen gas; and second, a substantial (and progressive) decrease was observed to follow addition of increasing amounts of mannitol to a glucose-limited culture. In both cases, however, there was no apparent change in the Y _ATP value.These results are discussed with respect to two imponder-ables, namely the mechanism(s) by which C. butyricum might partially or totally dissociate catabolism from anabolism, and how it might dispose of the excess reductant [as NAD(P)H] that attends both the formation of acetate from glucose and the fermentation of mannitol. With regards to the latter, evidence is presented that supports the conclusion that the ferredoxin-mediated oxidation of NAD(P)H, generating H₂, is neither coupled to, nor driven by, an energy-yielding reaction.     

Xanthan gum: an economical substitute for agar in plant tissue culture media

Xanthan gum, a microbial desiccation-resistant polysaccharide prepared commercially by aerobic submerged fermentation from Xanthomonas campestris, has been successfully used as a solidifying agent for plant tissue culture media. Its suitability as a substitute to agar was demonstrated for in vitro seed germination, caulogenesis and rhizogenesis of Albizzia lebbeck, androgenesis in anther cultures of Datura innoxia, and somatic embryogenesis in callus cultures of Calliandra tweedii. Culture media used for eliciting these morphogenic responses were gelled with either 1% xanthan gum or 0.9% agar. Xanthan gum, like agar, supported all these responses.     

Potential of marine lactic acid bacteria to ferment Sargassum sp. for enhanced anticoagulant and antioxidant properties

Aim

To evaluate the suitability of marine lactic acid bacteria (LAB) as starter cultures for Sargassum sp. fermentation to enhance its antioxidant and anticoagulation activity.

Methods and Results

LAB isolated from marine source were characterized for their ability to utilize seaweed as a sole carbon source and applied to Sargassum fermentation. Fermentation period was optimized by monitoring the fermented sample at regular interval for a period of 18 days. Results revealed that a fermentation period of 12 days was effective with maximum culture viability and other desirable characteristics such as pH, total titratable acidity, total and reducing sugars. Under optimum fermentation period, the sample fermented with P1‐2CB‐w1 (Enterococcus faecium) exhibited maximum anticoagulation activity and antioxidant activity.

Conclusions

The study reveals a novel well‐defined starter culture from marine origin intended for seaweed fermentation for recovery of bioactive molecules.

Significance and Impact of the study

The study provides information for the enhancement of bioactive molecules in an eco‐friendly manner and also paves a way towards the development of wide range of seaweed functional foods.     

Halobacterium sp. SP1(1) as a starter culture for accelerating fish sauce fermentation

Aims: Application of Halobacterium sp. SP1(1) for the acceleration of fish sauce fermentation. Methods and Results: Traditional fish sauce fermentation was mimicked using Halobacterium sp. SP1(1) as starter culture. Protease activity, peptide release and α‐amino content (parameters used to monitor the progress of the fermentation) were high at day 10 in tests and day 20 in un‐inoculated controls. The total protein and nitrogen contents were also high in tests compared with controls. The amino acid profile observed at the end of fermentation in experimental samples, when compared with the commercial sauce preparation, was found to be better with respect to flavour and aroma contributing amino acids as well as essential amino acid lysine. Microflora analysis of the final fish sauce revealed the absence of any nonhalophilic or halotolerant micro‐organisms. The protease‐producing halophilic isolates obtained from the fish sauce of eviscerated and uneviscerated controls were identified as Halobacterium sp. F1 and F2, respectively, by 16S rDNA sequence analysis. Conclusions: Exogenous augmentation of Halobacterium sp. SP1(1) accelerated the fish sauce fermentation process with an additive effect on the existing natural microflora present in the fish during fermentation. Halobacterium sp SP1(1), therefore, can be used as an important starter culture for accelerating the fish fermentation process, which is attributed to its extracellular protease. Significance and Impact of the Study: The present study is the first report on use of Halobacterium species as a starter culture for accelerating fish sauce fermentation. Use of halobacterial starter cultures may revolutionize the process in fish sauce industries by reducing the fermentation time and making the process more economical with improved nutritive value of product.     

Continuous cultures of Clostridium acetobutylicum: culture stability and low-grade glycerol utilisation

Continuous cultures of two strains of Clostridium acetobutylicum were stable for over 70 d when grown on glucose/glycerol mixtures. Butanol was the major fermentation end-product, accounting for 43 to 62% (w/w) of total products. Low-grade glycerol [65% (w/v) purity] could replace commercial glycerol [87% (w/v) purity], leading to a similar fermentation pattern: a butanol yield of 0.34 (mol/mol), a butanol productivity of 0.42 g l^–1 h^–1 and a 84% (w/w) glycerol consumption were attained when cultures were grown at pH 6 and D = 0.05 h^–1; butanol accounted for 94% (w/w) of total solvents. These values are among the highest reported in literature for C. acetobutylicum simple chemostats.     

Comparison of culture methods on exopolysaccharide production in the submerged culture of Cordyceps militaris and process optimization

Aims: To improve exopolysaccharides (EPS) production of Cordyceps militaris (C. militaris), effects of different culture method on mycelial biomass and EPS production in the submerged culture of C. militaris were investigated. Methods and Results: A new two‐stage fermentation process for EPS production of C. militaris was designed in this work. Central composite design (CCD) was utilized to optimize the two‐stage fermentation process. The results showed that the two‐stage fermentation process for EPS production was superior to other culture method (conventional static culture and shake culture). CCD revealed that the optimum values of the test variables for EPS production were shaken for 140 h followed by 130‐h static culture. The maximum EPS production reached 3·2 g l^?1 under optimized two‐stage culture and was about 2·3‐fold and 1·6‐fold in comparison with those of original static culture and shake culture. Conclusions: It was indicated that a new two‐stage culture method obtained in this work possessed a high potential for the industrial production for EPS of C. militaris. Significance and Impact of the Study: The fundamental information obtained in this work is complementary to those of previous investigations on the submerged culture of C. militaris for the production of bioactive metabolites.     

Multistrain probiotic production by co‐culture fermentation in a lab‐scale bioreactor

Most commercial probiotic products intended for pharmaceutical applications consist of combinations of probiotic strains and are available in various forms. The development of co‐culture fermentation conditions to produce probiotics with the correct proportion of viable microorganisms would reduce multiple operations and the associated costs. The aim of this study was to develop a fermentation medium and process to achieve biomass comprising the desired proportion of two probiotic strains in co‐culture. Initially, a quantification medium was developed, and the method was optimized to allow the quantification of each strain's biomass in a mixture. The specific growth rates of Lactobacillus delbrueckii spp. bulgaricus and Lactobacillus plantarum were determined in media with different carbon sources. The inoculum volume was optimized to achieve equal proportion of biomass in co‐culture fermentation in test tubes. Next, fermentation was carried out in a 3‐L bioreactor. A biomass concentration of 2.06 g/L, with L. delbrueckii spp. bulgaricus and L. plantarum in the ratio of 47%:53% (by weight), was achieved with concomitant production of 12.69 g/L of lactic acid in 14 h. The results show that with careful manipulation of process conditions, it is possible to achieve the desired proportion of individual strains in the final biomass produced by co‐culture fermentation. This process may serve as a model to produce multistrain probiotic drugs at industrial scale.     

Evaluating asymbiotic seed culture methods and establishing Disa (Orchidaceae) germinability in vitro: relationships, requirements and first-time reports

Many Disa species (80%) have not been germinated in vitro, fuelling conservation fears. Winter-rainfall species are typically germinable, whilst summer-rainfall species are exclusively intractable in vitro. We aimed to establish the in vitro germination requirements for previously ungerminated Disa species. Conventional asymbiotic seed culture protocols were reviewed and their efficacy in summer-rainfall Disa established. Mature seed was subjected to manipulations of media composition and viscosity, incubation temperature and illumination. Immature cultures were also established across four seed maturity classes. Four first-time germinations (D. cooperi, D. nervosa, D. pulchra and D. woodii) resulted from media with increased water availability. Germination rate (>12 weeks), percentage (<30%) and synchrony were not in accordance with values reported for winter-rainfall species (>80% germination in 8 weeks). Germination of immature seed under control conditions was similar to mature seed germination under modified conditions, but neither percentage approached the calculated germination potential (∼viability). Germination control in Disa is proposed as a trade-off between water availability and the presence of phyto-inhibitors in the environment of the embryo—two features typical of seeds exhibiting water-impermeable dormancy.     

In vitro culture provides additional variation for pigeonpea

Summary The present study is an attempt to exploit somaclonal variation for the varietal improvement of pigeonpea [Cajanus cajan (L) Millsp.]. The pigeonpea plants were regenerated from cotyledon explants, and their progeny was screened for variability. The regenerated R1 plants exhibited a spectrum of alterations in floral morphology and architecture that were absent in the control population. The field-sown R2 plants segregated for traits such as flower color, leaf shape, seed size, color and strophiolation, flowering habit, and fertility. Tissue culture produced different mutational events resulting in both dominant and recessive alleles. Significant variation was observed for plant height, seed mass, and damage due to the insect pest Helicoverpa armigera. The R3 plants, obtained from seed of R2 generation selected for traits such as white seed coat, strophiolation, reduced plant height, seed mass and low damage due to Helicoverpa, maintained the traits when compared with the seed-derived control populations. The results indicate a definite gene for white seed coat and the possibility of additional genes for pest tolerance and high seed mass in an adapted background. Submitted as Journal Article No. 1906 by International Crops Research Institute for the Semi-Arid Tropics (ICRISAT).     

Comparative assessment of fermentation techniques useful in the processing of ogi

Ogiis processed traditionally by the use of uncontrolled spontaneous fermentation of maize, sorghum and millet. In this study, traditionally applied spontaneous fermentation was compared with accelerated batch fermentation (or back-slopping) and the use of starter cultures to initiate fermentation. Lactic acid bacteria populations comprised 95 of the total viable bacteria and remained prominent throughout the fermentations, while number of moulds and coliform bacteria declined as the fermentation progressed. The fermentation method involving the application of starter culture helps most to control the prevalence of coliforms and moulds. Lactic acid bacteria, such as Lactococcus raffinolactis, Pediococcussp.,Pediococcus pentosaceus, Lactobacillus plantarum, Lb. suebicus and Lb. brevis,were isolated at different processing stages of ogi using accelerated batch fermentation (back-slopping) technique. Highest increase in acidity was observed immediately after wet-milling and sieving fermenting maize grains at 28 and 48 h. Sharp increases in the reducing sugar levels were noted between 24 and 28 h of fermentations during wet-milling and sieving processes.     

Fermentation of glycerol by Clostridium pasteurianum--batch and continuous culture studies

The fermentation of glycerol by Clostridium pasteurianum was studied with respect to product formation as influenced by the culture conditions. In the majority of batch cultures, butanol was the main fermentation product, but a varying fraction of glycerol was also converted to 1,3-propanediol, butyric and acetic acids and ethanol. More than 60 g/l glycerol was utilized, and up to 17 g/l butanol was produced. Fed-batch cultures did not offer an advantage. When molecular nitrogen was used as a nitrogen source, the fermentation time was prolonged by a factor of 1.5. Fermentations at constant pH values between 4.5 and 7.5 did not reveal significant differences in product formation except for an increase in the ethanol content starting at pH 6.5. Chemostat cultures also yielded predominantly n-butanol, but in some fermentations, the 1,3-propanediol fraction was relatively high. The pH auxostat cultures, which were operated at a glycerol excess, contained 1,3-propanediol as the main product. As a whole, the fermentations were characterized by a certain variability in product formation under seemingly equal or slightly varied conditions. It appears that the regulation of the numerous fermentation pathways occurring in this organism is not very strict. Journal of Industrial Microbiology & Biotechnology (2001) 27, 18–26. Received 25 September 2000/ Accepted in revised form 07 April 2001     

A multiplex PCR method for simultaneous species identification and strain typification of Oenococcus oeni

Selected starter cultures of Oenococcus oeni are widely used to initiate malolactic fermentation (MLF) in wine. Nevertheless, the inoculated culture does not always develop as expected and undesired strains can grow causing wine spoilage. Therefore, methods that can reliably differentiate O. oeni strains are essential to monitor the population dynamics of MLF. This work presents a new multiplex PCR method that allows the simultaneous species identification and strain typification of O. oeni, based on the combined use of species-specific PCR primers and a Random Polymorphic DNA (RAPD)-PCR primer. This method represents an useful tool for the control of wine MLF.