Morphology and viability analysis of<Emphasis Type="Italic"> Streptomyces clavuligerus</Emphasis> in industrial cultivation systems

The effects of varying inoculum age and production scale upon the morphology and viability of Streptomyces clavuligerus were studied by analyzing visible and fluorescent light images acquired throughout pilot-plant and pre-industrial scale fermentations. Changes in production scale reveal that in 5 m³ fermentors, the maximum hyphal area obtained is double the value obtained in 0.5 m³ fermentors. It is probably due to the higher shear stresses acting upon hyphae in the 0.5 m³ fermentor caused by higher tip speeds observed in these. The morphological quantification based on elongation and branching rates allowed fermentations to be pattern classified into distinct physiological time zones namely elongation, branching, fragmentation, etc. The general pattern observed for fermentations inoculated with late exponential phase inocula was similar to the pattern of fermentations run with stationary phase inocula except that both the elongation and branching periods started earlier in the former case. Using the available staining technique and image acquisition system, the viability seemed to be generally high and constant throughout the time course of all the studied fermentations.An erratum to this article can be found at     

Media and Fermentation Processes for the Rapid Production of High Concentrations of Stable Blastospores of the Bioinsecticidal Fungus Paecilomyces fumosoroseus

In shake flask and fermentor studies, various media components and culture inocula were tested to improve P. fumosoroseus spore production rates, yield and stability. To evaluate inoculum potential and inoculum scale-up for fermentor studies, conidia and liquid culture-produced spores of various strains of P. fumosoroseus were compared as inoculum. Inoculation of liquid cultures with blastospores at concentrations of at least 1×10⁶ spores mL^-1 resulted in the rapid production of high concentrations of blastospores (∼1×10⁹ spores mL^-1, 48 h fermentation time) for all strains tested. The rapid germination rate of blastospores (90% after 6 h incubation) compared to conidia (>90% after 16 h incubation) and the use of higher inoculum rates reduced the fermentation time from 96 to 48 h for maximal spore yields. A comparison of various complex nitrogen sources showed that liquid media supplemented with acid hydrolyzed casein or yeast extract supported the production of high concentrations of blastospores that were significantly more desiccation-tolerant (79-82% survival after drying) when compared to blastospores produced in media supplemented with other nitrogen sources (12-50% survival after drying). For rapid spore production, requirements for trace metals and vitamin supplementation were dependent on the type of hydrolyzed casein used in the medium. Fermentor studies with two strains of P. fumosoroseus showed that high concentrations (1.3-1.8×10⁹ spores mL^-1) of desiccation-tolerant blastospores could be produced in 48-h fermentations. These studies have demonstrated that the infective spores of various strains of the fungal bioinsecticide Paecilomyces fumosoroseus can be rapidly produced using deep-tank, liquid culture fermentation techniques.     

Morphology and clavulanic acid production of Streptomyces clavuligerus: Effect of stirrer speed in batch fermentations

Streptomyces clavuligerus ATCC 20764 was grown from spore-inocuia on a glycerol, malt extract, bacteriological peptone medium in 5-L batch fermentations at 490, 990, and 1300 rpm. Dry cell weights, clavulanic acid production, and the morphological parameters main hyphal length, total hyphal length, number of tips, and hyphal growth unit were measured. Growth and productivity were hardly dependent on stirrer speed. After early growth fragmentation of long, highly branched mycelia to shorter, less branched fragments occurred. This was followed by regrowth and, at 1300 rpm, a second fragmentation phase. The effect of increasing stirrer speed was to accelerate the initial fragmentation phase. It was clearly possible to obtain the same biomass concentration and clavulanic acid liter, with different morphologies depending on stirrer speed. This shows that for this fermentation at least there is no direct link between morphology and productivity and, hence, that it might be possible to manipulate them independently to improve fermentor performance.     

Changes of diversity and population of yeasts during the fermentations by pure and mixed inoculation of Saccharomyces cerevisiae strains

Mixed inoculation of Saccharomyces cerevisiae strains is used in winemaking for achieving high sensory quality of the wine. However, information on the diversity and population of yeasts during inoculated fermentation is very limited. In this study, we evaluated the effect of mixed inocula with different inoculation timing on the yeast community during fermentations of Cabernet Sauvignon. Grape must was inoculated with pure cultures of S. cerevisiae RC212 or S. cerevisiae R312, and simultaneous and sequential inoculation of both strains. Wallersterin Laboratory Nutrient (WLN) medium and sequence of the 26S rDNA D1/D2 domain were used to compare the diversity of yeast species. Five species, including Candida diversa, Hanseniaspora opuntiae, H. uvarum, Issatchenkia orientalis and I. terricola, were identified in the grape must, with Issatchenkia sp. being predominant (67.5 %). Three to four species were involved in each fermentation treatment. The fermentations by mixed inocula presented more yeast species than by pure inocula. Interdelta sequence typing was used to identify S. cerevisiae strains. Ten genotypes were identified among 322 isolated S. cerevisiae strains. Their distribution varied among different stages of fermentations and different inoculation treatments. The inoculated strains were not predominant, while indigenous genotypes I, III, and V showed strong competitiveness during fermentation. In general, this study provided information on the change of population structure and genetic diversity of yeasts in fermentations inoculated with pure and mixed S. cerevisiae strains.     

Evaluation of yeast diversity during wine fermentations with direct inoculation and pied de cuve method at an industrial scale

The diversity and composition of yeast populations may greatly impact wine quality. This study investigated the yeast microbiota in two different types of wine fermentations: direct inoculation of a commercial starter versus pied de cuve method at an industrial scale. The pied de cuve fermentation entailed growth of the commercial inoculum used in the direct inoculation fermentation for further inoculation of additional fermentations. Yeast isolates were collected from different stages of wine fermentation and identified to the species level using Wallersterin Laboratory nutrient (WLN) agar followed by analysis of the 26S rDNA D1/D2 domain. Genetic characteristics of the Saccharomyces cerevisiae strains were assessed by a rapid PCR-based method, relying on the amplification of interdelta sequences. A total of 412 yeast colonies were obtained from all fermentations and eight different WL morphotypes were observed. Non-Saccharomyces yeast mainly appeared in the grape must and at the early stages of wine fermentation. S. cerevisiae was the dominant yeast species using both fermentation techniques. Seven distinguishing interdelta sequence patterns were found among S. cerevisiae strains, and the inoculated commercial starter, AWRI 796, dominated all stages in both direct inoculation and pied de cuve fermentations. This study revealed that S. cerevisiae was the dominant species and an inoculated starter could dominate fermentations with the pied de cuve method under controlled conditions.     

The role of Bacillus species in the fermentation of cassava

Cassava dough inoculum is added to grated cassava in order to achieve a modification of texture during fermentation into the fermented cassava meal, agbelima. The microflora of two different types of inocula and subsequently inoculated cassava mash at 0, 24 and 48 h of fermentation were examined in order to determine the mechanism responsible for the breakdown of cassava tissue. Bacillus spp. occurred in high numbers, 10₇–10₈ cfu g_-1, in both types of inocula and persisted throughout the cassava dough fermentation. Bacillus spp. found were B. subtilis , B. mycoides , B. pumilus , B. cereus , B. amyloliquefaciens and B. licheniformis , with B. subtilis accounting for more than half of Bacillus isolates. All Bacillus isolates produced a wide spectrum of enzymes and showed similar enzymatic activities but only B. pumilus , B. licheniformis and B. amyloliquefaciens produced linamarase. Some isolates produced the tissue degrading enzymes polygalacturonase and pectin esterase and nearly all isolates hydrolysed starch. All isolates showed cellulase activity and were able to disintegrate cassava tissue. When cassava pieces were incubated in amylase, cellulase, pectin esterase and polygalacturonase solutions, only pieces in cellulase solution were dissolved revealing that the breakdown of cassava dough texture during fermentation with the inocula examined is brought about by Bacillus spp. through cellulase activity.     

Accelerated Fermentation of Brewer's Wort by Saccharomyces carlsbergensis

A rapid procedure for wort fermentation with Saccharomyces carlsbergensis at 12 C is described. Fermentation time was reduced from 7 to 4 days with normal inoculum by shaking. Increasing the inoculation to 5 to 10 times normal and shaking resulted in complete fermentation in 3 days. Maximum yeast population was reached rapidly with the large inocula, but fermentation proceeded at approximately the same rate when inoculations in excess of four times the normal were used. Similar results were obtained with both small-scale (100 ml) and microbrew (2.4 liters) fermentations.     

Optimization of Inocula Conditions for Enhanced Biosurfactant Production by Bacillus subtilis SPB1, in Submerged Culture, Using Box–Behnken Design

The inoculum age and density can influence considerably the production yield and cost of the fermentation process. Some literature studies report the use of two-stage inocula to enhance metabolite production. In the present study, optimization studies were done in order to define the best inocula conditions supporting a maximum biosurfactant production by Bacillus subtilis SPB1. Hence, by adjusting the levels of the two-stage inocula strategy, lipopeptide production was effectively enhanced to almost 3.4 g/l as estimated gravimetrically. The new defined parameters were as follows; a first inoculum age of 23 h followed by a second inoculum age and size of 4 h and 0.01, respectively. Thereby, we note an improved production as compared to the production yield described under non-optimized inocula conditions reported in our previous work.     

Batch ethanol fermentation of molasses: a correlation between the time necessary to complete the fermentation and the initial concentrations of sugar and yeast cells

Batch fermentations of sugar-cane blackstrap molasses to ethanol, using pressed yeast as inoculum, demonstrated an exponential relationship between the time necessary to complete the fermentation and the initial concentrations of sugar and yeast cells. The parameters of the derived exponential equations depended on the experimental conditions.     

Influence of the curing of the killer phenotype inSaccharomyces cerevisiae wine strains on their fermentative behaviour

Fermentative behaviour and cell growth have been studied in grape juice inoculated either with two killerSaccharomyces cerevisiae wild strains or with their Acridine Orange-cured isogenic counterparts. The number of viable cells/ml at the beginning of the fermentation, as well as during exponential growth, were higher in grape juices inoculated with the cured strains. The CO₂ production, fermentative rate and ethanol and acetic acid production were also higher in the cured strains, particularly during the stage of active fermentation. These differences, however, were minimal at the end of the fermentations.     

Ethanol production from corn cob hydrolysates by <Emphasis Type="Italic">Escherichia coli</Emphasis> KO11

Corn cob hydrolysates, with xylose as the dominant sugar, were fermented to ethanol by recombinant Escherichia coli KO11. When inoculum was grown on LB medium containing glucose, fermentation of the hydrolysate was completed in 163 h and ethanol yield was 0.50 g ethanol/g sugar. When inoculum was grown on xylose, ethanol yield dropped, but fermentation was faster (113 h). Hydrolysate containing 72.0 g/l xylose and supplemented with 20.0 g/l rice bran was readily fermented, producing 36.0 g/l ethanol within 70 h. Maximum ethanol concentrations were not higher for fermentations using higher cellular concentration inocula. A simulation of an industrial process integrating pentose fermentation by E. coli and hexose fermentation by yeast was carried out. At the first step, E. coli fermented the hydrolysate containing 85.0 g/l xylose, producing 40.0 g/l ethanol in 94 h. Baker's yeast and sucrose (150.0 g/l) were then added to the spent fermentation broth. After 8 h of yeast fermentation, the ethanol concentration reached 104.0 g/l. This two-stage fermentation can render the bioconversion of lignocellulose to ethanol more attractive due to increased final alcohol concentration. Journal of Industrial Microbiology & Biotechnology (2002) 29, 124–128 doi:10.1038/sj.jim.7000287 Received 20 February 2002/ Accepted in revised form 04 June 2002     

Effects of Inoculum Size on Solid-Phase Fermentation of Fodder Beets for Fuel Ethanol Production

This fuel ethanol study examined the effects of Saccharomyces cerevisiae inoculum size on solid-phase fermentation of fodder beet pulp. A 5% inoculum (wt/wt) resulted in rapid yeast and ethanol (9.1% [vol/vol]) production. Higher inocula showed no advantages. Lower inocula resulted in lowered final yeast populations and increased fermentation times.     

Use of disposable reactors to generate inoculum cultures for E. coli production fermentations

Disposable technology is being used more each year in the biotechnology industry. Disposable bioreactors allow one to avoid expenses associated with cleaning, assembly and operations, as well as equipment validation. The WAVE bioreactor is well established for Chinese Hamster Ovary (CHO) production, however, it has not yet been thoroughly tested for E. coli production because of the high oxygen demand and temperature maintenance requirements of that platform. The objective of this study is to establish a robust process to generate inoculum for E. coli production fermentations in a WAVE bioreactor. We opted not to evaluate the WAVE system for production cultures because of the high cell densities required in our current E. coli production processes. Instead, the WAVE bioreactor 20/50 system was evaluated at laboratory scale (10‐L) to generate inoculum with target optical densities (OD₅₅₀) of 15 within 7–9 h (pre‐established target for stainless steel fermentors). The maximum settings for rock rate (40 rpm) and angle (10.5) were used to maximize mass transfer. The gas feed was also supplemented with additional oxygen to meet the high respiratory demand of the culture. The results showed that the growth profiles for the inoculum cultures were similar to those obtained from conventional stainless steel fermentors. These inoculum cultures were subsequently inoculated into 10‐L working volume stainless steel fermentors to evaluate the inocula performance of two different production systems during recombinant protein production. The results of these production cultures using WAVE inocula showed that the growth and recombinant protein production was comparable to the control data set. Furthermore, an economic analysis showed that the WAVE system would require less capital investment for installation and operating expenses would be less than traditional stainless steel systems. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Aflatoxin B1 and fumosin B1 in mixed cultures of Aspergillus flavus and Fusarium proliferatum on maize

Production of aflatoxin B1 and fumonisin B1 in pure and mixed cultures of Aspergillus flavus and Fusarium proliferatum were determined on irradiated maize seeds inoculated with different spore concentrations at 0.97 water activity (a(w)) and a temperature of 25 degrees C. The highest levels of aflatoxin B1 were produced by A. flavus at the lowest levels of inoculum (10(3) spore ml(-1)). There was no spore concentration influence on fumonisin B1 production after 10, 20 and 35 days of incubation. When A. flavus was co-inoculated with F. proliferatum, aflatoxin B1 production was inhibited. The higher the inocula levels of Fusarium produced, the higher the inhibition and this inhibition increased during the incubation period. Total inhibition was reached at 35 days of incubation. There was no interaction influence on fumonisin B1 production at all inoculum levels assayed. These results suggest that under optimal environmental conditions of substrate, water activity and temperature, the interaction between A. flavus and F proliferatum could produce inhibition of aflatoxin B1 and stimulation of fumonisin B1.     

Enhancement of Cephalosporin C production by cultivation of Cephalosporium acremonium M25 using a mixture of inocula

AIMS: To enhance the productivity of Cephalosporin C (CPC) by cultivation of Cephalosporium acremonium M25 using a mixture of inocula. METHODS AND RESULTS: Inoculum age was classified into three stages (early, intermediate and late) by image analysis. A mixture of inocula, according to the inoculum ages, was used for efficient production of CPC in the main culture. The most effective mixing ratio of inocula for CPC production in shake flasks was a 3 : 7 volume ratio of early- and late-stage inocula. This was also the case in a 1.5 l stirred-tank reactor. CPC productivity was enhanced by about 32% and 34% when using an inoculum mixture in the shake flask and 1.5 l stirred-tank reactor, respectively. CONCLUSION: The morphological characteristics of C. acremonium M25 in the seed culture were quite different according to inoculum age. The compromise of different ages of inoculum showed better production of CPC. Significance and Impact of the Study: The productivity of CPC was enhanced considerably when using mixed inocula. The results of this study can be applied to fungal cultures for efficient production of various metabolites.     

Survival of Listeria monocytogenes in yogurt during storage at 4°C

Cows' milk was inoculated with ca 10³and 10⁷cfu/ml Listeria monocytogenes. After fermentation at 42°C for 0–5 h, the yogurt was stored at 4°C. Low and high inocula survived for 48 h and 7 d, respectively; L. monocytogenes cells were not detectable by direct plating or cold-enrichment after 5 and 15 d, respectively. In low inoculum samples, initial pH at the time of refrigeration was 4·9; the final pH at the time of last sampling was 4·2. In the samples with high inoculum the pH decreased from 5·0 to 4·2.     

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.     

Fermentation of cellodextrins to ethanol using mixed-culture fermentations

The potential for enhancing ethanol production from cellodextrins by employing mixed-culture (Candida wickerhamii-Saccharomyces cerevisiae) fermentations was investigated. Initially, ethanol production was monitored in fermentation medium containing 50 g/L glucose plus 45 g/L cellobiose. Inoculum levels and times of inoculum addition were varied. Of the conditions tested, the most rapid rates of ethanol formation occurred in fermentations in which either C. wickerhamii and S. cerevisiae were coinoculated at a ratio of 57 : 1 cell/mL or in fermentations in which a 10-fold-greater S. cerevisiae inoculum was added to a pure culture C. wickerhamii fermentation after 1 day incubation. These conditions were used to attempt to enhance fermentations in which cellodextrins produced by trifluoroacetic acid hydrolysis of cellulose served as the sole carbon source. Cellodextrins that were not further purified after cellulose hydrolysis contained compounds that were slightly inhibitory to C. wickerhamii. In this case the mixed-culture fermentations produced 12-45% more ethanol than a pure culture C. wickerhamii fermentation. However, if the substrate was treated with Darco G-60 charcoal, the toxic materials were apparently removed and the pure culture C. wickerhamii fermentations performed as well as the mixed-culture fermentations.     

Effect of different heterogeneous inocula in acidogenic fermentation of whey permeate

Summary The effect of different heterogeneous mixed bacterial inocula has been investigated on the acidogenic fermentation of cheese whey permeate. After a few months of continuous cultivation at pH 6 and at a retention time of 40 h, all mixed bacterial populations produced large amounts of lactate and butyrate, whereas the production of ethanol and acetate was largely depending on the source of the inoculum.     

Optimization of nutritional requirements and feeding strategies for clavulanic acid production by Streptomyces clavuligerus

The present work reports the nutritional requirements and environmental conditions for submerged culture of Streptomyces clavuligerus for clavulanic acid production using orthogonal matrix method (Taguchi L(16) design) and also fed-batch fermentation for clavulanic acid production by feeding glycerol, arginine and threonoine to the fermentation medium intermittently. Clavulanic acid production was increased by 18% with the span of feeding glycerol and reached a maximum at 1.30mg/ml with 120h glycerol feeding as compared to 1.10mg/ml in the control. The production also increased with the span of feeding amino acids and reached a maximum of 1.31 and 1.86mg/ml with feeding arginine and threonine, respectively in 120h. There was an overall increase of 18% and 9% in clavulanic acid production with arginine and threonine feeding as compared to the respective controls (1.10 and 1.70mg/ml, respectively).