The Composition of Jerusalem Artichoke (Helianthus tuberosus L.) Spirits Obtained from Fermentation with Bacteria and Yeasts

The composition of spirits distilled from fermentation of Jerusalem artichoke (Helianthus tuberosus L.) tubers was compared by means of gas chromatography. The microorganisms used in the fermentation processes were the bacterium Zymomonas mobilis, strains 3881 and 3883, the distillery yeast Saccharomyces cerevisiae, strains Bc16a and D₂ and the Kluyveromyces fragilis yeast with an active inulinase. The fermentation of mashed tubers was conducted using a single culture of the distillery yeast Saccharomyces cerevisiae and the bacterium Zymomonas mobilis (after acid or enzymatic hydrolysis) as well as Kluyveromyces fragilis (sterilized mashed tubers). The tubers were simultaneously fermented by mixed cultures of the bacterium or the distillery yeast with K. fragilis. The highest ethanol yield was achieved when Z. mobilis 3881 with a yeast demonstrating inulinase activity was applied. The yield reached 94 % of the theoretical value. It was found that the distillates resulting from the fermentation of mixed cultures were characterized by a relatively lower amount of by‐products compared to the distillates resulting from the single species process. Ester production of 0.30–2.93 g/L, responsible for the aromatic quality of the spirits, was noticed when K. fragilis was applied for ethanol fermentation both in a single culture process and also in the mixed fermentation with the bacterium. Yeast applied in this study caused the formation of higher alcohols to concentrations of 7.04 g/L much greater than those obtained with the bacterium. The concentrations of compounds other than ethanol obtained from Jerusalem artichoke mashed tubers, which were fermented by Z. mobilis, were lower than those achieved for yeasts.     

Exopolysaccharides produced by mixed culture of yeast Rhodotorula rubra GED10 and yogurt bacteria (Streptococcus thermophilus 13a + Lactobacillus bulgaricus 2-11)

AIMs: The studies of the production of exopolysaccharides by lactose-negative yeast and a yogurt starter co-cultivated in a natural substrate containing lactose may be considered of interest because they reveal the possibilities for high-efficiency synthesis of biopolymers by mixed cultivation. METHODS AND RESULTS: The mixed culture Rhodotorula rubra GED10 + (Streptococcus thermophilus 13a + Lactobacillus bulgaricus 2-11) was cultivated in cheese whey ultrafiltrate (WU) (44.0 g lactose l(-1)) at initial pH 6.0, 28 degrees C, under intensive aeration (air-flow rate 1.0 l l(-1) min(-1), agitation 220 rev min(-1)) in a MBR AG fermentor. The mixed culture manifested the highest activity for synthesis of exopolysaccharides (19.3 g l(-1)) and cell mass (21.0 g l(-1)) at the 84th hour. The yogurt starter synthesized neutral exopolysaccharides, while the mixed culture yeast + yogurt starter produced acidic exopolysaccharides containing uronic acid (6%). The neutral sugar composition was identified as mannose, glucose, galactose, xylose and arabinose. Mannose dominated in the polymer composition (83%) that was produced only by the yeast (97%). CONCLUSIONS: Lactose in the WU can be effectively utilized by a co-culture of lactose-negative yeast-yogurt starter for synthesis of exopolysaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: The present findings propose an alternative use of WU as a cost-effective carbohydrate substrate, and suggest that the lactose-negative yeast Rhodotorula rubra can have industrial application as producers of exopolysaccharides.     

Monoxenic liquid culture of the entomopathogenic nematode Steinernema carpocapsae using a culture medium containing whey kinetics and modeling

The submerged culture of the entomopathogenic nematode Steinernema carpocapsae and its symbiotic bacterium, Xenorhabdus nematophila, was carried out in orbitally agitated bottles using a culture medium containing whey (in grams per litre: 500 whey, 20 yeast extract, 10 dried egg yolk-food grade, 3 sodium chloride, 37 corn oil-food grade). Maximum total viable nematode concentrations of 198,333ml(-1) were achieved within fermentations of 24 days with 64% of the nematode population within the infective juvenile stage (IJ) (126,666ml(-1)) at the end. The kinetics of the bioprocess was well modelled using the four-parameter Sigmoidal model and the corresponding maximum specific rates of nematode production (0.47 day(-1)), carbohydrates consumption (0.0008g(carbohydrates)g(nematodes)(-1)day(-1)) and nitrogen consumption (4.44g(nitrogen)g(nematodes)(-1)day(-1)) are first proposed. Besides, X. nematophila appears to have the capacity of lactose hydrolysis.     

Bacterial degradation of emulsan.

Emulsan is a polyanionic heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. A mixed bacterial population was obtained by enrichment culture that was capable of degrading emulsan and using it as a carbon source. From this mixed culture, an emulsan-degrading bacterium, termed YUV-1, was isolated. Strain YUV-1 is an aerobic, gram-negative, non-spore-forming, rod-shaped bacterium which grows best in media containing yeast extract. When placed on preformed lawns of A. calcoaceticus RAG-1, strain YUV-1 produced translucent plaques which grew in size until the entire plate was covered. Plaque formation was due to solubilization of the emulsan capsule of RAG-1. Plaque formation was not observed on emulsan-negative mutants of RAG-1. As a consequence of the solubilization of the emulsan capsule, RAG-1 cells became more hydrophobic, as determined by adherence to hexadecane. Growth of YUV-1 on a medium containing yeast extract and emulsan was biphasic. During the initial 24 h, cell concentration increased 10-fold, but emulsan was not degraded; during the lag in growth (24 to 48 h), emulsan was inactivated and depolymerized but not consumed; during the second growth phase (48 to 70 h) the depolymerized emulsan products were consumed.     

Bacterial degradation of emulsan

Emulsan is a polyanionic heteropolysaccharide bioemulsifier produced by Acinetobacter calcoaceticus RAG-1. A mixed bacterial population was obtained by enrichment culture that was capable of degrading emulsan and using it as a carbon source. From this mixed culture, an emulsan-degrading bacterium, termed YUV-1, was isolated. Strain YUV-1 is an aerobic, gram-negative, non-spore-forming, rod-shaped bacterium which grows best in media containing yeast extract. When placed on preformed lawns of A. calcoaceticus RAG-1, strain YUV-1 produced translucent plaques which grew in size until the entire plate was covered. Plaque formation was due to solubilization of the emulsan capsule of RAG-1. Plaque formation was not observed on emulsan-negative mutants of RAG-1. As a consequence of the solubilization of the emulsan capsule, RAG-1 cells became more hydrophobic, as determined by adherence to hexadecane. Growth of YUV-1 on a medium containing yeast extract and emulsan was biphasic. During the initial 24 h, cell concentration increased 10-fold, but emulsan was not degraded; during the lag in growth (24 to 48 h), emulsan was inactivated and depolymerized but not consumed; during the second growth phase (48 to 70 h) the depolymerized emulsan products were consumed.     

Preparation and performance of immobilized yeast cells in columns containing no inert carrier

Schizosaccharomyces pombe was cultivated in a medium of glucose (10 g/L) malt extract (3 g/L), yeast extract (3 g/L), and bactopeptone (5 g/L) to form flocs. More than 95% of the cell population were flocculated. Variation in glucose concentration (from 10 to 100 g/L) did not affect flocculation. Yeast extract helped induce flocculation. Application of the immobilized yeast for the continuous production of ethanol was tested in a column reactor. Soft yeast flocs (50-200 mesh) underwent morphological changes to heavy particles (0.1-0.3 cm diameter) after continuously being fed with fresh substrates in the column. Productivity as high as 87 g EtOH L(-1) h(-1) was obtained when a 150 g/L glucose medium was fed. The performance of this yeast reactor was stable over a two-month period. The ethanol yield was 97% of the theoretical maximum based upon glucose consumed.     

Decolorization of azo dyes by Shewanella sp. under saline conditions

Wastewaters from textile processing and dye-stuff manufacture industries contain substantial amounts of salts in addition to azo dye residues. To examine salinity effects on dye-degrading bacteria, a study was carried out with four azo dyes in the presence of varying concentrations of NaCl (0-100 g l(-1)) with a previously isolated bacterium, Shewanella putrefaciens strain AS96. Under static, low oxygen conditions, the bacterium decolorized 100 mg dye l(-1) at salt concentrations up to 60 g NaCl l(-1). There was an inverse relationship between the velocity of the decolorization reaction and salt concentration over the range between 5 and 60 g NaCl l(-1) and at dye concentrations between 100 and 500 mg l(-1). The addition of either glucose (C source) or NH(4)NO(3) (N source) to the medium strongly inhibited the decolorization process, while yeast extract (4 g l(-1)) and Ca(H(2)PO(4))(2).H(2)O (1 g l(-1)) both enhanced decolorization rates. High-performance liquid chromatography analysis demonstrated the presence of 1-amino-2-naphthol, sulfanilic acid and nitroaniline as the major metabolic products of the azo dyes, which could be further degraded by a shift to aerobic conditions. These findings show that Shewanella could be effective for the treatment of dye-containing industrial effluents containing high concentrations of salt.     

Isolation of a β-Carotene Over-Producing Soil Bacterium, Sphingomonas sp.

A carotenoid-accumulating bacterium isolated from soil, identified as a Sphingomonas sp., grew at 0.18 h(-1) and produced 1.7 mg carotenoids g(-1) dry cell, among which beta-carotene (29% of total carotenoids) and nostoxanthin (36%). A mutant strain, obtained by treatment with ethyl methanesulfonate, accumulated up to 3.5 mg carotenoids g(-1) dry cell. Accumulation of beta-carotene by this strain depended on the oxygenation of the growth medium, with maximal accumulation (89%) occurring under limiting conditions. Beta-carotene accumulation could be further enhanced by incubating the cells in the presence of glycerol (either not or only slowly assimilated) and yeast extract resulting in an accumulation of 5.7 mg beta-carotene g(-1) dry cell wt. The strain used lactose as carbon source with similar biomass and carotenoid production, providing a viable alternative use for cheese whey ultra-filtrate.     

Mixed culture of killer and sensitive Saccharomyces cerevisiae strains in batch and continuous fermentations

This paper presents a kinetic study of the dynamics of the population of two Saccharomyces cerevisiae strains (designated K1 and 522D) in mixed culture. These two strains are commonly used in wine making. The K1 strain (killer yeast) secretes a glycoprotein (killer toxin) which causes the death of the 522D strain (sensitive yeast). Initially, the mixed cultures were realized in batch fermentations. Initial concentrations of killer yeast were 5 and 10% of the total population. The influence of the killer strain on the sensitive cultures was measured in comparison with a reference fermentation. The reference fermentation was inoculated only with the sensitive strain. Results show that an initial concentration of 10% of killer strain affects the microbial population balance and the rate of ethanol production. However the fermentation was only slightly disturbed when the proportion of killer to sensitive yeast at the beginning of mixed culture was 5%. To achieve total displacement by the killer yeast at low concentrations, the mixed cultures were carried out in a continuous system. The results obtained in continuous fermentations with the same strains have shown that a level of contamination as low as 0.8% of killer strain was sufficient to completely displace the original sensitive population after 150 h incubation.     

Mixed culture of killer and sensitive Saccharomyces cerevisiae strains in batch and continuous fermentations

This paper presents a kinetic study of the dynamics of the population of two Saccharomyces cerevisiae strains (designated K1 and 522D) in mixed culture. These two strains are commonly used in wine making. The K1 strain (killer yeast) secretes a glycoprotein (killer toxin) which causes the death of the 522D strain (sensitive yeast). Initially, the mixed cultures were realized in batch fermentations. Initial concentrations of killer yeast were 5 and 10% of the total population. The influence of the killer strain on the sensitive cultures was measured in comparison with a reference fermentation. The reference fermentation was inoculated only with the sensitive strain. Results show that an initial concentration of 10% of killer strain affects the microbial population balance and the rate of ethanol production. However the fermentation was only slightly disturbed when the proportion of killer to sensitive yeast at the beginning of mixed culture was 5%. To achieve total displacement by the killer yeast at low concentrations, the mixed cultures were carried out in a continuous system. The results obtained in continuous fermentations with the same strains have shown that a level of contamination as low as 0.8% of killer strain was sufficient to completely displace the original sensitive population after 150 h incubation.     

Desulfacinum subterraneum sp.nov.--a new thermophilic sulfate-reducing bacterium isolated from a high temperature oil field

A new thermophilic sulfate-reducing bacterium isolated from the high-temperature White Tiger oil field (Vietnam) is described. Cells of the bacterium are oval (0.4-0.6 by 0.6-1.8 microns), nonmotile, non-spore-forming, and gram-negative. Growth occurs at 45 to 65 degrees C (with an optimum at 60 degrees C) at NaCl concentrations of 0 to 50 g/l. In the course of sulfate reduction, the organism can utilize lactate, pyruvate, malate, fumarate, ethanol, salts of fatty acids (formate, acetate, propionate, butyrate, caproate, palmitate), yeast extract, alanine, serine, cysteine, and H2 + CO2 (autotrophically). In addition to sulfate, the bacterium can use sulfite, thiosulfate, and elemental sulfur as electron acceptors. In the absence of electron acceptors, the bacterium can ferment pyruvate and yeast extract (a yet unrecognized capacity of sulfate reducers) with the formation of acetate and H2. The G + C content of DNA is 60.8 mol %. The level of DNA-DNA hybridization of the isolate (strain 101T) and Desulfacinum infernum (strain B alpha G1T) is as low as 34%. Analysis of the nucleotide sequence of 16S rDNA places strain 101T in the phylogenetic cluster of the Desulfacinum species within the sulfate reducer subdivision of the delta subclass of Proteobacteria. All these results allowed the bacterium studied to be described as a new species, Desulfacinum subterraneum sp. nov., with strain 101 as the type strain.     

Anaerobic degradation of chlorophenol by an enrichment culture

Summary An anaerobic mixed culture from sewage sludge was enriched in a yeast extract and peptone-containing medium; it was able to degrade 2-cholorophenol completely to methane and CO₂. Degradation rates of 2-chlorophenol of up to 0.18 g/l per day were observed in suspended cultures without biomass retention and of 0.375 g/l per day in cultures immobilized on Liapor clay beads. Attempts to isolate the dechlorinating organism failed. The mixed culture was reduced to three morphologically distinctive microorganisms using a medium with limited amounts of yeast extract and peptone and n-butyrate as a co-substrate. Under these conditions the phenol-degrading bacterium was lost and phenol accumulated in the medium. No growth and no dehalogenation of 2-chlorophenol was obtained when yeast extract and peptone were omitted completely. Besides serving as a source of supplementary components, yeast extract and peptone were apparently required as the main source of carbon, wereas reducing equivalents for reductive dehalogenation were obtained by oxidation of n-butyrate. A spirochaete-like organism was presumably the dechlorinating bacterium. The mixed culture lost its dehalogenation capability if this organism was lost. n-Butyrate could be replaced by n-valerate, hexanoate, heptanoate, octanoate, pelargonic acid, n-decanoic acid or palmitate as co-substrates for dehalogenation of either 2-chlorophenol, 2-bromophenol or complete dechlorination of 2,6-dichlorophenol, whereas from 2,4-dichlorophenol only the substituent in the ortho-position could be eliminated.Dedicated to Professor O. Kandler on the occassion of his 70th birthdayOffprint requests to: J. Winter     

Ethanol production by immobilized Saccharomyces cerevisiae, Saccharomyces uvarum, and Zymomonas mobilis

Saccharomyces cerevisiae NRRL Y-2034, S, uvarum NRRL Y-1347, and Zymomonas mobilis NRRL B-806 each were separately immobilized in a Ca-alginate matrix and incubated in the presence of a free-flowing and continuous 1, 3, 5, 10, or 20% (w/w) glucose solution. In general, the yeast cells, converted 100percnt; of the 1, 3, and 5% glucose to alcohol within 48 h and maintained such a conversion rate for at least two weeks. The bacterium converted ca. 90% (w/w) of the 1, 3, and 5% glucose to alcohol continuously for one week. However, both the yeast and bacterium were inhibited in the highest glucose (20% w/w) solution. All of the immobilized cultures produced some alcohol for at least 14 days. Immobilized S. cerevisiae was the best alcohol producer of all of the glucose concentrations; the yeast yielded 4.7 g ethanol/100 g solution within 72 h in the 10% glucose solution. After 7-8 days in the 10% solution, S. cerevisiae produced ethanol at 100% of theoretical yield (5.0 g ethanol/100 g solution), with a gradual decrease in alcohol production by 14 days. Immobillized S. uvarum produced a maximum of 4.0 g ethanol/100 g solution within 2 days and then declined to ca. 1.0 g ethanol/100 g solution after 7 days continuous fermentation in the 10% glucose solution. Zymomonas mobilis reached its maximum ethanol production at 4 days (4.7 g/100 g solution), and then diminished similarly to S. uvarum. The development of a multiple disk shaft eliminated the problem both of uneven distribution of alginate-encapsulated cells and of glucose channeling within the continuous-flow fermentor column. This invention improved alcohol production about threefold for the yeast cells.     

Measuring growth of a phenanthrene-degrading bacterial inoculum in soil with a quantitative competitive polymerase chain reaction method

We measured growth of a phenanthrene-degrading bacterium, Arthrobacter, strain RP17, in Forbes soil, amended with 500 μg g(-1) phenanthrene using a quantitative competitive polymerase chain reaction method. The inoculum, which was not indigenous to Forbes soil, grew from 5.55x10(5) colony forming units (cfu) g(-1) to 1.97x10(7) cfu g(-1) within 100 h after the cells were added to the soil. Maximum population density was reached before the highest degradation rate was observed 150 h after the cells were added to soil. Population density remained stable even after 56% of the phenanthrene had mineralized. This study is one of the few documented examples of growth by a non-indigenous bacterium in a non-sterile soil amended with a pollutant.     

Association of bacteria and yeasts in hot springs.

The thermophilic bacterium Bacillus sp. strain TB-1 was isolated in association with the yeast Debaryomyces vanriji from hot springs at 46 degrees C. It was shown that TB-1 excreted thiamine into the culture broth, which not only promoted D. vanriji growth in mixed culture but also increased the maximal temperature for yeast growth.     

Influence of culture medium composition, dissolved oxygen concentration and harvesting time on the production of Serratia entomophila, a microbial control agent of the New Zealand grass grub

The bacterium Serratia entomophila (Enterobacteriaceae) has been developed as a commercially available biopesticide for control of the pasture pest Costelytra zealandica. The influence of culture medium composition, dissolved oxygen (DO) concentration and harvesting time were investigated in order to optimise the production of S. entomophila. In batch fermentations, highest yields were achieved using sucrose (40 g L^-1) as the carbon source, followed closely by fructose and molasses. The effect of yeast extract (YE), marmite and bakery yeast as cell growth enhancers was also examined in both batch and fed-batch mode. Culture medium containing 20 g L^-1 of YE (fed-batch) produced the highest cell density. No significant effect on cell yield was detected when cultures were supplemented with bakery yeast or marmite. The DO concentration influenced biomass production: a 5-fold increase in cell density was achieved when the concentration of DO was maintained in the range of 20-50% (5.7×10¹⁰ CFUs mL^-1) in comparison with 1% (1.2×10¹⁰ CFUs mL^-1). In cultures maintained at 1 and 20% DO concentration, cells harvested from the exponential growth phase survived for less than 2 weeks when stored at 4°C. In contrast, high cell survival (85-100%) was achieved when cells were harvested after they had entered the stationary growth phase. Recommendations are provided for the production of robust, high cell density cultures of S. entomophila.     

Synergistic action of both Aspergillus niger and Burkholderia cepacea in co-culture increases phosphate solubilization in growth medium

Co-inoculation of the fungus Aspergillus niger and the bacterium Burkholderia cepacia was undertaken to understand the interaction between different species of phosphate-solubilizing microorganisms (PSM). PSM were inoculated in a single or mixed (A. niger-B. cepacia) culture. During 9 days of incubation, microbial biomass was enhanced, accompanied with increases in the levels of soluble phosphate and titratable acidity, as well as increased acid phosphatase activity. Production of acids and levels of phosphate solubilization were greater in the co-culture of A. niger-B. cepacia than in the single culture. The quantity of phosphate solubilized by the co-culture ranged from 40.51 ± 0.60 to 1103.64 ± 1.21 μg PO(4) 3- mL(-1) and was 9-22% higher than single cultures. pH of the medium dropped from 7.0 to 3.0 in the A. niger culture, 3.1 in the co-culture, and 4.2 in the B. cepacia culture. On the third day of postinoculation, acid production by the co-culture (mean 5.40 ± 0.31 mg NaOH mL(-1)) was 19-90% greater than single cultures. Glucose concentration decreased almost completely (97-99% of the starting concentration) by the ninth day of the incubation. These results show remarkable synergism by the co-culture in comparison with single cultures in the solubility of CaHPO(4) under in vitro conditions. This synergy between microorganisms can be used in poor available phosphate soils to enhance phosphate solubilization.     

Simultaneous saccharification and L-(+)-lactic acid fermentation of protease-treated wheat bran using mixed culture of lactobacilli

Protease-treated wheat bran (20% w/v) of particle size less than 300 μm containing 65% (w/w) starch was used for the simultaneous saccharification and l-(+)-lactic acid fermentation by the mixed cultures of Lactobacillus casei and Lactobacillus delbrueckii. Maximum lactate yield after various process optimizations was 123 gl⁻¹ with a productivity of 2.3 gl⁻¹ h⁻¹ corresponding to a conversion of 0.95 g lactic acid per gram starch after 54 h at 37°C. By using protease-treated wheat bran around tenfold decrease in supplementation of the costly medium component, like yeast extract, was achieved together with a considerable increase in the production level.     

Production of a novel transfructosylating enzyme from Bacillus macerans EG-6

A new bacterium producing a novel transfructosylating enzyme was isolated from soil and designated as Bacillus macerans EG-6. Various culture conditions for enzyme production were optimized in a flask culture. 1% (w/v) sucrose as a carbon source and a mixed nitrogen source (1% yeast extract, 1% polypeptone, and 0.5% ammonium chloride) gave the best enzyme production. Addition of phosphate and magnesium ion into the medium enhanced the enzyme yield. Optimum culture pH and temperature were 7.0 and 37?°C, respectively. Under optimal culture conditions, transfructosylating enzyme was rapidly produced in the early growth period, thereafter invertase activity was predominant as the culture proceeded. Using the culture filtrate, production of fructooligosaccharides from sucrose was preliminarily carried out. In a low sucrose concentration (200?g/l), transfructosylating activity competes with invertase activity in sucrose utilization. Subsequently, low fructooligosaccharide yield (20%) was achieved due to liberation of high amounts of glucose and fructose. The best oligosaccharide yield (43%) was achieved when 500?g/l sucrose was utilized.     

Very high expression of an anti-carcinoembryonic antigen single chain Fv antibody fragment in the yeast Pichia pastoris

In this paper we report the development of a recombinant strain of the yeast Pichia pastoris, which secretes an anti-carcinoembryonic antigen single chain Fv (scFv) antibody fragment to the culture supernatant as a biologically active protein, at levels of 1.2 g l(-1). The yeast scFv was purified by IMAC, with a final yield of approximately 0.440 g of 93% pure scFv per liter of culture supernatant. The specific activity in ELISA of the yeast scFv was almost three times higher than that of a bacterial periplasmic counterpart. These results reaffirm that the yeast P. pastoris is a suitable host for high level production of scFv antibody fragments with potential in vivo diagnostic and therapeutic applications.