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Optimization of the culture medium for growth and the kinetics of lactate fermentation by Pediococcus sp. ISK-1.

The growth of a newly isolated strain of Pediococcus sp., designated ISK-1, was very slow and the concentration of cells in the medium remained low. Fermentation with an initial 30 g/liter glucose required about 60 h. To stimulate fermentation, we attempted to optimize the medium by flask culture and jar fermentation tests. Mevalonic acid and mieki (soy bean hydrolyzate) stimulated fermentation and increased the rate of formation of DL-lactate. Kinetic analysis of the fermentation showed that mevalonic acid markedly increased the specific glucose consumption rate and the specific lactate production rate. Mieki and mevalonic acid had a synergistic effect, but the effect of mevalonic acid was different from that of mieki.

Cell Division↗

Isolation and characterization of thermotolerant Gluconobacter strains catalyzing oxidative fermentation at higher temperatures.

Thermotolerant acetic acid bacteria belonging to the genus Gluconobacter were isolated from various kinds of fruits and flowers from Thailand and Japan. The screening strategy was built up to exclude Acetobacter strains by adding gluconic acid to a culture medium in the presence of 1% D-sorbitol or 1% D-mannitol. Eight strains of thermotolerant Gluconobacter were isolated and screened for D-fructose and L-sorbose production. They grew at wide range of temperatures from 10 degrees C to 37 degrees C and had average optimum growth temperature between 30-33 degrees C. All strains were able to produce L-sorbose and D-fructose at higher temperatures such as 37 degrees C. The 16S rRNA sequences analysis showed that the isolated strains were almost identical to G. frateurii with scores of 99.36-99.79%. Among these eight strains, especially strains CHM16 and CHM54 had high oxidase activity for D-mannitol and D-sorbitol, converting it to D-fructose and L-sorbose at 37 degrees C, respectively. Sugar alcohols oxidation proceeded without a lag time, but Gluconobacter frateurii IFO 3264T was unable to do such fermentation at 37 degrees C. Fermentation efficiency and fermentation rate of the strains CHM16 and CHM54 were quite high and they rapidly oxidized D-mannitol and D-sorbitol to D-fructose and L-sorbose at almost 100% within 24 h at 30 degrees C. Even oxidative fermentation of D-fructose done at 37 degrees C, the strain CHM16 still accumulated D-fructose at 80% within 24 h. The efficiency of L-sorbose fermentation by the strain CHM54 at 37 degrees C was superior to that observed at 30 degrees C. Thus, the eight strains were finally classified as thermotolerant members of G. frateurii.

Catalysis↗

Effects of fermentation temperature on the content and composition of isoflavones and beta-glucosidase activity in sufu.

Sufu is a popular fermented tofu product in China. The low quality of sufu produced in the hot summer is a big problem in sufu manufacture, so we prepared sufu at two different temperatures, 26 degrees C as normal and 32 degrees C as high temperature, and the effects of temperature on isoflavones and beta-glucosidase activity were investigated. Fermentation temperature did not cause significant differences in the recovery of isoflavones, but resulted in a different redistribution of isoflavone isomers in sufu. Sufu fermented at 26 degrees C was richer in isoflavone aglycones than at 32 degrees C; the enrichment of isoflavone aglycones might have the advantage of enhancing the physiological function. No 6''-O-malonyl-glucosides were detected in sufu fermented at 26 degrees C, whereas some 6''-O-malonyl-glucosides were found at 32 degrees C. A fermentation temperature of 26 degrees C benefited the beta-glucosidase production by fungi, which contributed to valid conversion from beta-glucosides to aglycones. It was also found that beta-glucosidase converted beta-glucosides more effectively than 6''-O-malonyl-glucosides and 6''-O-acetyl-glucosides into aglycones.

Fermentation↗

Conversion of municipal solid waste into carboxylic acids by anaerobic countercurrent fermentation: effect of using intermediate lime treatment.

Municipal solid waste (MSW) and sewage sludge (SS) were combined and anaerobically converted into carboxylate salts by using a mixed culture of acid-forming microorganisms. MSW is an energy source and SS is a source of nutrients. In this study, MSW and SS were combined, so they complemented each other. Four fermentors were arranged in series for a countercurrent fermentation process. In this process, the solids and liquid were transferred in opposite directions, with the addition of fresh biomass to fermentor 1 and fresh liquid media to fermentor 4. An intermediate lime treatment of solids exiting fermentor 3 before entering fermentor 4 was applied to improve the product acid concentration from the untreated MSW/SS fermentations. All fermentations were performed under anaerobic conditions at 40 degrees C. Calcium carbonate was added to neutralize the carboxylic acids and to control the pH. Iodoform was used as a methanogen inhibitor. Carboxylic acid concentration and gas composition were determined by gas chromatography. Substrate conversion was measured by volatile solids loss, and carboxylic acid productivity was calculated as the function of the total carboxylic acids produced, the amount of liquid in all fermentors, and time. The addition of intermediate lime treatment increased product concentration and conversion by approx 30 and 15%, respectively. The highest carboxylic acid concentrations for untreated MSW/SS fermentations with and without intermediate lime treatment were 22.2 and 17.7 g of carboxylic acid/L of liquid, respectively. These results confirm that adding a treatment step between fermentor 3 and fermentor 4 will increase the digestibility and acid productivity of the fermentation.

Anaerobiosis↗

Inhibition of microbial xylitol production by acetic acid and its relation with fermentative parameters.

Precipitated sugarcane bagasse hemicellulosic hydrolysate containing acetic acid was fermented by Candida guilliermondii FTI20037 under different operational conditions (pH 4.0 and 7.0, three aeration rates). At pH 7.0 and kLa of 10 (0.75 vvm) and 22.5/h (3.0 vvm) the acetic acid had not been consumed until the end of the fermentations, whereas at the same pH and kLa of 35/h (4.5 vvm) the acid was rapidly consumed and acetic acid inhibition was not important. On the other hand, fermentations at an initial pH of 4.0 and kLa of 22.5 and 35/h required less time for the acid uptake than fermentations at kLa of 10/h. The acetic acid assimilation by the yeast indicates the ability of this strain to ferment in partially detoxified medium, making possible the utilization of the sugarcane bagasse hydrolysate in this bio-process. The effects on xylitol yield and production are reported.

Acetic Acid↗

Comparative ethanol productivities of different Zymomonas recombinants fermenting oat hull hydrolysate.

Iogen Corporation of Ottawa, Canada, has recently built a 50 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. Iogen has partnered with the University of Toronto to test the C6/C5 cofermentation performance characteristics of National Renewable Energy Laboratory's metabolically engineered Zymomonas mobilis using its biomass hydrolysates. In this study, the biomass feedstock was an agricultural waste, namely oat hulls, which was hydrolyzed in a proprietary two-stage process involving pretreatment with dilute sulfuric acid at 200-250 degrees C, followed by cellulase hydrolysis. The oat hull hydrolysate (OHH) contained glucose, xylose, and arabinose in a mass ratio of about 8:3:0.5. Fermentation media, prepared from diluted hydrolysate, were nutritionally amended with 2.5 mL/L of corn steep liquor (50% solids) and 1.2 g/L of diammonium phosphate. The estimated cost for large-scale ethanol production using this minimal level of nutrient supplementation was 4.4cents/gal of ethanol. This work examined the growth and fermentation performance of xylose-utilizing, tetracycline-resistant, plasmid-bearing, patented, recombinant Z. mobilis cultures: CP4:pZB5, ZM4:pZB5, 39676:pZB4L, and a hardwood prehydrolysate-adapted variant of 39676:pZB4L (designated as the "adapted" strain). In pH-stat batch fermentations with unconditioned OHH containing 6% (w/v) glucose, 3% xylose, and 0.75% acetic acid, rec Zm ZM4:pZB5 gave the best performance with a fermentation time of 30 h, followed by CP4:pZB5 at 48 h, with corresponding volumetric productivities of 1.4 and 0.89 g/ (L x h), respectively. Based on the available glucose and xylose, the process ethanol yield for both strains was 0.47 g/g (92% conversion efficiency). At 48 h, the process yield for rec Zm 39676:pZB4L and the adapted strain was 0.32 and 0.34 g/g, respectively. None of the test strains was able to ferment arabinose. Acetic acid tolerance appeared to be a major determining factor in cofermentation performance.

Acetic Acid↗

Fermentation and costs of fuel ethanol from corn with Quick-Germ process.

The Quick-Germ process developed at the University of Illinois at Urbana-Champaign is a way to obtain corn oil, but with lower capital costs than the traditional wet-milling process. Quick-Germ has the potential to increase the coproduct credits and profitability of the existing dry-grind fuel ethanol process, but the fermentability of the corn remaining after oil recovery has not been tested. Therefore, a series of pilot scale (50 L) fermentations was carefully controlled and monitored with unique methods for standard inoculation and automatic sampling. It was found that the concentration of suspended solids was significantly reduced in the Quick-Germ fermentations. When compared at the same concentration of fermentable sugars, the fermentation rate and yield were not statistically different from controls. When Quick-Germ was integrated into a state-of-the-art dry-grind fuel ethanol process, computer simulation and cost models indicated savings of approx $0.01/L of ethanol ($0.04/gal) with the Quick-Germ process. Additional savings associated with the lower suspended solids could not be quantified and were not included. However, the savings are sensitive to the price of corn oil.

Biotechnology↗

Laboratory method for high-solids countercurrent fermentations.

Equipment and procedures were developed to study the conversion of lignocellulosic biomass to carboxylic acids using high-solids countercurrent fermentations. Countercurrent fermentations of cattle manure yielded a rapid fermentation (maximum 2.98 g of total acid/[L x d]) with high acid concentrations (maximum of 32.5 g of total acid/L), but the acid yield tended to be low (maximum of 0.24 g of total acid/g of volatile solids). Countercurrent fermentations of a mixture of 80% municipal solid waste/20% sewage sludge fermented more slowly (maximum of 1.98 g of total acid/[L x d]) with a lower acid concentration (maximum of 26.5 g of total acids/L), but higher acid yields were achieved (maximum of 0.34 g of total acid/g of volatile solids).

Animals↗

Effects of a novel fermented soy product on the serum lipids of hypercholesterolemic rabbits.

OBJECTIVE: To assess the effect of a new feed soy product fermented by Enterococcus faecium and Lactobacillus jugurti on the serum lipid levels of rabbits with induced hypercholesterolemia. METHODS: Thirty-two rabbits were divided into 4 groups as follows: 1) control (C); 2) hypercholesterolemic (H); 3) hypercholesterolemic + fermented product (HPF); and 4) control + fermented product (CPF). The H and HPF groups were fed with a diet with 0.15% (p/p) cholesterol in the first 15 days. C and CPF groups received regular food preparation. The HPF and CPF groups received 10 mL daily of the fermented 30 days. Blood samples were drawn at the beginning of the study and at the 15th and 30th days. Concentrations of total cholesterol, HDL-cholesterol, and triglycerides were analyzed. RESULTS: After 15 days, the HPF group showed a total cholesterol concentration lower (18.4%) than that of the H group (p = 0.05), but this difference disappeared after 30 days. No change was observed in total cholesterol levels of C and CPF groups. After 15 days, the HDL-cholesterol was higher (17.8%) in the HPF group, but the triglyceride levels remained unchanged in all groups during the same period of time. CONCLUSION: The soy fermented product caused an 18.4% reduction in total cholesterol and a 17.8% increase in the HDL-fraction. It may, therefore, be a possible coadjutor in the treatment of hypercholesterolemia.

Animals↗

Application technique and slurry co-fermentation effects on ammonia, nitrous oxide, and methane emissions after spreading: II. Greenhouse gas emissions.

The aim of this study was to investigate the effect of different application techniques on greenhouse gas emission from co-fermented slurry. Ammonia (NH3), nitrous oxide (N2O), and methane (CH4) emissions were measured in two field experiments with four different application techniques on arable and grassland sites. To gather information about fermentation effects, unfermented slurry was also tested, but with trail hose application only. Co-fermented slurry was applied in April at a rate of 30 m3 ha(-1). Measurements were made every 4 h on the first day after application and were continued for 6 wk with gradually decreasing sampling frequency. Methane emissions were <150 g C ha(-1) from co-fermentation products and seemed to result from dissolved CH4. Only in the grassland experiment were emissions from unfermented slurry significantly higher, with wetter weather conditions probably promoting CH4 production. Nitrous oxide emission was significantly increased by injection on arable and grassland sites two- and threefold, respectively. Ammonia emissions were smallest after injection or trail shoe application and are discussed in the preceding paper. We evaluated the climatic relevance of the measured gas emissions from the different application techniques based on the comparison of CO2 equivalents. It was evident that NH3 emission reduction, which can be achieved by injection, is at least compensated by increased N2O emissions. Our results indicate that on arable land, trail hose application with immediate shallow incorporation, and on grassland, trail shoe application, bear the smallest risks of high greenhouse gas emissions when fertilizing with co-fermented slurry.

Ammonia↗

Identification of mesophilic and thermophilic fermentative species in anaerobic granular sludge.

Large quantities of biodegradable food waste in the form of fruit and vegetables are still being deposited in landfill sites in Ireland. The development of an anaerobic digestion process using fermentative species which degrade the carbohydrate-rich waste could divert the food waste from landfills. We identified fermentative species grown on glucose and sucrose at mesophilic and thermophilic temperatures using molecular biology techniques. The dominating fermentative bacteria of the mesophilic sludge were of the Bacteroidetes and Spirochaetes classes. Although both groups of bacteria are typically fermentative their substrate range appears to be limited. The dominating fermentative bacteria in the thermophilic sludge was Thermoanaerobacterium aotearoense of the Clostridia class. The indications are that Thermoanaerobacterium aotearoense may be highly suitable to biodegrade a carbohydrate-rich influent feed due to its possibly very rapid growth rate and also an extensive substrate range.

Anaerobiosis↗

Effect of fermentation system on the production and properties of tannase of Aspergillus niger van Tieghem MTCC 2425.

The tannase-producing efficiency of liquid-surface fermentation (LSF) and solid-state fermentation (SSF) vis-à-vis submerged fermentation (SmF) was investigated in a strain of Aspergillus niger, besides finding out if there was a change in the activity pattern of tannase in these fermentation processes. The studies on the physicochemical properties were confined to intracellular tannase as only this form of enzyme was produced by A. niger in all three fermentation processes. In LSF and SmF, the maximum production of tannase was observed by 120 h, whereas in SSF its activity peaked at 96 h of growth. SSF had the maximum efficiency of enzyme production. Tannase produced by the SmF, LSF and SSF processes had similar properties except that the one produced during SSF had a broader pH stability of 4.5-6.5 and thermostability of 20 degrees-60 degrees C.

Aspergillus niger↗

Fermentation of raw poultry byproducts for animal nutrition.

In this study, the fermentation of raw, inedible poultry byproducts mixed with sugarbeet pulp and dextrose and inoculated with Lactobacillus plantarum and(or) Enteroccocus faecium resulted in a drop of pH in the byproducts to approximately 4.0 to 4.5 within 48 h. To keep the fermented product stable for a period of 21 d, the addition of > or = 3% (wt/wt) of a fermentable carbohydrate was necessary. With a high inoculation level of approximately 10(8) to 10(9) L. plantarum per gram, or with acidification of the initial mixture with .4% lactic acid, the number of Enterobacteriaceae decreased faster than with inoculation at 10(5) L. plantarum per gram, or without initial acidification. After 21 d of fermentation, a high level of enzymatic breakdown of proteins and amino acids was observed: the nonprotein N level increased from 5% to between 15 and 40% of total N and the volatile N level increased from 1% to between 3 and 11% of total N. An increase in histamine, cadaverine, and putrescine was also observed. Despite the technological measures taken, such as the application of a high inoculum of starter culture and initial acidification with .4% lactic acid, this amino acid breakdown could not be reduced to an acceptable level. These results suggest that, because of biochemical deterioration, fermentation alone is not a useful method of preservation of raw poultry byproducts.

Amino Acids↗

Influence of non-fibrous carbohydrate and degradable intake protein on fermentation by ruminal microorganisms in continuous culture.

Four continuous culture fermenters were used in a 4 x 4 Latin square design to evaluate the effects of dietary non-fibrous carbohydrate (NFC) and ruminally degradable intake protein (DIP) on fermentation by ruminal microorganisms. Four diets, arranged in a 2 x 2 factorial, were formulated to contain either 25 or 40% NFC and 50 or 70% of dietary CP as DIP. Dietary DM contained 32% corn silage, 20% alfalfa-grass hay, and 48% concentrate. Solvent-extracted or lignosulfonate-treated soybean meal were used to alter DIP and contributed 40% of dietary CP. Corn or soybean hulls were included at 28% of dietary DM to alter NFC levels. Percentage of true OM digestion was similar (P > .05) among diets but NDF and total nonstructural carbohydrate digestion were inversely related depending on NFC content of the diet. Amylolytic bacterial concentrations (cells/milliliter) were lower (P = .03) in fermenters supplied with 25% NFC diets, resulting in less (P = .0001) total nonstructural carbohydrate digestion. Cellulolytic concentrations were similar (P > .05) among diets despite an increase (P = .002) in NDF digestion with 25% NFC diets. Total viable bacterial concentrations tended to decrease (P = .11) with 50% DIP diets, inducing a decline (P = .03) in total VFA production (millimoles/day). Reduced degradation of CP in 50% DIP diets (P = .008) increased outflow of total amino acid (P = .07) and individual outflows (P < .05) of glutamic acid, aspartic acid, arginine, histidine, and lysine. Few interactions occurred for the parameters measured despite the controlled nature of the fermentation in the current experiment. The preponderance of significant main effects illustrates that ruminal fermentation may not be improved by synchronization of energy and N release but may more likely be limited by either energy or N alone.

Amino Acids↗

In vitro fermentation of cellulose, beet pulp, citrus pulp, and citrus pectin using fecal inoculum from cats, dogs, horses, humans, and pigs and ruminal fluid from cattle.

We evaluated the influence of gastrointestinal tract microflora from several species on fiber fermentation characteristics in vitro. Selected fibrous substrates (cellulose, beet pulp, citrus pulp, and citrus pectin) were incubated for 6, 12, 24, and 48 h with ruminal fluid from cattle or feces from dogs, cats, pigs, horses, or humans. When data were pooled across all substrates and fermentation times, OM disappearance (29.4%) and acetate, propionate, butyrate, and total short-chain fatty acid (SCFA) production (1.09, .41, .12, and 1.61 mmol/g of OM, respectively) were lowest (P < .05), and lactate production (.23 mmol/g of OM) was greatest (P < .05) for horse fecal microflora compared with samples from the other species. The greatest (P < .05) acetate production resulted when substrates were fermented by cat fecal microflora (2.38 mmol/g of OM). The greatest (P < .05) propionate productions resulted from pig fecal and cattle ruminal microflora (.88 and .83 mmol/g of OM, respectively), and the greatest (P < .05) butyrate productions resulted from human and pig fecal microflora (.39 and .40 mmol/g of OM, respectively). Total SCFA production was greatest (P < .05) for cat fecal microflora (3.38 mmol/g of OM). When data were pooled across the species, substrate OM disappearance and SCFA production ranked from least to greatest in the following order: cellulose < beet pulp < citrus pulp < citrus pectin. The fermentability of different fibrous substrates by fecal or ruminal microflora from various species seems to be dependent not only on the fermentative activity of the microbial population but on other factors as well, perhaps lag time and rate of digesta passage.

Adult↗

The effect of in vitro fermentation on specific gravity and sedimentation measurements of forage particles.

Dry matter degradability (DMD), gas production (GP), functional specific gravity (FSG), volume of gas associated (GA), water-holding capacity (WHC), and sedimentation measurements of orchard-grass (OG) and alfalfa (AA) hays (ground through a 8-mm screen) were studied before and after in vitro incubation with ruminal fluid for 2, 4, 8, 24, 48, and 72 h. The DMD was higher for AA than for OG (P < .001), but GP did not differ. The FSG of unfermented OG and AA was .59 and .73, respectively (P < .01). During fermentation, the FSG of OG increased more than did that of AA (from .93 to 1.39 for OG and from .97 to 1.27 for AA after 2 and 72 h, respectively), and GA decreased more rapidly (from .94 to -.04 mL/g DM and from .74 to .15 mL/g DM, respectively). The DMD was positively correlated with FSG (r = .83; P < .001) and, therefore, negatively with GA (r = -.72; P < .01). The WHC increased similarly in the two forages with fermentation time. Unfermented and fermented samples were incubated in sedimentation columns filled with distilled water for 19, 37, 75, 150, and 300 s. After 300 s of sedimentation time, the unfermented AA and OG samples tended to float (91.1 and 72.7% of DM, respectively). In contrast, fermented samples tended to sediment (90.7 and 90.9% of DM, respectively). There were only small effects of forage species and fermentation time on sedimentation tendency. Correlations between sedimentation measurements and DMD and FSG were not significant, with the only exception of DM recovered in the lower section of sedimentation columns after 75 s, which was particularly correlated with DMD (P < .01) and FSG (P < .05). The results suggest that degradation rate of fibrous particles is related to changes in FSG and GA and, therefore, could influence ruminal transit. However, FSG was unable to predict accurately the sedimentation behavior of samples.

Animal Feed↗

Fermentation substrate and dilution rate interact to affect microbial growth and efficiency.

The effect of dilution rate (D) on carbohydrate, fibrous and nonfibrous, and protein fermentation by ruminal microorganisms was studied using a single-effluent continuous-culture system. The diets of fibrous carbohydrate, nonfibrous carbohydrate, or protein were formulated with soybean hulls (FC), ground corn (NFC), or isolated soy protein (PR) as the primary ingredient, respectively. Six dilution rates (.025, .050, .075, .10, .15, and .20/h of fermenter volume) were used. Digestibilities of DM, OM, and CP for the three diets and of NDF and ADF for the FC diet decreased (P<.001) as D increased, although the response of the digestibility to D varied with diet. Increasing D resulted in an increase in pH (P<.001) and a decrease (P<.001) in ammonia concentration. Daily volatile fatty acid production increased (quadratic; P<.01) for the FC and NFC diets, but decreased (quadratic; P<.001) for the PR diet. Increasing D quadratically increased (P<.001) the molar percentage of acetate and propionate, but quadratically decreased (P<.001) butyrate and valerate for the FC and NFC diets. For the PR diet, the molar percentage of propionate and valerate increased (quadratic; P<.01), whereas acetate and butyrate decreased (linear; P<.001) in response to increasing D. Molar percentage of isobutyrate and isovalerate decreased (P<.01) with increasing D for all three diets. As D increased, daily microbial N production showed quadratic responses with maximum values achieved at .126, .143, and .187/h D for the FC, NFC, and PR diet, respectively. There was a positive correlation between microbial growth efficiency (MOEFF) and D. A quadratic model fit the data of MOEFF as affected by D, and maximum MOEFF of 37.3, 59.6, and 71.4 g of bacterial N/kg OM truly fermented were calculated to be achieved at .177, .314, and .207/h D for the FC, NFC, and PR diet, respectively. Dilution rate significantly influenced the ruminal microbial fermentation of fibrous and nonfibrous carbohydrates and proteins, and was positively related to microbial yield and growth efficiency. In addition, microbial nitrogen composition, and therefore efficiency, was affected by substrate fermented.

Animal Feed↗

Changes in energy metabolism in relation to physical activity due to fermentable carbohydrates in group-housed growing pigs.

Fermentable nonstarch polysaccharides (dietary fiber) affect energy retention in group-housed growing pigs by reducing physical activity. This study assessed the effects of fermentation and bulkiness of dietary carbohydrates on physical activity in relation to energy metabolism. Eight clusters of 14 pigs were fed one of four diets in a 2x2 factorial arrangement. Factors included 1) gastrointestinal fermentation and 2) dietary bulkiness. Contrasts in fermentation were created by exchanging gelatinized maize starch with raw potato starch on a volume basis. Bulkiness was altered by adding 15% milled wheat straw to the diets. Apart from these differences, amounts of other dietary ingredients fed to the pigs were similar. Pigs were housed in groups. Nitrogen and energy balances were measured per cluster during a 14-d period. Dietary bulkiness did not affect ME intake, heat production, or energy retention. Metabolizability decreased when maize starch was replaced with raw potato starch (P< .01), resulting in a lower energy retention on the potato starch diets (P<.01). However, the lower ME intake on the potato diets was partially compensated by a reduced energy expenditure on physical activity (P< .01), which was 17.6% lower than that of pigs fed the maize starch diets. Dietary bulkiness did not affect physical activity. The effect of fiber-rich diets (nonstarch polysaccharides) on activity in growing group-housed pigs seems to be related to fermentation in the gastrointestinal tract, and not to bulkiness (volume).

Animal Feed↗