Fermentation Balance Research Articles

Cinétique de la croissance mycélienne de Conidiobolus obscurus

The growth of C. obscurus was studied in solid and liquid media. In agar media, the hyphal growth was linear, but the number of apices increased exponentially; total growth was consequently exponential but characterized by a low growth rate. In batch cultures in a fermenter, mycelial growth was continuously linear following a short exponential phase. The absence of branching induced a linear development of the fungus in liquid culture. Fermentation balances showed that the linear growth could not result from the presence of a specific inhibitor, nor from the existence of a limiting concentration of a nutrient in the medium. Hypotheses concerning the non-branching of the fungus in liquid culture are discussed.

Fermentation Characteristics of Heterolactic Acid Bacteria in Green Bean Juice

ABSTRACTGreen bean juice was fermented with 10 species (14 strains) of heterofermentative and two homofermentative lactic acid bacteria to select organisms which might be used to carry out a complete fermentation. Lactobacillus cellobiosus was the only organism to remove all fermentable sugars from bean juice with or without 2.5% NaCI. Nine other cultures used from 75–95% of the sugars. Lactobacillus cellobiosus also produced the lowest final pH among the 14 strains. A complete analysis of the major fermentation substrates and products was done for each of the organisms. Fermentation balance calculations showed a range from 74–132% carbon recovery. These bacteria showed considerable variation in the ability to degrade malic acid and to form mannitol and acetic acid.

Commensalistic Interaction Between Lactobacillus acidophilus and Propionibacterium shermanii

Propionibacterium shermanii and Lactobacillus acidophilus were grown in batch mixed culture in a 5-liter fermenter under controlled conditions of pH 5.8 and 35 degrees C on a semisynthetic medium with glucose as an energy source. Cellular efficiencies and fermentation balances were developed for this pair and compared with P. shermanii grown in pure culture on glucose, lactate, and a mixture of these substrates and with L. acidophilus grown on glucose. P. shermanii had ATP yield coefficient values of 17 for each substrate alone but had an average value of 30 for substrate mixtures. Growth rates were similar for P. shermanii on glucose or lactate but higher cell yields were observed for glucose. P. shermanii used both lactate and glucose in mixed substrate until lactate was exhausted, and growth rates slowed thereafter. L. acidophilus had a similar ATP yield coefficient of 15 but produced lower cell yields than did P. shermanii on glucose. Mixed culture of both microorganisms on glucose resulted in much faster and nearly equal growth rates for both and no lactate accumulation in the medium. Acetic acid production rates per generation were lower in mixed culture, suggesting use by the growing culture. The cause of the synergistic effect was not determined but may be due to the rapid production and removal of lactate or CO(2) enhancement in mixed culture.

Effect of environmental pH on fermentation balance of Lactobacillus bulgaricus.

When Lactobacillus bulgaricus NLS-4 was grown anaerobically in continuous culture with limiting glucose, a shift in the pH of the medium from the acidic to the alkaline range caused this normally homofermentative bacterium to catabolize glucose in a heterofermentative fashion. The change in the nature of the fermentation was accompanied by a decrease in lactate dehydrogenase biosynthesis in alkaline conditions. The lactate dehydrogenase from this organism did not require fructose 1,6-diphosphate or manganese ions (Mn2+) for catalytic activity. Involvement of the phosphoroclastic split in the pyruvate conversion in an alkaline environment was also confirmed. The high lactate dehydrogenase synthesis in acidic medium together with the participation of the phosphoroclastic split under alkaline conditions may explain the shift from homolactic to heterolactic fermentation of L. bulgaricus NLS-4 with the change of environmental pH.

Anaerobic glucose and serine metabolism in Staphylococcus epidermidis.

Anaerobically grown Staphylococcus epidermidis fermented glucose with the production of lactate and trace amounts of acetate, formate and CO2. Isotopic and inhibitor studies, assays for key enzymes of different metabolic pathways, and fermentation balances, all indicated that glucose was metabolized principally via glycolysis and to a very limited extent by the hexose monophosphate oxidative pathway. Serine fermentation proceeded via deamination and dismutation yielding NH3 and equimolar amounts of lactate, acetate and CO2; small amounts of formate arose by the operation of pyruvate-formate lyase. Incorporation of 0.5% (w/v) glucose in the growth medium depressed serine metabolism by repressing the activities of serine dehydratase and pyruvate dehydrogenase but, conversely, enhanced the activities of phosphofructokinase and lactate dehydrogenase. Glucose-grown organisms at various stages of anaerobic batch growth showed an inverse relationship between the rates of fermentation of serine and glucose. L-Lactate dehydrogenase activity in crude extracts depended on fructose 1,6-bisphosphate, and fructose 1,6-bisphosphate aldolase was found to be a class I aldolase. Despite the presence of ribokinase, D-ribose-5-phosphate isomerase, transaldolase and transketolase, the organisms utilized ribose only after growth aerobically in basal medium, and then at a slow rate after an initial lag period.

Anaerobic fish spoilage by bacteria. I. Biochemical changes in herring extracts.

Water extracts of herring fillets were used as laboratory model substrates for the study of anaerobic bacterial spoilage of fish stored in bulk. The organisms studied (Enterobacter sp., str. NTHC 151, Proteus sp., str. NTHC 153, and Aeromonas sp., str. NTHC 154) were selected for by an enrichment method designed to isolate the bacteria having the highest anaerobic growth yields in these herring extracts. The extracts were then inoculated with pure cultures of the bacteria and incubated anaerobically at 15°C. Chemical analyses of the extracts were carried out at the start and at the end of the incubation period. The compounds determined accounted for 88% of total carbon and 93% of total nitrogen of the sterile extracts. Ribose (free and bound), free hexoses and lactate were the main substrates for the growth of the bacteria. These compounds were converted to acetate and CO2; hydrogen liberated by these processes was used for the quantitative reduction of trimethylamine oxide to trimethylamine. Fermentation balances support the contention that the biochemical changes observed represent the quantitative dominating chemical events in the herring extracts as a result of the development of the spoilage bacteria.

Intracellular polysaccharide of Bacteroides fragilis.

Formation of iodophilic polysaccharide (IPS) from glucose was demonstrated in 27 strains of Bacteroides fragilis. Synthesis was dependent on the glucose concentration of the medium, the pH and the growth phase. When glucose was in short supply the cellular polysaccharide was degraded rapidly at pH 4.5 to 6.5 and fatty acids accumulated in the medium. Storage of IPS was not responsible for the low carbon recoveries observed in fermentation balance studies. In electron micrographs of thin sections, the IPS was observed as cytoplasmic granules dispersed throughout the whole cell. After extraction and purification the IPS was characterized as a glycogen.

Growth yields and fermentation balance of Bacteroides fragilis cultured in glucose-enriched medium

Bacteroides fragilis is an obligate anaerobic bacterium classified with the gram-negative, non-sporeforming bacilli and is the Bacteroides species most frequently isolated from human infections. In the present study, experiments were designed to investigate growth characteristics of B. fragilis in a complex medium. In a minimal defined medium, which was employed for comparison purposes, B. fragilis grew with a generation time of 2 h. Growth of the organism in glucose-enriched medium used in the present study was superior. Maximum generation time was 60 min. Total and viable cells (colony-forming units) were 8.9 x 10(9) and 2.1 x 10(9), respectively, at maximum measurable growth. The molar growth yield (Ym) was 51.5. Growth yields were found to reach a maximum 2 to 3 h before maximum growth and to vary with respect to the phase of growth. Estimates of the fermentation products indicated that glucose was the sole energy substrate. Major products included acetic acid, propionic acid, lactic acid, and succinic acid. Other products included ethyl alcohol, pyruvic acid, and fumaric acid. No attempt was made to recover CO2 or formic acid. The OR balances from two experiments were 0.013 and -0.093 and the respective carbon recoveries were 6.268 and 6.241. The results of the present study show that B. fragilis is capable of rapid rates of growth in vitro by using glucose as the sole energy source.

Oxygen metabolism by the anaerobic bacterium veillonella alcalescens.

Veillonella alcalescens contained a membrane-bound lactate oxidase system. Studies on the effect of inhibitors on lactate oxidase showed the participation of non-heme iron, quinone and cytochromesb andd. Superoxide anion radicals (\(O_2^{\bar \cdot } \)) and H2O2 were shown to be formed at lactate oxidation and presumably arose from cyanide- and azide-resistant side chains of the respiratory system. The →H+/O ratio withL-lactate as a hydrogen donor was 2.3. When an anaerobic culture growing on lactate was shifted to a high dissolved oxygen tension (d.o.t.=15 kPa) rapid inhibition of growth and lactate conversion occurred. This could be correlated with a rapid inactivation of lactate dehydrogenase. The effects of high d.o.t.'s on lactate dehydrogenase, lactate conversion and growth were reversible. After a shift to low d.o.t.'s (<2.5 kPa) growth ofV. alcalescens continued for one or two doublings whereafter lysis did occur. Acetate and pyruvate were the main fermentation products. P/O ratio's were calculated from molar growth yields and fermentation balances. A P/O value of 0.66 was found after a shift to a very low oxygen supply at which the d.o.t. presumably was zero. Shifts to higher d. o. t.'s gave much lower growth yields. Presumably, under these conditions uncoupling between growth and energy production occurred. Accumulation of toxic oxygen compounds was given as an explanation for the behaviour ofV. alcalescens at low d.o.t.'s.

Fermentation of fumarate and L-malate by Clostridium formicoaceticum.

The fermentation of fumarate and L-malate by Clostridium formicoaceticum was investigated. Growing and nongrowing cells degraded fumarate by dismutation to succinate, acetate, and CO2; on the other hand, only small amounts of succinate were detected when the organism was grown on L-malate. This dicarboxylic acid was mainly converted to acetate and CO2. The fermentation balances were modified if bicarbonate or formate were present in the medium. When C. formicoaceticum was grown in the presence of both dicarboxylic acids, fumarate was consumed before L-malate. The latter was mainly converted to acetate, whereas fumarate was fermented to acetate and succinate. Molar growth yields were determined to be 6 g of dry weight per mol of fumarate and 8 g of dry weight per mol of L-malate fermented.

ATP formation associated with fumarate and nitrate reduction in growing cultures of Veillonella alcalescens

Molar growth yields, fermentation balances and enzyme activities were measured in Veillonella alcalescens grown anaerobically with different substrates in the absence or presence of fumarate or nitrate. The molar growth yields on malate (14.3 g dry wt bacteria/mole substrate) and citrate (19.3) were higher than that on lactate (8.6). The molar growth yield on lactate was increased to 15.5 or 19.8 by the addition of fumarate or nitrate, respectively, to the growth medium, and the molar growth yield on citrate was increased to 25.3 by addition of nitrate. Active growth on pyruvate was only observed in the presence of nitrate, and the molar growth yield was 25.5. From fermentation balances and fermentation systems similar YATP values (g dry wt bacteria/mole ATP) were calculated for all substrates or mixtures of substrates assuming that one mole of ATP is generated at the electron transport from pyruvate, NADH and NADPH to nitrate or fumarate whereas ATP is not produced in the electron transport from lactate to fumarate or nitrate, and, therefore, this assumption was considered to reflect the actual situation. The mean YATP value at a doubling time of 1 h was 16.5 g dry wt bacteria/mole ATP for growth without an added hydrogen acceptor, 14.4 for growth with fumarate, and 14.2 for growth with nitrate.

Fermentation of cellulose and cellobiose by Clostridium thermocellum in the absence of Methanobacterium thermoautotrophicum.

The fermentation of cellulose and cellobiose by Clostridium thermocellum monocultures and C. thermocellum/Methanobacterium thermoautotrophicum cocultures was studied. All cultures were grown under anaerobic conditions in batch culture at 60 degrees C. When grown on cellulose, the coculture exhibited a shorter lag before initiation and growth and celluloysis than did the monoculture. Cellulase activity appeared earlier in the coculture than in the monoculture; however, after growth had ceased, cellulase activity was greater in the monoculture. Monocultures produced primarily ethanol, acetic acid, H2 and CO2. Cocultures produced more H2 and acetic acid and less ethanol than did the monoculture. In the coculture, conversion of H2 to methane was usually complete, and most of the methane produced was derived from CO2 reduction rather than from acetate conversion. Agents of fermentation stoppage were found to be low pH and high concentrations of ethanol in the monoculture and low pH in the coculture. Fermentation of cellobiose was more rapid than that of cellulose. In cellobiose medium, the methanogen caused only slight changes in the fermentation balance of the Clostridium, and free H2 was produced.

Dissimilatory nitrate reduction inClostridium tertium

Fermentation balance studies were carried out on Clostridium tertium grown with and without nitrate in the medium. Nitrate reduction increased the efficiency of energy produced from glucose by permitting the utilization of additional sites of substrate level phosphorylation. The effect was even more dramatic in C. tertium than in C. perfringens, with increased cell yields of about 30% being observed in the former compared with 20% in the latter. Unlike C. perfringens, C. tertium responded to the presence of nitrate in the medium with an increased growth rate. A slight increase in the Y ATP of these cultures was also observed, and quantitatively, this appeared to be consistent with the prediction of Stouthammer and Bettenhaussen that Y ATP will vary with the growth rate. Thus, C. tertium, like C. perfringens, was able to use nitrate as an electron acceptor in conjunction with its energy metabolism, suggesting that this may be widespread among the nitrate-reducing anaerobes.

End products and fermentation balances for lactic streptococci grown aerobically on low concentrations of glucose

Maximum acetate produced aerobically by Streptococcus diacetilactis and Streptococcus cremoris was 14% of 1 to 7 mumol of glucose/ml in a partially defined medium that contained lipoic acid. Y (glucose) values were 35.3 (S. diacetilactis) and 31.4 (S. cremoris) with low concentrations (1 to 7 mumol/ml) of glucose in the medium and 21 (S. diacetilactis) with higher concentrations (6 to 15 mumol/ml). Y (adenosine 5'-triphosphate) values for the bacteria, determined by taking into account the end products produced, were 15.6 and 13.9 for S. diacetilactis and S. cremoris, respectively, in the partially defined medium containing 1 to 7 mumol of glucose/ml and higher (21.5 and 18.9, respectively) in a complex medium that contained 2 mumol of glucose/ml. Addition of citrate in addition to glucose did not result in higher molar growth yields.

Net utilization of roughage and concentrate diets by sheep.

1. Two diets, an all-roughage diet and a high-concentrate diet, were fed at two levels, a low level of estimated 1-5 times maintenance energy requirement and a higher level of estimated two times maintenance energy requirement, to South African Mutton Merino castrated male sheep, aged 13 months and in fairly lean condition at the start of the 93 d experimental period. 2. Body composition and energy retention were determined using the comparative slaughter technique and two series of digestibility and balance studies were done during the course of the experiment. Metabolizability of each diet was estimated and corrected for fermentation heat using the fermentation balance approach. 3. Although there were significantly different rates of energy gain on different diets and feeding levels, fat energy gained (% total energy gained) was similar for the four groups, i.e. 78-80. 4. Regression of energy gain v. corrected metabolizable energy (ME) intake indicated that the maintenance energy requirements of sheep used in this experiment were 310-2 and 302-3 kJ ME/kg body-weight0-75 per d and the values for net utilization of ME for body energy gain were 0-411 and 0-479 with the roughage and concentrate diets respectively. 5. It was concluded that the estimated maintenance energy requirements of sheep obtained in this study are realistic values and that the efficiency of utilization of surplus ME for the two diets did not differ significantly.

Vergärung von Pyruvat durch sieben Arten phototropher Purpurbakterien

The dark, anaerobic fermentation of pyruvate under growth conditions was examined with the following species of phototrophic purple bacteria: Rhodospirillum rubrum strains Ha and S1, Rhodopseudomonas gelatinosa strain 2150, Rhodopseudomonas acidophila strain 7050, Rhodopseudomonas palustris strain ATCC 17001, Rhodopseudomonas capsulata strains Kb1 and 6950, Rhodopseudomonas sphaeroides strain ATCC 17023, and Chromatium vinosum strain D. Fermentation balances were established for all experiments. Under fermentative conditions cell protein and dry weight increased only slightly, if at all. The species differed considerably in their fermentative activity; R. rubrum and R. gelatinosa exhibited the highest rates (2-8 mumoles pyruvate/mg protein-h). R. acidophila and R. capsulata showed an intermediate fermentation rate (0.4--2.0 mumoles pyruvate/mg protein-h), while the other strains tested fermented at quite low rates (0.2-0.4 mumoles pyruvate/mg protein-h). The extremes of fermentation times were from 30-380 hours. Based on the products of fermentation which were formed in addition to acetate, formate, and CO2, the species can be grouped as follows: a) R. rubrum, R. gelatinosa, and R. sphaeroides additionally form propionate. b) R. gelatinosa, R. palustris, R. capsulata, R. sphaeroides, and C. vinosum additionally form lactate. R. palustris also produces butyrate. c) R. acidophila and R. capsulata additionally form much 2,3-butanediol, acetoin, and diacetyl. Small amounts of acetoin were formed by the rest of the strains. A comparison of the fermentation of pyruvate by normal and starved cells (4 days in the light without a carbon source) of R. rubrum and R. gelatinosa shows that the latter ferment more slowly and produce less acetate and formate, but more propionate or lactate. The fermentation of pyruvate by R. rubrum was also studied in cultures in which the pH fell (7.2--6.6). Compared with the fermentation at neutral pH (7.3, 7.4), the following differences were found: a slower fermentation rate, an increased production of dry weight, an increased formation of propionate, but a reduced formation of acetate and a very low production of formate.

Microcalorimetric study of the anaerobic growth of Escherichia coli: growth thermograms in a synthetic medium.

A microcalorimetric technique was used for studying the growth of Escherichia coli during anaerobiosis. The growth thermograms obtained are complex and the shape of curves is dependent on the hydrogen lyase activity of the cells. Fermentation balances are given for different culture conditions, and simple growth thermograms are obtained when the hydrogen lyase activity is inhibitied.

Vergärung von Pyruvat durch sieben Arten phototropher Purpurbakterien

AbstractThe dark, anaerobic fermentation of pyruvate under growth conditions was examined with the following species of phototrophic purple bacteria: Rhodospirillum rubrum strains Ha and S1, Rhodopseudomonas gelatinosa strain 2150, Rhodopseudomonas acidophila strain 7050, Rhodopseudomonas palustris strain ATCC 17001, Rhodopseudomonas capsulata strains Kb1 and 6950, Rhodopseudomonas sphaeroides strain ATCC 17023, and Chromatium vinosum strain D. Fermentation balances were established for all experiments.Under fermentative conditions cell protein and dry weight increased only slightly, if at all. The species differed considerably in their fermentative activity; R. rubrum and R. gelatinosa exhibited the highest rates (2–8 μmoles pyruvate/mg protein · h). R. acidophila and R. capsulata showed an intermediate fermentation rate (0.4–2.0 μmoles pyruvate/mg protein · h), while the other strains tested fermented at quite low rates (0.2–0.4 μmoles pyruvate/mg protein · h). The extremes of fermentation times were from 30–380 hours.Based on the products of fermentation which were formed in addition to acetate, formate, and CO2, the species can be grouped as follows: R. rubrum, R. gelatinosa, and R. sphaeroides additionally form propionate. R. gelatinosa, R. palustris, R. capsulata, R. sphaeroides, and C. vinosum additionally form lactate. R. palustris also produces butyrate. R. acidophila and R. capsulata additionally form much 2,3‐butanediol, acetoin, and diacetyl. Small amounts of acetoin were formed by the rest of the strains. A comparison of the fermentation of pyruvate by normal and starved cells (4 days in the light without a carbon source) of R. rubrum and R. gelatinosa shows that the latter ferment more slowly and produce less acetate and formate, but more propionate or lactate.The fermentation of pyruvate by R. rubrum was also studied in cultures in which the pH fell (7.2–6.6). Compared with the fermentation at neutral pH (7.3, 7.4), the following differences were found: a slower fermentation rate, an increased production of dry weight, an increased formation of propionate, but a reduced formation of acetate and a very low production of formate.

Comparative Biochemical Studies of Litomosoides carinii, Dipetalonema viteae, and Brugia pahangi Adults

The metabolisms and effects of several anthelmintics on 3 adult filarial nematodes, Litomo- soides carinii, Dipetalonema viteae, and Brugia pahangi, have been compared. The latter 2 differ from the obligate aerobe, L. carinii, in that they survive for extended periods of time anaerobically, indicating that they obtain their metabolic energy by utilizing anaerobic pathways. In accord with their apparent anaerobic nature, fermentation balance studies indicate that D. viteae and B. phangi are homolactate fermenters. L. carinii, on the other hand, forms appreciable quantities of acetate in addition to lactate and C02. Isotope studies indicate that the tricarboxylic acid cycle does not con- stitute a quantitatively significant energy-yielding pathway in L. carinii. Instead, findings suggest that the aerobic requirement of L. carinii may reside completely in one system, the oxidative decarboxyla- tion of pyruvate to acetate and CO2. Cytochrome c oxidase activity could not be demonstrated in any of the parasites as assayed by the oxidation of reduced cytochrome c. 1-Tetramisole at low concen- trations (4.2 X 10-7 M) completely stops motility of all 3 species within 1.5 min. d-Tetramisole also inhibits motility effectively, but it is considerably more readily reversible than the corresponding levo-isomer.

Generation of ATP during cytochrome-linked anaerobic electron transport in propionic acid bacteria.

Summary: Enzyme determinations in bacteria-free extracts and dual-wavelength experiments with membrane suspensions established that Propionibacterium freudenreichii converted glycerol into triose phosphate via glycerol kinase and NAD-independent glycerol 1-phosphate dehydrogenase which is closely linked to cytochrome b. Glycerol 1-phosphate dehydrogenase uses fumarate as a final hydrogen acceptor. The enzyme system catalysing fumarate reduction with glycerol 1-phosphate as a hydrogen donor, is membrane bound and is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO). Fumarate reduction with reduced benzyl-viologen is not inhibited by HOQNO. Cytochrome b is therefore probably involved in the anaerobic electron transport from glycerol 1-phosphate to fumarate. Molar growth yields and fermentation balances were determined for P. freudenreichii and P. pentosaceum growing on glucose, fructose, glycerol and lactate and ATP yields (mol of ATP formed/mol of substrate fermented) were calculated assuming that 1 mol ATP is formed in the electron transport from glycerol 1-phosphate and lactate to fumarate, and that 2 mol ATP are formed in the electron transport from NADH to fumarate. Mean Y ATT values (g dry wt bacteria/mol ATP) were 15.2 and 12.9 for P. freudenreichii, and 16.4 and 11.8 for P. pentosaceum each growing on complex or synthetic medium respectively. The observation that for each strain YATP values were constant for the same medium, supported our assumptions on energy generation in propionic acid bacteria.