End-product induced metabolic shifts in <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> ATCC 27405

When attempting to increase yields of desirable end-products during fermentation, there is the possibility that increased concentrations of one product redirects metabolism towards the synthesis of less desired products. Changes in growth, final end-product concentrations, and activities of enzymes involved in pyruvate catabolism and fermentative end-product formation were studied in Clostridium thermocellum in response to the addition of individual end-products (H₂, acetate, ethanol, formate, and lactate) to the growth medium. These were added to the growth medium at concentrations ten times greater than those found at the end of growth in cultures grown under carbon-limited conditions using cellobiose (1.1 g l⁻¹) as model soluble substrate. Although growth rate and final cell biomass decreased significantly with the addition of all end-products, addition of individual end-products had less pronounced effects on growth. Metabolic shifts, represented by changes in final end-product concentrations, were observed; H₂ and acetate yields increased in the presence of exogenous ethanol and lactate, while ethanol yields increased in the presence of exogenous hydrogen (H₂), acetate, and lactate. Late exponential phase enzyme activity data of enzymes involved in pyruvate catabolism and end-product formation revealed no changes in enzyme levels greater than 2-fold in response to the presence of any given end-product, with the exception of pyruvate:formate lyase (PFL), ferredoxin-dependent hydrogenase (Fd-H₂ase), and pyruvate:ferredoxin oxidoreductase (PFO): PFL and Fd-H₂ase activities increased 2-fold in the presence of ethanol, while PFO activity decreased by 57% in the presence of sodium formate. Changes in enzyme levels did not necessarily correlate with changes in final end-product yields, suggesting that changes in final end-product yields may be governed by thermodynamic considerations rather than levels of enzyme expressed under the conditions tested. We demonstrate that bacterial metabolism may be manipulated in order to selectively improve desired product yields.     

Identification by quantitative gas chromatography of common Fusobacterium species isolated from clinical specimens

Two unusual hydroxy acids have recently been described in broth cultures of certain anaerobes. Based on these and other known metabolic end-products, an identification scheme from quantitative gas chromatography for common Fusobacterium species was established. After examining 137 isolates and eight type strains the scheme agrees with conventional procedures. This was assisted by only three simple tests: gas, indole and lipase production. No other subculture was needed.     

Development of media to accelerate the isolation of indigo-reducing bacteria,which are difficult to isolate using conventional media

Indigo-reducing bacteria perform natural fermentation in indigo fermentation fluid. Owing to the stochastic nature of the process, the constituent in indigo fermentation fluid differ depending on the prepared batch and fermentation period. To identify new indigo-reducing bacteria, isolation of the bacteria is indispensable. However, isolation of indigo-reducing bacteria is difficult because conventional media are often unsuitable to isolate these slow-growing bacteria that also exist in low numbers. Hydrolysates of polysaccharides and mixtures of plant base constituents are candidates to accelerate the isolation of indigo-reducing bacteria that cannot be isolated using conventional media. In this current study, wheat bran hydrolysate and composted indigo leaves (sukumo) were used as ingredients in the fermentation fluid in the selective medium for indigo-reducing bacteria in anaerobic culture. The results suggested that obligate and oxygen-non-metabolizing facultative anaerobes are difficult to isolate using conventional media, whereas oxygen-metabolizing facultative anaerobes, relatively rapid-growing and major bacterial strains are relatively easy to isolate. Media containing sukumo hydrolysate facilitated the isolation of novel species of Bacillus pseudofirmus-related strains, whereas media containing wheat bran hydrolysate facilitated the isolation of Amphibacillus spp. (including new species). Seven species (including two new species) of indigo-reducing bacteria were isolated using wheat bran hydrolysate-containing media, whereas six species (including three new species) of indigo-reducing bacteria were isolated using media containing both wheat bran and sukumo hydrolysates. These newly developed culture media will facilitate the isolation of unknown bacteria in indigo fermentation and in environments similar to indigo fermentation fluid.     

Influence of end-products inhibition and nutrient limitations on the growth of Lactococcus lactis subsp. lactis

Lactococcus lactis was grown in a simple synthetic medium with glucose as substrate, enabling the precise quantification of each nutrient's contribution to growth. As expected, for the growth of lactic acid bacteria, the growth rate decreased progressively during the cultivation after a short period of exponential growth. End-products of fermentation, predominantly lactate and in minor amounts formate, acetate and ethanol, accumulated within the medium. Growth of the bacterium in fresh media supplemented with these end-products showed that the concentrations attained in the fermentor had no significant influence on the growth rate. As regards nutrients, vitamins and magnesium were never limiting during the culture. On the other hand, amino acid concentrations decreased, some of them being totally consumed and exhausted from the medium before growth ceased. However, growth in reconstituted media constructed with the amino acid concentrations remaining at different times of cultivation showed that amino acid depletion could not account for the observed growth decrease. Batch culture supernatant fluid was used as cultivation medium. Growth rates observed in supernatant cultures supplemented with various nutrients, compared to non-supplemented supernatant, showed that no addition improved growth. Finally, it was concluded that in the experimental conditions used in this study, growth inhibition was predominantly due to phenomena other than lactate inhibition and nutritional limitations, and hence associated with unidentified compounds produced in the fermentation.     

Isolation, Culture Characteristics, and Identification of Anaerobic Bacteria from the Chicken Cecum

Studies on the anaerobic cecal microflora of the 5-week-old chicken were made to determine a suitable roll-tube medium for enumeration and isolation of the bacterial population, to determine effects of medium components on recovery of total anaerobes, and to identify the predominant bacterial groups. The total number of microorganisms in cecal contents determined by direct microscope cell counts varied (among six samples) from 3.83 x 10(10) to 7.64 x 10(10) per g. Comparison of different nonselective media indicated that 60% of the direct microscope count could be recovered with a rumen fluid medium (M98-5) and 45% with medium 10. Deletion of rumen fluid from M98-5 reduced the total anaerobic count by half. Colony counts were lower if chicken cecal extract was substituted for rumen fluid in M98-5. Supplementing medium 10 with liver, chicken fecal, or cecal extracts improved recovery of anaerobes slightly. Prereduced blood agar media were inferior to M98-5. At least 11 groups of bacteria were isolated from high dilutions (10(-9)) of cecal material. Data on morphology and physiological and fermentation characteristics of 90% of the 298 isolated strains indicated that these bacteria represented species of anaerobic gram-negative cocci, facultatively anaerobic cocci and streptococci, Peptostreptococcus, Propionibacterium, Eubacterium, Bacteroides, and Clostridium. The growth of many of these strains was enhanced by rumen fluid, yeast extract, and cecal extract additions to basal media. These studies indicate that some of the more numerous anaerobic bacteria present in chicken cecal digesta can be isolated and cultured when media and methods that have been developed for ruminal bacteria are employed.     

Isolation and identification of fecal bacteria from adult swine.

An examination of the fecal microflora of adult swine was made with regard to the efficiency of several roll tube media in enumeration and recovery of anaerobes, the effects of medium constituents on recovery, and the isolation and identification of the predominant kinds of bacteria. Total number of organisms by microscopic bacterial counts varied among fecal samples from 4.48 X 10(10) to 7.40 X 10(10) bacteria/g (wet weight). Comparison of different nonselective roll tube media indicated that about 30% of the fecal bacteria could be recovered with a rumen fluid (40%, vol/vol) medium (M98-5). Recoveries of 21 and 15%, respectively, were obtained with M10 and rumen fluid-glucose-cellobiose agar (RGCA) media. Rumen fluid, Trypticase, sugars, and CO2 gas phase were important components required for maximum recovery with this medium. Similar high recoveries of anaerobes were also obtained with M98-5 containing swine cecal extract of place in rumen fluid or M10 plus swine cecal extract. Significantly lower recoveries were observed with RCGA, media supplemented with swine fecal extracts, reinforced clostridial medium, brain heart infusion agar, and prereduced blood agar. Ninety percent of the bacteria isolated from roll tube media were gram positive and consisted of facultatively anaerobic streptococci, Eubacterium sp., Clostridium sp., and Propionibacterium acnes. The remainder of the flora (8%) included several other species of anaerobes and Escherichia coli. Rumen fluid (or volatile fatty acids), Trypticase, and yeast extract additions to basal media stimulated the growth of anaerobic strains. Variation in the relative proportions of the predominant fecal microflora was observed. This work indicates that satisfactory enumeration, isolation and cultivation of the predominant microflora in swine feces can be obtained when strict anaerobic culture methods and a rumen fluid medium are used.     

Identification of equine cecal bacteria producing amines in an in vitro model of carbohydrate overload

Acute laminitis has been associated with the overgrowth of gram-positive bacteria within the equine hindgut, causing the release of factor(s) leading to ischemia-reperfusion of the digits. The products of fermentation which trigger acute laminitis are, as yet, unknown; however, vasoactive amines are possible candidates. The objectives of this study were to use an in vitro model of carbohydrate overload to study the change in populations of cecal streptococci and lactobacilli and to establish whether certain species of these bacteria were capable of producing vasoactive amines from amino acids. Cecal contents from 10 horses were divided into aliquots and incubated anaerobically with either corn starch or inulin (fructan; both at 1 g/100 ml). Samples were taken at 6-h intervals over a 24-h period for enumeration of streptococci, lactobacilli, and gram-negative anaerobes by a dilution method onto standard selective growth media. The effects of the antibiotic virginiamycin (1 mg/100 ml) and calcium hydrogen phosphate (CaHPO(4); 0.3 g/100 ml) were also examined. Fermentation of excess carbohydrate was associated with increases in numbers of streptococci and lactobacilli (2- to 3.5-log unit increases; inhibited by virginiamycin) but numbers of gram-negative anaerobes were not significantly affected. A screening agar technique followed by 16S rRNA gene sequence analysis enabled the identification of 26 different bacterial strains capable of producing one or more vasoactive amines. These included members of the species Streptococcus bovis and five different Lactobacillus spp. These data suggest that certain bacteria, whose overgrowth is associated with carbohydrate fermentation, are capable of producing vasoactive amines which may play a role in the pathogenesis of acute laminitis.     

Continuous hydrogen production during fermentation of alpha-cellulose by the thermophillic bacterium Clostridium thermocellum

Continuous hydrogen (H2) production during fermentation of alpha-cellulose was established using the thermophillic, anaerobic bacterium Clostridium thermocellum ATCC 27405. The objectives of this work were to characterize growth of C. thermocellum, quantify H2 production and determine soluble end-product synthesis patterns during fermentation of a cellulosic substrate under continuous culture conditions. A 5 L working volume fermentor was established and growth experiments were maintained for over 3,000 h. Substrate concentrations were varied from 1 to 4 g/L and the feed was introduced with continuous nitrogen gas sparging to prevent clogging of the feed-line. The pH and temperature of the reactor were maintained at 7.0 and 600 degrees C, respectively, throughout the study. At concentrations above 4 g/L, the delivery of alpha-cellulose was impaired due to feed-line clogging and it became difficult to maintain a homogenous suspension. The highest total gas (H2 plus CO2) production rate, 56.6 mL L(-1) h(-1), was observed at a dilution rate of 0.042 h(-1) and substrate concentration of 4 g/L. Under these conditions, the H2 production rate was 5.06 mmol h(-1). Acetate and ethanol were the major soluble end-products, while lactate and formate were greatly reduced compared to production in batch cultures. Concentrations of all metabolites increased with increasing substrate concentration, with the exception of lactate. Despite a number of short-term electrical and mechanical failures during the testing period, the system recovered quickly, exhibiting substantial robustness. A carbon balance was completed to ensure that all end-products were accounted for, with final results indicating near 100% carbon recovery. This study shows that long-term, stable H2 production can be achieved during direct fermentation of an insoluble cellulosic substrate under continuous culture conditions.     

Influence of moisture content and cultivation duration on Clostridium thermocellum 27405 end-product formation in solid substrate cultivation on Avicel

Avicel serves as a model microcrystalline cellulose substrate for investigations of cellulolytic microbial performance and cellulase enzyme systems in submerged liquid cultures. Clostridium thermocellum is a thermophilic, anaerobic bacterium capable of degrading lignocellulose and fermenting it to ethanol and other products, suggesting the native growth environment is similar to that supported by solid substrate cultivation. Few studies have examined the effects of process parameters on the metabolism of thermophilic anaerobes in solid substrate cultivation, however. The effects of solid substrate cultivation (SSC) substrate moisture content (30%, 50% and 70% wet-basis) and cultivation duration (2, 4 and 8 days) on the metabolic activity of C. thermocellum 27405 on Avicel was studied. The 70% substrate moisture content SSC culture yielded total end-product concentrations that were comparable to submerged liquid cultures. The SSC cultivation conditions with the highest end-product formation on Avicel were the combination of 70% substrate moisture content and cultivation duration period of 4 days, producing approximately 100mM of total end-products. The ethanol and lactate concentrations were fairly constant and did not change significantly over time in SSC. Acetate production was more dependent on the cultivation conditions in SSC and was significant for both the 70% substrate moisture content SSC and liquid cultivation experiments, making up on average 56% and 86% of total end-products, respectively. Performance of C. thermocellum 27405 in SSC was more dependent on the kinetic properties rather than the thermodynamic properties of substrate moisture content. High substrate loadings in C. thermocellum cultivation affected product ratios, resulting in the higher observed acetate production. In addition, cessation of metabolism was observed prior to complete Avicel conversion; the mechanisms involved need further investigation.     

Comparative fermentation studies of industrial strains belonging to four species of solvent-producing clostridia

Industrial and culture collection strains of solvent-producing clostridia, classified as Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharobutylicum, and Clostridium saccharoperbutylacetonicum were utilised in a comparative study of fermentation performance in a laboratory fermentation medium, a molasses fermentation medium, and a maize fermentation medium under standardised culture conditions. At least one representative strain was selected from each of the sub-groups within the four species. Preliminary evaluations were first undertaken for the three different fermentation media to determine the most appropriate media formulations, carbohydrate concentrations, and culture conditions for comparison of the solvent-producing ability of these strains. Standardised fermentation media and culture conditions were then selected for each of the comparative fermentation studies. These included TYA medium containing 4% glucose, a supplemented molasses medium containing 6% fermentable sugars, and a supplemented maize mash medium containing 8% maize. Additional comparative fermentation studies on industrial strains belonging to two species of solvent-producing clostridia were carried out in molasses containing higher concentrations of fermentable sugars, and the sugar concentrations supporting maximum levels of solvent production were determined. Although all the strains tested grew in the maize fermentation medium and degraded starch, only a few strains produced consistently high solvent levels. Optimum starch utilisation and solvent production was obtained at a maize concentration of 80 g/l. Pretreatment of the maize by milling or saccharification decreased the buffering capacity of the medium and resulted in decreased solvent production. Decreasing the time used to gelatinise the starch had little effect. Solvent yields and concentrations obtained in this study were compared with various published data in the scientific and patent literature and appeared to closely simulate the results obtained in the industrial fermentation process. The fermentation performances of individual strains could provide useful comparative data for the selection and development of strains for use on various commercial fermentation substrates.     

The influence of glucose concentration and culture incubation time on end-product formation during aerobic growth of Brochothrix thermosphacta

Fifteen strains of Brochothrix thermosphacta , isolated from various meat sources, produced the same end-products during aerobic growth in a tryptone-based medium containing 0.2% (w/v) glucose. Growth of one strain at different glucose concentrations showed that 2-methylpropanol and 2,3-butanediol were also produced. Formation of the branched chain acids was favoured by low glucose concentrations, that of the alcohols and the diol by higher concentrations. The traces of the diol, but not those of the alcohols, produced during the early stages of growth at low, limiting concentrations of glucose, gradually disappeared following glucose depletion. These findings provide adequate explanations for the observed differences in end-product formation during growth of Broc. thermosphacta on non-processed and corned beef.     

Identification of Equine Cecal Bacteria Producing Amines in an In Vitro Model of Carbohydrate Overload

Acute laminitis has been associated with the overgrowth of gram-positive bacteria within the equine hindgut, causing the release of factor(s) leading to ischemia-reperfusion of the digits. The products of fermentation which trigger acute laminitis are, as yet, unknown; however, vasoactive amines are possible candidates. The objectives of this study were to use an in vitro model of carbohydrate overload to study the change in populations of cecal streptococci and lactobacilli and to establish whether certain species of these bacteria were capable of producing vasoactive amines from amino acids. Cecal contents from 10 horses were divided into aliquots and incubated anaerobically with either corn starch or inulin (fructan; both at 1 g/100 ml). Samples were taken at 6-h intervals over a 24-h period for enumeration of streptococci, lactobacilli, and gram-negative anaerobes by a dilution method onto standard selective growth media. The effects of the antibiotic virginiamycin (1 mg/100 ml) and calcium hydrogen phosphate (CaHPO₄; 0.3 g/100 ml) were also examined. Fermentation of excess carbohydrate was associated with increases in numbers of streptococci and lactobacilli (2- to 3.5-log unit increases; inhibited by virginiamycin) but numbers of gram-negative anaerobes were not significantly affected. A screening agar technique followed by 16S rRNA gene sequence analysis enabled the identification of 26 different bacterial strains capable of producing one or more vasoactive amines. These included members of the species Streptococcus bovis and five different Lactobacillus spp. These data suggest that certain bacteria, whose overgrowth is associated with carbohydrate fermentation, are capable of producing vasoactive amines which may play a role in the pathogenesis of acute laminitis.     

Fatty acids synthesized by oral treponemes in chemically defined media

OMIZ-W68, a chemically defined medium that contains no long-chain fatty acids and yet supports in vitro proliferation of a wide range of fastidious oral anaerobes, is described. The type strains of Treponema denticola, Treponema lecithinolyticum, Treponema maltophilum, Treponema pectinovorum, Treponema socranskii, and an as yet unpublished canine Treponema species could be propagated indefinitely in this medium with sugar supplements for the saccharolytic species. Analysis of the cellular fatty acids (CFA) of these treponemes by gas chromatography demonstrated the synthesis of C14, C15, C16, and C17 fatty acids (linear-, iso-, and anteiso-forms) in various proportions, but neither hydroxy- nor unsaturated fatty acids. However, between 0% and 40% of the eluted material could not be identified. The proportions of CFAs differed not only between species but also between the eight strains of Treponema denticola investigated. Replacing OMIZ-W68 by a derivative minimal essential medium (OMIZ-M/TDCDK) developed for Treponema denticola had little effect on the CFA profiles. In contrast, the CFA profiles of treponemes grown in OMIZ-W68 showed at best minor similarity to the strains from the Moore library of the Virginia Polytechnic Institute, which had been grown in media containing serum, peptones, and yeast extract.     

APPLICATIONS FOR BACTERIAL MEMBRANE FRACTIONS IN STERILE PHARMACEUTICAL QUALITY CONTROL

The commercially available bacterial membrane preparation Oxyrase was examined for use in quality control laboratory procedures for culturing anaerobic microorganisms, and as a media additive for parenteral product filling line validation by media fill to detect anaerobes. Comparison studies between Oxyrase products and conventional anaerobic culturing methods were performed. The results from the studies showed Oxyrase for Broth to be effective in promoting the growth of anaerobes in nonprereduced media not normally used for anaerobic cultivation.     

Use of an in vitro gas production method to investigate interactions between veld hay and Napier hay or groundnut hay supplements

Measurement of gas produced during in vitro fermentation was used to investigate the fermentability of poor quality natural pasture (veld) hay mixed with different amounts of Napier hay or groundnut hay. In vitro fermentations were conducted in nitrogen-rich (NR) and nitrogen-free (NF) media. Groundnut hay was more rapidly fermented than Napier hay, the nitrogen content of the medium making little difference to the fermentation characteristics of either hay. Veld hay was the least fermentable substrate, particularly when NF medium was used. Statistically significant positive interactive effects were observed between both supplements and veld hay fermented in both media as gauged by gas production and dry matter disappearance. Evidence of significant interactions had not been obtained from earlier measurements of in vivo digestibility using the same feeds, but they may have been obscured by increased rates of passage with increased supplementation. Differences between gas productions in NR and NF media were explored as possible indicators of nitrogen deficiency in feed mixtures. Both Napier hay and groundnut hay appeared to be balanced in terms of energy and nitrogen fermentable by rumen microbes.     

Recovery and Identification of Anaerobes: a System Suitable for the Routine Clinical Laboratory

A system for the isolation of anaerobes based upon the use of reducible solid media is described. Plates of reducible media prepared and stored aerobically were reduced before use by incubation in a GasPak jar for 24 h. Clinical specimens for culture were carefully selected. The value of Amies transport medium was confirmed. Selective and nonselective formulations of reducible media were used for primary isolation. Abbreviated identification schemes based in part on gas-liquid chromatography are presented. The suitability of this system for improving the recovery and identification of anaerobes in a routine clinical laboratory is documented.     

Comparison of Methods for Isolation of Anaerobic Bacteria from Clinical Specimens

Five different anaerobic culture methods and several different media were compared for their ability to recover anaerobes from clinical specimens. Specimens were obtained from patients with documented infections, avoiding contamination with normal flora, and immediately placed in an anaerobic transporter. Each specimen was cultured by all methods and on all the various media. The comparative data indicate that anaerobic jars (GasPak and evacuation-replacement types) are just as effective in the recovery of clinically significant anaerobes as the more complex roll-tube and chamber methods employing prereduced media. Liquid media were disappointing as a "back-up" system but chopped-meat glucose was superior to two thioglycolate formulations. Growth of all anaerobes was poorer on selective media, but these media were very helpful in the workup of specimens containing mixed growth of anaerobic and facultative organisms. A variety of different anaerobes was isolated, but no very fastidious or extremely oxygen-sensitive organisms were recovered. This suggests that such organisms may not play a significant role in causing clinical infections.     

Glucose metabolism of Haemonchus contortus adults: effects of thiabendazole on susceptible versus resistant strain

Analyses of the in vitro glucose metabolism of Haemonchus contortus adults have confirmed a complex series of end-products: ethanol, n-propanol, propionate, acetate, and CO2 as the major end-products of catabolism. No difference in end-product accumulation was seen between the cambendazole sensitive (BPL) and cambendazole resistant (CR) strains. Thiabendazole (5 mM) in vitro depressed ethanol, propanol, acetate, and propionate accumulation by approximately 42% in the BPL strain. However, in the resistant strain (CR), these end-product accumulations increased by 50% when the worms were exposed to drug in vitro and 80% when exposed in vivo. Resistance manifested by the CR strain appeared to be associated with the ability to increase its carbon flow in the presence of thiabendazole.