Showing papers in "Applied and Environmental Microbiology in 1977"

Use of nuclepore filters for counting bacteria by fluorescence microscopy.

Abstract: Polycarbonate Nuclepore filters are better than cellulose filters for the direct counting of bacteria because they have uniform pore size and a flat surface that retains all of the bacteria on top of the filter. Although cellulose filters also retain all of the bacteria, many are trapped inside the filter where they cannot be counted. Before use, the Nuclepore filters must be dyed with irgalan black to eliminate autofluorescence. Direct counts of bacteria in lake and ocean waters are twice as high with Nuclepore filters as with cellulose filters.

Fermentation of mucin and plant polysaccharides by strains of Bacteroides from the human colon.

Abstract: Ten Bacteroides species found in the human colon were surveyed for their ability to ferment mucins and plant polysaccharides ("dietary fiber"). A number of strains fermented mucopolysaccharides (heparin, hyaluronate, and chondroitin sulfate) and ovomucoid. Only 3 of the 188 strains tested fermented beef submaxillary mucin, and none fermented porcine gastric mucin. Many of the Bacteroides strains tested were also able to ferment a variety of plant polysaccharides, including amylose, dextran, pectin, gum tragacanth, gum guar, larch arabinogalactan, alginate, and laminarin. Some plant polysaccharides such as gum arabic, gum karaya, gum ghatti and fucoidan, were not utilized by any of the strains tested. The ability to utilize mucins and plant polysaccharides varied considerably among the Bacteroides species tested.

Determination of bacterial number and biomass in the marine environment.

Abstract: Three techniques for the measurement of bacterial numbers and biomass in the marine environment are described. Two are direct methods for counting bacteria. The first employs an epifluorescence microscope to view bacteria that have been concentrated on membrane filters and stained with acridine orange. The second uses a transmission electron microscope for observing replicas of bacteria that are concentrated on membrane filters. The other technique uses Limulus amebocyte lysate, an aqueous extract from the amebocytes of the horseshoe crab, Limulus polyphemus, to quantitate lipopolysaccharide (LPS) in seawater samples. The biomass of gram-negative (LPS containing) bacteria was shown to be related to the LPS content of the samples. A factor of 6.35 was determined for converting LPS to bacterial carbon.

Epichloë typhina from toxic tall fescue grasses.

Abstract: Epichloe typhina, a clavicipitaceous systemic phytopathogen, was isolated from two varieties and three hybrids of tall fescue (Festuca arundinaceae). The morphology of the fescue isolates was compared with E. typhina isolated from bent grass (Agrostis perennans). In all isolates, conidia were identical and were typical of E. typhina. In fescue grasses the endophyte failed to produce stromata, but on bent grass the fungus seasonally produced stromata, typical of the genus. Cattle grazing the fescue grasses showed signs of the fescue toxicity syndrome, the E. typhina was found in frequencies of 100%; in grasses from pastures in which cattle showed no signs of the syndrome, frequencies were 0 to 50%. Nutritional factors in vitro were more complex for the isolates from fescue than for the isolate from bent grass. These studies suggested that E. typhina includes biotypes that might be involved in the toxicity syndrome. The fescue biotypes grew poorly on media, and yields were inadequate for toxicity studies. However, the bent grass isolate grew well on three media, and extracts from two of these were toxic to chicken embryos. All isolates produced in vitro the nontoxic fungal steroid tetraenone [ergosta-4,6,8(14),22-tetraen-3-one], which has been isolated from toxic fescue grasses.

Effect of sulfate on carbon and electron flow during microbial methanogenesis in freshwater sediments.

Abstract: The effect of sulfate on methane production in Lake Mendota sediments was investigated to clarify the mechanism of sulfate inhibition of methanogenesis. Methanogenesis was shown to be inhibited by the addition of as little as 0.2 mM sulfate. Sulfate inhibition was reversed by the addition of either H2 or acetate. Methane evolved when inhibition was reversed by H2 additions was derived from 14CO2. Conversely, when acetate was added to overcome sulfate inhibition, the evolved methane was derived from [2-14C]acetate. A competition for available H2 and acetate was proposed as the mechanism by which sulfate inhibited methanogenesis. Acetate was shown to be metabolized even in the absence of methanogenic activity. In the presence of sulfate, the methyl position of acetate was converted to CO2. The addition of sulfate to sediments did not result in the accumulation of significant amounts of sulfide in the pore water. Sulfate additions did not inhibit methanogenesis unless greater than 100 mug of free sulfide per ml was present in the pore water. These results indicate that carbon and electron flow are altered when sulfate is added to sediments. Sulfate-reducing organisms appear to assume the role of methanogenic bacteria in sulfate-containing sediments by utilizing methanogenic precursors.

Fermentation of mucins and plant polysaccharides by anaerobic bacteria from the human colon.

Abstract: A total of 154 strains from 22 species of Bifidobacterium, Peptostreptococcus, Lactobacillus, Ruminococcus, Coprococcus, Eubacterium, and Fusobacterium, which are present in high concentrations in the human colon, were surveyed for their ability to ferment 21 different complex carbohydrates. Plant polysaccharides, including amylose, amylopectin, pectin, polygalacturonate, xylan, laminarin, guar gum, locust bean gum, gum ghatti, gum arabic, and gum tragacanth, were fermented by some strains from Bifidobacterium, Peptostreptococcus, Ruminococcus, and Eubacterium species. Porcine gastric mucin, which was fermented by some strains of Ruminococcus torques and Bifidobacterium bifidum, was the only mucin utilized by any of the strains tested.

Growth of desulfovibrio in lactate or ethanol media low in sulfate in association with H2-utilizing methanogenic bacteria.

Abstract: In the analysis of an ethanol-CO2 enrichment of bacteria from an anaerobic sewage digestor, a strain tentatively identified as Desulfovibrio vulgaris and an H2-utilizing methanogen resembling Methanobacterium formicicum were isolated, and they were shown to represent a synergistic association of two bacterial species similar to that previously found between S organism and Methanobacterium strain MOH isolated from Methanobacillus omelianskii. In lowsulfate media, the desulfovibrio produced acetate and H2 from ethanol and acetate, H2, and, presumably, CO2 from lactate; but growth was slight and little of the energy source was catabolized unless the organism was combined with an H2-utilizing methanogenic bacterium. The type strains of D. vulgaris and Desulfovibrio desulfuricans carried out the same type of synergistic growth with methanogens. In mixtures of desulfovibrio and strain MOH growing on ethanol, lactate, or pyruvate, diminution of methane produced was stoichiometric with the moles of sulfate added, and the desulfovibrios grew better with sulfate addition. The energetics of the synergistic associations and of the competition between the methanogenic system and sulfate-reducing system as sinks for electrons generated in the oxidation of organic materials such as ethanol, lactate, and acetate are discussed. It is suggested that lack of availability of H2 for growth of methanogens is a major factor in suppression of methanogenesis by sulfate in natural ecosystems. The results with these known mixtures of bacteria suggest that hydrogenase-forming, sulfate-reducing bacteria could be active in some methanogenic ecosystems that are low in sulfate.

Methods for Growing Spirillum lipoferum and for Counting It in Pure Culture and in Association with Plants.

Abstract: Methods are described for growing Spirillum lipoferum in quantities sufficient to serve as inoculant in field trials of its associative N(2)-fixing ability with higher plants and as a source of cells for the preparation of nitrogenase, cytochromes, respiratory enzymes, etc A heavy inoculum of S lipoferum grown on NH(4) was transferred to a medium of minimal nitrogen content, and initial rapid growth at the expense of residual combined nitrogen was replaced later by slower growth on N(2) Conversion to N(2) fixation was prompt upon exhaustion of fixed nitrogen; growth on N(2) was most rapid at a pO(2) of 0005 to 0007 atm Numbers of S lipoferum can be estimated by diluting soil, crushed roots, or other material, and inoculating diluted samples into a stagnant semisolid medium Development of a characteristic subsurface layer of organisms and demonstration the these organisms can reduce C(2)H(2) are presumptive evidence that they are S lipoferum With most-probable-number tables the observations can be converted to numbers of S lipoferum in the samples The most-probable-number method indicated that numbers of S lipoferum may increase 100-fold or more in roots of maize removed from the plant and incubated for 24 h at 30 degrees C at a pO(2) initially adjusted to 001 atm

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

Abstract: 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.

Numerically Dominant Denitrifying Bacteria from World Soils

Abstract: Nineteen soils, three freshwater lake sediments, and oxidized poultry manure were examined to determine the dominant denitrifier populations. The samples, most shown or expected to support active denitrification, were from eight countries and included rice paddy, temperate agricultural, rain forest, organic, and waste-treated soils. Over 1,500 organisms that could grow anaerobically on nitrate agar were isolated. After purification, 146 denitrifiers were obtained, as verified by production of N2 from NO3-. These isolates were characterized by 52 properties appropriate for the Pseudomonas-Alcaligenes group. Numerical taxonomic procedures were used to group the isolates and compare them with nine known denitrifier species. The major group isolated was representative of Pseudonomas fluorescens biotype II. The second most prevalent group was representative of Alcaligenes. Other Pseudomonas species as well as members of the genus Flavobacterium, the latter previously not known to denitrify, also were identified. One-third of the isolates could not utilize glucose or other carbohydrates as sole carbon sources. Significantly, none of the numerically dominant denitrifiers we isolated resembled the most studied species: Pseudomonas denitrificans, Pseudomonas perfectomarinus, and Paracoccus denitrificans. Denitrification appears to be a property of a very diverse group of gram-negative, motile bacteria, as shown by the large number (22.6%) of ungrouped organisms. The diversity of denitrifiers from a given sample was usually high, with at least two groups present. Denitrifiers, nitrite accumulators, and organisms capable of anaerobic growth were present in the ratio of 0.20±0.23:0.81±0.23:1. There were few correlations between their numbers and the sample characteristics measured. However, the temperatures at which isolates could grow were significantly related to the temperatures of the environments from which they were isolated. Regression analysis revealed few relationships between physical parameters and bacterial types, save for the anaerobe numbers, in which 94% of the variance could be accounted for.

Microbial metabolic activity in soil as measured by dehydrogenase determinations

Abstract: The dehydrogenase technique for measuring the metabolic activity of microorganisms in soil was modified to use a 6-h, 37 degrees C incubation with either glucose of yeast extract as the electron-donating substrate. The rate of formazan production remained constant during this time interval, and cellular multiplication apparently did not occur. The technique was used to follow changes in the overall metabolic activities of microorganisms in soil undergoing incubation with a limiting concentration of added nutrient. The sequence of events was similar to that obtained by using the Warburg respirometer to measure O2 consumption. However, the major peaks of activity occurred earlier with the respirometer. This possibly is due to the lack of atmospheric CO2 during the O2 consumption measurements.

Factors affecting the cellulolytic activity of rumen contents.

Abstract: The cellulolytic activity of rumen contents was assayed by measuring losses in weight and tensile strength of cotton yarn incubated in rumen contents in the presence of dietary additives (barley, tallow) and at different pH values. The addition of barley depressed cellulolysis and the titer of filter paper-degrading bacteria only if the pH was allowed to fall. Lowering the pH from 7.0 to 6.0 by addition of HCl almost completely inhibited attack of cotton and greatly reduced the titer of filter paper-degrading bacteria. The layering of tallow on cotton inhibited attack of cotton but did not decrease the titer of filter paper-degrading bacteria. The results are discussed with special reference to the importance of the study of cellulosic substrates, which require a known enzyme or mixture of enzymes for attack.

Preparation of mutants of Trichoderma reesei with enhanced cellulase production.

Abstract: The development of an agar plate screening technique has allowed the isolation of a range of mutants of Trichoderma reesei capable of synthesizing cellulase under conditions of high catabolite repression. The properties of one of these mutants (NG-14) is described to illustrate the use of this technique. NG-14 produced five times the filter paper-degrading activity per ml of culture medium and twice the specific activity per mg of excreted protein in submerged culture when compared with the best existing mutant, QM9414. NG-14 also showed enhanced endo-beta-glucanase and beta-glucosidase production. Although these mutants were isolated as cellulase producers in the presence of 5% glycerol on agar plates, in similar liquid medium, NG-14 exhibits only partial derepression of the cellulase complex. Since the proportions of filter paper activity, endo-beta-glucanase, and cellobiase were not the same in mutants NG-14 and QM9414, and the yields of each enzyme under conditions repressive for cellulase synthesis were different, differential control of each enzyme of the cellulase complex is implied. These initial results suggest that the selective technique for isolating hyper-cellulase-producing mutants of Trichoderma will be of considerable use in the development of commercially useful cellulolytic strains.

Effect of monensin on rumen metabolism in vitro.

Abstract: The effect of Monensin (Rumensin, Eli Lilly & Co.) in incubations with mixed rumen microorganisms metabolizing carbohydrate or protein substrates was investigated. Monensin partly inhibited methanogenesis and increased propionate production, although the effect was not always statistically significant. Incubations with substrates specific for methane bacteria suggest that inhibition of methanogenesis by Monensin was not due to a specific toxic action on the methanogenic flora, but rather to an inhibition of hydrogen production from formate. Total and net microbial growth were considerably decreased by addition of Monensin, although the amount of substrate fermented was not altered, resulting in lowered values of microbial growth efficiency. In incubations with casein, Monensin lowered protein degradation in line with a lowered ammonia production, whereas a slight accumulation of alpha-amino nitrogen was observed. The results suggest that besides an influence of Monensin on the rumen carbohydrate fermentation pattern, another reason for the beneficial effects observed in vivo might be decreased food protein degradation in the rumen, altering the final site of protein digestion in the animal. Also, the possibility of a decrease in rumen microbial growth efficiency has to be considered when using Monensin as a food additive.

Deconjugation of bile acids by intestinal lactobacilli.

Abstract: Lactobacillus species normally found in the intestinal tract of humans varied in the ability to deconjugate bile acids, whereas laboratory strains of Lactobacillus acidophilus deconjugated both glycocholate and taurocholate. All isolates of L. acidophilus from human feces deconjugated taurocholate, whereas only one of six deconjugated glycocholate. None of 13 isolates identified as L. casei deconjugated taurocholate, whereas 9 deconjugated glycocholate. The deconjugating system of L. acidophilus appeared to be constitutive, required low oxidation-reduction potential, and was most active at pH 6. No degradation beyond deconjugation was detected.

Microtechnique for Most-Probable-Number Analysis

Abstract: A microtechnique based on the most-probable-number (MPN) method has been developed for the enumeration of the ammonium-oxidizing population in soil samples. An MPN table for a research design ([8 by 12] i.e., 12 dilutions, 8 replicates per dilution) is presented. A correlation of 0.68 was found between MPNs determined by the microtechnique and the standard tube technique. Higher MPNs were obtained with the microtechnique with increased accuracy in endpoint determinations being a possible cause. Considerable savings of time, space, equipment, and reagents are observed using this method. The microtechnique described may be adapted to other microbial populations using various types of media and endpoint determinations.

Generalized indicator plate for genetic, metabolic, and taxonomic studies with microorganisms.

Abstract: We have developed an indicator plate that works well for diverse types of substrates and microorganisms. The plates are inexpensive and easy to prepare. The essential components are agar, buffer, growth-supporting nutrients, a test substrate, and 2,3,5-triphenyl tetrazolium chloride (TTC). Using various strains of Salmonella typhimurium and Escherichia coli, we have studied and defined the contribution of each component to the satisfactory function of the plate. Colonies capable of catabolizing the test substrate reduce TTC and produce a deep red formazan, whereas colonies failing to catabolize the substrate remain uncoloured. Those with intermediate rates of catabolism differ in rate and/or extent of color formation. In all cases the color is stable because TTC reduction is essentially irreversible. Since the mode of action of these plates is fairly well understood, alternative formulations can be devised to meet specific needs. The general applicability of this TTC indicator system makes it an extremely useful tool in microbial genetics, metabolism, and taxonomy.

Survival of a psychrophilic marine Vibrio under long-term nutrient starvation.

Abstract: Ant-300, a psychrophilic marine vibrio isolated from the surface water of the Antarctic convergence, was starved for periods of more than 1 year. During the first week of starvation, cell numbers increased from 100 to 800% of the initial number of cells. Fifty percent of the starved cells remained viable for 6 to 7 weeks while a portion of the population remained viable for more than 1 year. During the first 2 days of starvation, the endogenous respiration of the cells decreased over 80%. After 7 days, respiration had been reduced to 0.0071% total carbon respired per hour and remained constant thereafter. After 6 weeks of starvation, 46% of the cellular deoxyribonucleic acid had been degraded. Observation of the cellular deoxyribonucleic acid with Feulgen staining before starvation showed the average number of nuclear bodies per cell varied from 1.44 to 4.02 depending on the age of the culture. A linear relationship was found between the number of nuclear bodies per cell and the increase in cell numbers upon starvation. Our data suggest that Ant-300 is capable of surviving long periods of time with little or no nutrients and is therefore well adapted for the sparse nutrient conditions of the colder portions of the open ocean.

Emetic and refusal activity of deoxynivalenol to swine.

Abstract: The minimum emetic dose of deoxynivalenol to swine weighing 9 to 10 kg was 0.05 mg/kg of body weight intraperitoneally and 0.1 to 0.2 mg/kg orally. There was no emesis by undosed pigs consuming vomitus from pigs orally dosed with deoxynivalenol or penned with such pigs without access to vomitus. Analysis by gas-liquid chromatography of a sample of Gibberella zeae-infected corn containing about 25% visually damaged kernels indicated 12 ppm of deoxynivalenol. Deoxynivalenol added to feed reduced feed consumption of 20- to 45-kg pigs, ranging from a 20% decrease with 3.6 ppm to 90% reduction with 40 ppm. Loss in weight was associated with feed refusal. Feed refusal, however, was much greater for naturally infected corn samples than for feeds with equal concentrations of the pure compound added, indicating the involvement of an additional factor(s) in the swine refusal response.

Toxicity and Mutagenicity of Hexavalent Chromium on Salmonella typhimurium

Abstract: Four hexavalent and two trivalent chromium compounds were tested for toxicity and mutagenicity by means of the Salmonella typhimurium/mammalian-microsome test All hexavalent compounds yielded a complete inhibition of bacterial growth at doses of 400 to 800 mug/plate, a significant increase of his/sup +/ revertant colonies at doses ranging from 10 to 200 mug, and no effect at doses of less than 10 mug The distinctive sensitivity of the four Salmonella strains tested (TA1535, TA1537, TA98, and TA100) suggested that hexavalent chromium directly interacts with bacterial deoxyribonucleic acid by causing both frameshift mutations and basepair substitutions The latter mutations, which are prevalent, are amplified by an error-prone recombinational repair of the damaged deoxyribonucleic acid On the average, 1 mumol of hexavalent chromium yielded approximately 500 revertants of the TA100 strain, irrespective of the compound tested (sodium dichromate, calcium chromate, potassium chromate, or chromic acid) The mutagenic potency of the hexavalent metal was not enhanced by adding the microsomal fraction of rat hepatocytes, induced either with sodium barbital or with Aroclor 1254 The two trivalent compounds (chromium potassium sulfate and chromic chloride), with or without the microsomal fraction, were neither toxic nor mutagenic for the bacterial tester strains

Construction of a kit of reference strains for rapid genetic mapping in Bacillus subtilis 168.

Abstract: A set of nine reference strains bringing convenient markers in the genetic background of Bacillus subtilis Marburg 168 has been prepared to allow rapid mapping of new markers.

Lipid accumulation in an oleaginous yeast (Candida 107) growing on glucose in single-stage continuous culture.

Abstract: Lipid accumulation of Candida 107, grown at dilution rates from 0.03 to the maximum of 0.21/h, with carbon, nitrogen, phosphate, and magnesium limitations in a chemostat, was maximal at about 40% (wt/wt) with nitrogen-limited medium at a dilution rate of 0.06/h, giving an efficiency of substrate conversion of 22 g of lipid per g of glucose consumed. At higher dilution rates the lipid content decreased. With carbon-limited growth, the highest lipid content (14%, wt/wt) was at the maximum dilution rate. High lipid contents also occurred with phosphate + nitrogen as double limitations of growth, with the lipid content of the yeast (about 35%, wt/wt) continuing to be near maximum at dilution rates also near maximum (0.17/h), thus giving the highest specific rate of lipid formation of any growth conditions (0.59 g of lipid/g of yeast per h). However, the efficiency of substrate utilization was only 5.2 g of lipid formed per 100 g of glucose consumed. The composition of the fatty acyl residues within the lipid remained constant over many weeks if the steady-state conditions remained unchanged. With carbon-limited growth, the degree of unsaturation of the fatty acids markedly decreased as the dilution rate was increased, but with nitrogen limitation the reverse trend was seen. In all cases, linoleic and oleic acids were the principal fatty acyl residues affected, and their relative proportions always varied in opposite directions. When magnesium was a limiting nutrient, there was a considerable increase in the proportion of myristic acid produced within the lipid. Neutral lipids (predominantly triglycerides) varied from 66 to 92% of the total lipid from carbon- and nitrogen-limited growth; phospholipids (varying from 2 to 25%) were highest in nitrogen-limited growth. The fatty acyl residues within each lipid fraction showed the same variations with changing growth rates.

Effect of Temperature, Aeration, and Moisture on CO(2) Formation in Bench-Scale, Continuously Thermophilic Composting of Solid Waste.

Abstract: A compost production system was employed to supply uniform material for controlled experiments of factorial design. Over a 96-h composting period, the cumulative amount of CO2 evolved was maximal at 56 to 60°C, an aeration rate that left an O2 residual of 10 or 18% in the exhaust gas and a moisture content of 60% wet weight. Carbon dioxide evolution was submaximal at 64°C and higher.

Sensitivity of Various Bacteria, Including Actinomycetes, and Fungi to Cadmium and the Influence of pH on Sensitivity.

Abstract: A variety of microorganisms, including gram-negative and gram-positive eubacteria, actinomycetes, yeasts, and filamentous fungi, were tested for their sensitivity to cadmium (Cd). In general, the actinomycetes were more tolerant to Cd than were the eubacteria; gram-negative eubacteria were more tolerant to Cd than were gram-positive eubacteria. The period of exponential growth of the eubacteria and actinomycetes was extended in the presence of Cd. Wide extremes in sensitivity to Cd were noted among the fungi; there was no correlation between the class of fungus and tolerance to Cd. Fungal sporulation was more sensitive to Cd than was mycelial growth, as spore formation was inhibited at Cd concentrations that were noninhibitory to mycelial proliferation. The toxicity of Cd to the eubacteria, actinomycetes, and fungi appeared to be pH dependent, as toxicity was generally potentiated at pH 8 or 9.

Occurrence and distribution of bacterial indicators and pathogens in canal communities along the Texas coast.

Abstract: Increased construction of residential canal communities along the southern coastline of the United States has led to a concern about their impact on water quality. Pollution of such dead-end canals is potentially hazardous because of their heavy usage for recreational activities. Coliforms, fecal coliforms, and salmonellae in the surface water and bottom sediments of six selected residential coastal canals were monitored over a period of 17 months. No statistically significant relationship was observed between the organism concentrations and temperature, pH, turbidity, and suspended solids content of water. An inverse relationship between the concentration of indicator organism and salinity of water was found, however, to occur at a 99.9% level of significance. All of the microorganisms studied were found to be present in greater numbers in sediments than in the overlying water, often by a factor of several logs. Heavy rainfall resulted in large increases in the number of organisms in both water and sediment samples. Our results indicate that bottom sediments in the shallow canal systems can act as reservoirs of enteric bacteria, which may be resuspended in response to various environmental factors and recreational activities.

Fermentation of cellulose by Ruminococcus flavefaciens in the presence and absence of Methanobacterium ruminantium.

Abstract: The anaerobic cellulolytic rumen bacterium Ruminococcus flavefaciens normally produces succinic acid as a major fermentation product together with acetic and formic acids, H2, and CO2. When grown on cellulose and in the presence of the methanogenic rumen bacterium Methanobacterium ruminantium, acetate was the major fermentation product; succinate was formed in small amounts; little formate was detected; H2 did not accumulate; and large amounts of CH4 were formed. M. ruminantium depends for growth on the reduction of CO2 to CH4 by H2, which it can obtain directly or by producing H2 and CO2 from formate. In mixed culture, the methanobacterium utilized the H2 and possibly the formate produced by the ruminococcus and in so doing stimulated the flow of electrons generated during glycolysis by the ruminococcus toward H2 formation and away from formation of succinate. This type of interaction may be of significance in determining the flow of cellulose carbon to the normal rumen fermentation products.

Changes in lipid composition of Escherichia coli resulting from growth with organic solvents and with food additives.

Abstract: Cells of Escherichia coli contain an altered fatty acid and phospholipid composition when grown in the presence of sublethal concentrations of a variety of organic solvents and food additives. The diversity of compounds examined which caused these changes indicates that no single catabolic pathway is involved. Many of the observed changes are consistent with the hypothesis that cells adapt their membrane lipids to compensate for the presence of these compounds in the environment. Both sodium benzoate and calcium propionate caused the synthesis of unusual fatty acids.

Thermophilic methane production from cattle waste.

Abstract: Methane production from waste of cattle fed a finishing diet was investigated, using four 3-liter-working volume anaerobic digestors at 60 degrees C. At 55 degrees C a start-up culture, in which waste was the only source of bacteria, was generated within 8 days and readily adapted to 60 degrees C, where efficiency of methanogenesis was greater. Increasing the temperature from 60 to 65 degrees C tended to drastically lower efficiency. When feed concentrations of volatile solids (VS, organic matter) were increased in steps of 2% after holding for 1 months at a given concentration, the maximum concentrations for efficient fermentation were 8.2, 10.0, 11.6, and 11.6% for the retention times (RT) of 3, 6, 9, and 12 days, respectively. The VS destructions for these and lower feed concentrations were 31 to 37, 36 to 40, 47 to 49 and 51 to 53% for the 3-, 6-, 9-, and 12-day RT digestors, respectively, and the corresponding methane production rates were about 0.16, 0.18, 0.20, and 0.22 liters/day per g of VS in the feed. Gas contained 52 to 57% methane. At the above RT and feed concentrations, alkalinity rose to 5,000 to 7,700 mg of CaCo3 per liter (pH to 7.5 to 7.8), NH3 plus NH4+ to 64 to 90 mM, and total volatile acids to 850 to 2,050 mg/liter as acetate. The 3-day RT digestor was quite stable up to 8.2% feed VS and at this feed concentration produced methane at the very high rate of 4.5 liters/day per liter of digestor. Increasing the percentage of feed VS beyond those values indicated above resulted in greatly decreased organic matter destruction and methane production, variable decrease in pH, and increased alkalinity, ammonia, and total volatile acid concentrations, with propionate being the first to accumulate in large amounts. In a second experiment with another lot of waste, the results were similar. These studies indicate that loading rates can be much higher than those previously thought useful for maximizing methanogenesis from cattle waste.

Metabolism of dibenzothiophene by a Beijerinckia species.

Abstract: Beijerinckia B8/36 when grown with succinate in the presence of dibenzothiophene, accumulated (+)-cis-1,2-dihydroxy-1,2-dihydrodibenzothiophene and dibenzothiophene-5-oxide in the culture medium. Each metabolite was isolated in crystalline form and characterized by a variety of chemical techniques, cis-Naphthalene dihydrodiol dehydrogenase, isolated from Pseudomonas putida, oxidized (+)-cis-1,2-dihydroxy-1,2-dihydrodibenzothiophene to a compound that was tentatively identified as 1,2-dihydroxydibenzothiophene. The same product was formed when crude cell extracts of the parent strain of Beijerinckia oxidized (+)-cis-1,2-dihydroxy-1,2-dihydrodibenzothiophene under anaerobic conditions. Further metabolism of 1,2-dihydroxydibenzothiophene by heat-treated cell extracts led to the formation of 4[2-(3-hydroxy)-thionaphthenyl]-2-oxo-3-butenoic acid. The latter compound was metabolized by crude cell extracts to 3-hydroxy-2-formylthionaphthene. Further degradation of this metabolite was not observed.

Use of Adenosine 5′-Triphosphate as an Indicator of the Microbiota Biomass in Rumen Contents

Abstract: A number of techniques were tested for their efficiency in extracting adenosine 5′-triphosphate (ATP) from strained rumen fluid (SRF). Extraction with 0.6 N H2SO4, using a modification of the procedure described by Lee et al. (1971), was the most efficient and was better suited for extracting particulate samples. Neutralized extracts could not be stored frozen before assaying for ATP because large losses were incurred. The inclusion of internal standards was necessary to correct for incomplete recovery of ATP. The ATP concentration in rumen contents from a cow receiving a ration of dried roughage (mainly alfalfa hay) ranged from 31 to 56 μg of ATP per g of contents. Approximately 75% of the ATP was associated with the particulate material. The ATP was primarily of microbial origin, since only traces of ATP were present in the feed and none was found in “cell-free” rumen fluid. Fractionation of the bacterial and protozoal populations in SRF resulted in the isolation of an enriched protozoal fraction with a 10-fold higher ATP concentration than that of the separated rumen bacteria. The ATP pool sizes of nine functionally important rumen bacteria during the exponential phase of growth ranged from 1.1 to 17.6 μg of ATP per mg of dry weight. This information indicates that using ATP as a measure of microbial biomass in rumen contents must be done with caution because of possible variations in the efficiency of extraction of ATP from rumen contents and differences in the concentration of ATP in rumen microbes.