Decomposition of the various chemical constituents etc. of complex plant materials by pure cultures of fungi and bacteria

Summary A series of experiments are reported which deal with the decomposition of complex plant materials by pure and mixed cultures of microorganisms.A complete proximate analysis has been made of the plant material undergoing decomposition.The results show that different organisms attack different chemical constituents, some preferring the water-soluble substances, hemicelluloses and nitrogenous complexes; others attack the cellulose and a few organisms are capable of decomposing the lignin.The processes of decomposition are accompanied by synthetic processes, new complexes being built up side by side with those that are being decomposed.The loss in weight as a result of decomposition is usually not a true index of the amount of decomposition that has taken place. Only an analysis of the various organic constituents will help to visualize the nature of the changes produced in the decomposition of plant material by microorganisms.The author is indebted to Miss F. G. Tenney and Mr. K. R. Stevens for assistance in carrying out some of these experiments.     

Anaerobic degradation of toluene by pure cultures of denitrifying bacteria

Several denitrifying Pseudomonas spp., isolated with various aromatic compounds, were tested for the ability to degrade toluene in the absence of molecular oxygen. Four out of seven strains were able to degrade toluene in the presence of N₂O. More than 50% of the ¹⁴C from ring-labelled toluene was released as CO₂, and up to 37% was assimilated into cell material. Furthermore it was demonstrated for two strains that they were able to grow on toluene as the sole carbon and energy source in the presence of N₂O. Suspensions of cells pre-grown on toluene degraded toluene, benzaldehyde or benzoate without a lag phase and without accumulation of intermediates. p-Cresol, p-hydroxybenzylalcohol, p-hydroxybenzaldehyde or p-hydroxybenzoate was degraded much slower or only after distinct lag times. In the presence of fluoroacetate [¹⁴C]toluene was transformed to [¹⁴C]benzoate, which suggests that anaerobic toluene degradation proceeds through oxidation of the methyl side chain to benzoate.     

A note on the fermentation of pectin by pure strains and combined cultures of rumen bacteria

Bacteroides ruminicola, pure or combined with Selenomonas ruminantium, and Lachnospira multiparus, pure or combined with Succinivibrio dextrinosolvens, were grown on a medium with pectin as energy source. There was a difference in fermentation products between the pure and combined cultures and efficiency of substrate utilization was better with the combined cultures.     

Degradation of trans-1,2-dichloroethene by mixed and pure cultures of methanotrophic bacteria

Summary Out of seven chlorinated aliphatic hydrocarbons tested, only trans-1,2-dichloroethene was relatively non-toxic for a mixed methanotrophic culture. The compound was degraded at a rate of 0.4 mol/mg protein·h^-1 and liberation of inorganic chloride was observed. Trans-2,3-dichlorooxirane was formed as an intermediate which was converted further only by chemical transformation with a half life of 31 h. From the consortium, a pure culture was isolated and found to be capable of degradation of trans-1,2-dichloroethene when grown in the presence of methane or methanol. The ability of cometabolic degradation of this compound was not specific for this isolate, since Methylomonas methanica NCIB11130 and Methylosinus trichosporium OB3b also showed degradation of trans-1,2-dichloroethene when grown with methane as sole carbon source.Abbreviations t12DCE trans-1,2-dichloroethene - t23DCO trans-2,3-dichlorooxirane - c23DCO cis-2,3-dichlorooxirane - ¹H-NMR proton nuclear magnetic resonance     

Rate of isolated hemicellulose degradation and utilization by pure cultures of rumen bacteria

Rate studies on the utilization or degradation (or both) of isolated hemicelluloses were conducted with six strains of rumen cellulolytic bacteria. Utilization was estimated by total pentose loss, and degradation values were based on solubilization of the hemicellulose in acidified 80% ethyl alcohol. With the various strains of ruminococci, degradation of flax and fescue grass hemicellulose was near the maximum within the first 12 hr of incubation. However, where applicable, the rates of utilization were considerably slower. Both degradation and utilization of corn hull hemicellulose occurred at much slower rates than observed with the other two substrates. With flax and fescue grass hemicellulose, the rates of degradation did not appear to be influenced by the organism's ability, or inability, to utilize the substrate as an energy source. The rates and extent of isolated hemicellulose degradation and utilization were compared between the cellulolytic ruminococci and three strains of bacteria isolated from the rumen with a xylan medium. Similar values were obtained with both types of bacteria. These observations would suggest that the cellulolytic ruminococci may be important in the overall fermentation of forage hemicelluloses in the rumen. The acidified 80% ethyl alcohol supernatant fluids, obtained from fermentations of isolated fescue grass hemicellulose by two strains of Ruminococcus flavefaciens, of which only one was able eventually to utilize the substrate, were investigated by thin-layer chromatography. Results indicated that soluble oligosaccharides were produced, which were observed to disappear gradually with time in fermentations with the utilizing strain and to accumulate in fermentations with the nonutilizing strain. Examination of the acidified 80% ethyl alcohol-insoluble residue hydrolysates, obtained from fermentations with the utilizing strain, revealed that the concentration of all the constituent sugars decreased uniformly.     

Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria

The rate of cellulose digestion in the presence of either glucose or cellobiose was studied for the three predominant species of cellulolytic rumen bacteria: Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes. When a soluble carbohydrate was added to cellulose broth, the lag phase of cellulose digestion was shortened. Presumably, this was due to greater numbers of bacteria, because increasing the size of the inoculum had a similar effect. Cellulose digestion occurred simultaneously with utilization of the soluble carbohydrate. The rate of cellulose digestion slowed markedly for B. succinogenes and R. flavefaciens and slowed less for R. albus after the cellobiose or glucose had been utilized, and was accompanied by a decrease in pH. Both the rate and the extent of cellulose digestion were partially inhibited when the initial pH of the medium was 6.3 or below. R. albus appeared to be less affected by a low-pH medium than were B. succinogenes and R. flavefaciens. When a soluble carbohydrate was added to the fermentation during the maximum-rate phase of cellulose digestion, the rate of cellulose digestion was not affected until after the soluble carbohydrate had been depleted and the pH had decreased markedly. Prolonged exposure of the bacteria to a low pH had little if any effect on their subsequent ability to digest cellulose. Cellulase activity of intact bacterial cells appeared to be constitutive in nature for these three species of rumen bacteria.     

Fermentative degradation of nonionic surfactants and polyethylene glycol by enrichment cultures and by pure cultures of homoacetogenic and propionate-forming bacteria

Linear alkyl ethoxylates (polyethylene glycol alkyl ethers) were fermented completely to methane and CO2 in enrichment cultures inoculated with anoxic sewage sludge. Long-chain fatty acids were released as intermediates. No degradation was found with polypropylene glycol and polypropylene glycol-containing surfactants. Two types of primary ethoxylate-degrading bacteria were isolated and characterized. Both degraded polyethylene glycols with molecular weights of 1,000 completely. Strain KoB35 fermented polyethylene glycol, ethoxyethanol, and lactate to acetate and propionate and was assigned to the described species Pelobacter propionicus. Strain KoB58 converted polyethylene glycol and many other substrates to acetate only and was assigned to the genus Acetobacterium. The pathways of anaerobic degradation of nonionic surfactants are discussed with respect to their limitations and the various groups of bacteria involved.     

Transformation of mercuric chloride and methylmercury by the rumen microflora

The microflora in strained rumen fluid did not methylate or volatilize 203Hg2+ at detectable rates. However, there was an exponential decay in the concentration of added CH3Hg+, which was attributed to demethylation. The major product of demethylation was metallic mercury (Hg0), and it was released as a volatile product from the reaction mixture. Demethylation occurred under both anaerobic and aerobic conditions. The rate of demethylation was proportional to the concentration of added CH3Hg+-Hg from 0.02 to 100 microgram of Hg per ml. The presence of HgCl2 had almost no inhibitory effect on the rate of cleavage of the carbon-mercury bond of CH2HgCl, but it completely inhibited volatilization of the Hg formed, when the concentration of HgCl2-Hg reached 100 micrograms/ml. Three of 11 species of anaerobic rumen bacteria catalyzed demethylation. These were Desulfovibrio desulfuricans, Selenomonas ruminantium, and Megasphaera elsdenii. None of the 11 species caused detectable methylation, and only two caused limited volatilization of Hg2+. Three species of bacteria out of 90 fresh aerobic isolates from rumen contents were demethylators: two were identified as Pseudomonas sp., and the third was a Micrococcus sp. Demethylation by the rumen microflora appeared to be carried out by both aerobic and anaerobic bacteria and, on the basis of Hg2+ sensitivity, probably resulted from the activity of two enzymes, a CH3-Hg+ hydrolase and a Hg2+ reductase.     

Effect of soluble carbohydrates on digestion of cellulose by pure cultures of rumen bacteria.

The rate of cellulose digestion in the presence of either glucose or cellobiose was studied for the three predominant species of cellulolytic rumen bacteria: Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes. When a soluble carbohydrate was added to cellulose broth, the lag phase of cellulose digestion was shortened. Presumably, this was due to greater numbers of bacteria, because increasing the size of the inoculum had a similar effect. Cellulose digestion occurred simultaneously with utilization of the soluble carbohydrate. The rate of cellulose digestion slowed markedly for B. succinogenes and R. flavefaciens and slowed less for R. albus after the cellobiose or glucose had been utilized, and was accompanied by a decrease in pH. Both the rate and the extent of cellulose digestion were partially inhibited when the initial pH of the medium was 6.3 or below. R. albus appeared to be less affected by a low-pH medium than were B. succinogenes and R. flavefaciens. When a soluble carbohydrate was added to the fermentation during the maximum-rate phase of cellulose digestion, the rate of cellulose digestion was not affected until after the soluble carbohydrate had been depleted and the pH had decreased markedly. Prolonged exposure of the bacteria to a low pH had little if any effect on their subsequent ability to digest cellulose. Cellulase activity of intact bacterial cells appeared to be constitutive in nature for these three species of rumen bacteria.     

Fermentative degradation of nonionic surfactants and polyethylene glycol by enrichment cultures and by pure cultures of homoacetogenic and propionate-forming bacteria.

Linear alkyl ethoxylates (polyethylene glycol alkyl ethers) were fermented completely to methane and CO2 in enrichment cultures inoculated with anoxic sewage sludge. Long-chain fatty acids were released as intermediates. No degradation was found with polypropylene glycol and polypropylene glycol-containing surfactants. Two types of primary ethoxylate-degrading bacteria were isolated and characterized. Both degraded polyethylene glycols with molecular weights of 1,000 completely. Strain KoB35 fermented polyethylene glycol, ethoxyethanol, and lactate to acetate and propionate and was assigned to the described species Pelobacter propionicus. Strain KoB58 converted polyethylene glycol and many other substrates to acetate only and was assigned to the genus Acetobacterium. The pathways of anaerobic degradation of nonionic surfactants are discussed with respect to their limitations and the various groups of bacteria involved.     

Esterase activity of pure cultures of rumen bacteria as expressed by the hydrolysis of p-nitrophenylpalmitate

Seventy-four strains of rumen bacteria comprising 20 genera were tested for the ability to hydrolyze p-nitrophenylpalmitate (PNPP-C16). This ability was detectable in all cultures tested, but the level of activity was quite variable. Known lipolytic strains of these bacteria showed generally low levels of activity in this assay, which suggests that the hydrolysis of this artificial substrate indicates a general esterase activity and not a lipase activity, as reported in the literature. The highest activity was found to occur in strains known to be feed-particle-associated digesters of starch, pectin and cellulose. In fractionated rumen contents, p-nitrophenylpalmitase activity was largely associated with feed particles. Although the in vivo role of the enzymes that hydrolyze PNPP-C16 remains obscure, it appears that they are primarily of microbial origin, and may be important in hydrolyzing ester bond-containing compounds from plant material.     

Ammonia-induced injury in pure cultures and natural populations of coliform bacteria

Ammonia-induced injury was investigated in pure cultures of Escherichia coli and Enterobacter aerogenes, and in natural coliform populations obtained from the oligotrophic Luxapallila and the eutrophic Sunflower Rivers in northern Mississippi. Pure cultures were affected by ammonia exposure as indicated by changes in the injury ratio (IR) of CFU on m-T7 agar/CFU on m-Endo agar. Ammonia concentrations between 0 and 20 (mg NH3-N/1) had little or no effect and concentrations between 40 and 80 caused the greatest injury. Natural coliform populations from the oligotrophic river were more prone to ammonia-induced injury than those from the eutrophic river. The results stress the need for the routine use of m-T7 media and the enumeration of injured cells when using the membrane filter procedure to ascertain domestic water quality.     

Studies on the mechanism of fungicidal action of mercuric chloride on mycelial felts of Rhizoctonia solani

Summary Mercuric chloride induced strong inhibitory effect on the growth, respiration and carbohydrate synthesis by mycelial felts of R. solani. Such inhibitory effects can be antagonised by the amino acid cysteine when mixed with the toxin in the nutritive medium. Methionine failed to do so. The possible explanations for the inhibitory actions of mercuric chloride are thoroughly discussed.     

Transformation of mercuric chloride and methylmercury by the rumen microflora.

The microflora in strained rumen fluid did not methylate or volatilize 203Hg2+ at detectable rates. However, there was an exponential decay in the concentration of added CH3Hg+, which was attributed to demethylation. The major product of demethylation was metallic mercury (Hg0), and it was released as a volatile product from the reaction mixture. Demethylation occurred under both anaerobic and aerobic conditions. The rate of demethylation was proportional to the concentration of added CH3Hg+-Hg from 0.02 to 100 microgram of Hg per ml. The presence of HgCl2 had almost no inhibitory effect on the rate of cleavage of the carbon-mercury bond of CH2HgCl, but it completely inhibited volatilization of the Hg formed, when the concentration of HgCl2-Hg reached 100 micrograms/ml. Three of 11 species of anaerobic rumen bacteria catalyzed demethylation. These were Desulfovibrio desulfuricans, Selenomonas ruminantium, and Megasphaera elsdenii. None of the 11 species caused detectable methylation, and only two caused limited volatilization of Hg2+. Three species of bacteria out of 90 fresh aerobic isolates from rumen contents were demethylators: two were identified as Pseudomonas sp., and the third was a Micrococcus sp. Demethylation by the rumen microflora appeared to be carried out by both aerobic and anaerobic bacteria and, on the basis of Hg2+ sensitivity, probably resulted from the activity of two enzymes, a CH3-Hg+ hydrolase and a Hg2+ reductase.     

Inhibition by methyl mercury chloride and mercuric chloride of the in vitro polymerization of microtubules

Mercury was tested at the same concentration but under two different forms, organic CH3HgCl and inorganic HgCl2, in order to compare its relative inhibitory effect on in vitro microtubules polymerization. Induced by GTP and glycerol 8 M, tubulin polymerization was completely inhibited by HgCl2 10(-3) M while a 75.8% inhibition was measured for CH3HgCl2 10(-3) M.