Evidence of hydrolytic route for anaerobic cyanide degradation.

Products observed during anaerobic cyanide transformation are consistent with a hydrolytic pathway (HCN + H2O <--> HCONH2 + H2O <--> HCOOH + NH3). Formate, the most frequently observed product, was generally converted to bicarbonate. Formamide was rapidly hydrolyzed to formate upon exposure to the anaerobic consortium but was not detected as an intermediate of cyanide transformation.     

Evidence for anaerobic syntrophic benzoate degradation threshold and isolation of the syntrophic benzoate degrader.

An anaerobic, motile, gram-negative, rod-shaped, syntrophic, benzoate-degrading bacterium, strain SB, was isolated in pure culture with crotonate as the energy source. Benzoate was degraded only in association with an H2-using bacterium. The kinetics of benzoate degradation by cell suspensions of strain SB in coculture with Desulfovibrio strain G-11 was studied by using progress curve analysis. The coculture degraded benzoate to a threshold concentration of 214 nM to 6.5 microM, with no further benzoate degradation observed even after extended incubation times. The value of the threshold depended on the amount of benzoate added and, consequently, the amount of acetate produced. The addition of sodium acetate, but not that of sodium chloride, affected the threshold value; higher acetate concentrations resulted in higher threshold values for benzoate. When a cell suspension that had reached a threshold benzoate concentration was reamended with benzoate, benzoate was used without a lag. The hydrogen partial pressure was very low and formate was not detected in cell suspensions that had degraded benzoate to a threshold value. The Gibbs free energy change calculations showed that the degradation of benzoate was favorable when the threshold was reached. These studies showed that the threshold for benzoate degradation was not caused by nutritional limitations, the loss of metabolic activity, or inhibition by hydrogen or formate. The data are consistent with a thermodynamic explanation for the existence of a threshold, but a kinetic explanation based on acetate inhibition may also account for the existence of a threshold.     

Microbes and microbial enzymes for cyanide degradation

Cyanide is an important industrial chemical produced on a grand scale each year. Although extremely toxic to mammalian life, cyanide is a natural product generated by fungi and bacteria, and as a result microbial systems have evolved for the degradation of cyanide to less toxic compounds. The enzymes which utilize cyanide as a substrate can be categorized into the following reaction types: substitution/addition, hydrolysis, oxidation, and reduction. Each of these categories is reviewed with respect to the known biochemistry and feasibility for use in treatment of cyanide containing wastes.     

Biological degradation of cyanide compounds

Cyanide compounds are produced as waste products of a number of industrial processes and several routes for their removal from the environment are under investigation, including the use of biodegradation. The most recent developments in this area have come from studies of the hydrolytic and oxidative pathways for biodegradation and the conditions that affect their activity. The biodegradation of cyanide under anaerobic conditions has also recently demonstrated the feasibility for concomitant biogas generation, a possible economic benefit of the process. Significant advances have been reported in the use of plants for the phytoremediation of cyanide compounds and evidence for the biodegradation of thiocyanate and metal-cyanide complexes has become available. Despite these advances, however, physical and economic factors still limit the application of cyanide biodegradation, as do competing technologies.     

Kinetics of enzymatic degradation of cyanide

CYANIDASE(@) is a new enzyme preparation capable of degrading cyanide in industrial wastewaters to ammonia and formate in an apparently one-step reaction, down to very low concentrations. This enzyme has both a high selectivity and affinity toward cyanide. A granular form of the biocatalyst was used in a recirculation fixed bed reactor in order to characterize the new biocatalyst with respect to pH, ionic strength, common ions normally present in wastewaters, mass transfer effects, and temperature. Long term stability was investigated. The kinetics of the enzymatic degradation of cyanide were studied in a batch reactor using the powdered immobilized enzyme preparation and modeled using a simple Michaelis-Menten equation.     

Microbial degradation of cyanide and thiocyanate

The role played by a bacterial community composed ofPseudomonas putida, strain 21;Pseudomonas stutzeri, strain 18; andPseudomonas sp., strain 5, and by physical and chemical factors in the degradation of CN⁻ and SCN⁻ was studied. It was shown that the degradation of CN⁻ is determined both by the action of bacteria and by abiotic physical and chemical factors (pH, O₂, temperature, the medium agitation rate, etc.). The contribution of chemical degradation was found to increase drastically at pH below 9.0; when air was blown through the medium (irrespective of the pH value); under active agitation of the medium; and when the medium surface interfacing air was increased. Even at elevated pH values (9.0-9.2), suboptimal for bacterial growth, the microbial degradation could account for at most 20–25 mg/1 of CN⁻, regardless of its initial concentration. When CN⁻ and SCN⁻ were concurrently present in the medium, the former compound was the first to be degraded by microorganisms. The rate of bacterial degradation of SCN⁻ under continuous cultivation in a chain of reactors was found to depend on its concentration, the medium flow rate, agitation rate, and the pattern of carbon source supply and could exceed 1 g/(l day). CN⁻ and SCN⁻ are utilized by bacteria solely as nitrogen sources. The mechanism of CN⁻ and SCN⁻ degradation by the microbial community is discussed. Deceased.     

Evidence for cyanide and mercury inactivation of endogenous plastocyanin

Cyanide and mercury treatment of chloroplast membranes inactivates plastocyanin as shown by the inability of the extracted plastocyanin to restore electron transport in a bioassay on chloroplasts depleted of their endogenous plastocyanin by digitonin treatment. The extraction procedure did remore the enzyme from cyanide and mercury treated chloroplasts as shown by sodium dodecyl sulfate polyacrylamide electrophoresis of the extracts. This procedure normally shows a plastocyanin band at 11,000 dalton molecular weight and the band was present in extracts from control and cyanide or mercury treated membranes.     

Antibiotic bacitracin induces hydrolytic degradation of nucleic acids

Background

Bacitracin is a polypeptide antibiotic active against Gram-positive bacterial strains. Its mechanism of action postulates disturbing the cell wall synthesis by inhibiting dephosphorylation of the lipid carrier. We have discovered that bacitracin induces degradation of nucleic acids, being particularly active against RNA.

Methods

In the examination of the nucleolytic activity of bacitracin several model RNA and DNA oligomers were used. The oligomers were labeled at their 5′ ends with ³²P radioisotope and following treatment with bacitracin the cleavage sites and efficiency were determined.

Results and conclusions

Bacitracin induces degradation of RNA at guanosine residues, preferentially in single-stranded RNA regions. Bacitracin is also able to degrade DNA to some extent but comparable effects to those observed with RNA require its 10-fold higher concentration. The sites of degradation in DNA are very infrequent and preferentially occur near cytidine residues. Free radicals are not involved in the reaction, and which probably proceeds via a hydrolytic mechanism. The phosphate groups at the cleavage sites are present at the 3' ends of RNA products and at the 5' ends of DNA fragments. Importantly, the presence of EDTA does not influence RNA degradation but completely inhibits the degradation of DNA. For DNA degradation divalent metal ions like Mg^2 +, Mn^2 + or Zn^2 + are absolutely necessary.

General significance

The ability of bacitracin to degrade nucleic acids via a hydrolytic mechanism was a surprising observation, and it is of interest whether these properties can contribute to its mechanisms of action during antibiotic treatment.     

Limited degradation of chlorophenols by anaerobic sludge granules.

To better understand the fate of chlorophenols treated in upflow anaerobic sludge bed reactors, we examined the ability of sludge granules from such bioreactors to degrade two trichlorophenols and one dichlorophenol in batch incubations under controlled conditions. Biodegradation was primarily limited to two distinct activities, reductive dehalogenation of ortho- and of meta-chlorine substituents. Both 3- and 4-monochlorophenol were persistent degradation products, while 2-monochlorophenol was further degraded. We also examined factors potentially affecting the rate and extent of 2,3,6-trichlorophenol degradation. An initial concentration of up to 1.75 mM (346 mg/liter) was dehalogenated. At that concentration, dehalogenation was partially inhibited but methanogenesis from formate was not. The initial concentration affected both the extent of dehalogenation and which products were detected. The maximum dechlorination rate observed was 1.4 mumol of Cl- h-1 g of volatile suspended solids-1. Dechlorination had a temperature optimum of 50 degrees C, was inhibited by added electron acceptors, and was not appreciably affected by added electron donors. The availability of electron acceptors and electron donors did not affect the extent of chlorophenol degradation. These particular sludge granules do not appear to be capable of mineralizing phenols with meta- or para-chlorine substituents.     

Limited degradation of chlorophenols by anaerobic sludge granules.

To better understand the fate of chlorophenols treated in upflow anaerobic sludge bed reactors, we examined the ability of sludge granules from such bioreactors to degrade two trichlorophenols and one dichlorophenol in batch incubations under controlled conditions. Biodegradation was primarily limited to two distinct activities, reductive dehalogenation of ortho- and of meta-chlorine substituents. Both 3- and 4-monochlorophenol were persistent degradation products, while 2-monochlorophenol was further degraded. We also examined factors potentially affecting the rate and extent of 2,3,6-trichlorophenol degradation. An initial concentration of up to 1.75 mM (346 mg/liter) was dehalogenated. At that concentration, dehalogenation was partially inhibited but methanogenesis from formate was not. The initial concentration affected both the extent of dehalogenation and which products were detected. The maximum dechlorination rate observed was 1.4 mumol of Cl- h-1 g of volatile suspended solids-1. Dechlorination had a temperature optimum of 50 degrees C, was inhibited by added electron acceptors, and was not appreciably affected by added electron donors. The availability of electron acceptors and electron donors did not affect the extent of chlorophenol degradation. These particular sludge granules do not appear to be capable of mineralizing phenols with meta- or para-chlorine substituents.     

Analeptic agent from microbes upon cyanide degradation

Applied Microbiology and Biotechnology - Microbes being the initial form of life and ubiquitous in occurrence, they adapt to the environment quickly. The microbial metabolism undergoes alteration...     

Sequential anaerobic degradation of 2,4-dichlorophenol in freshwater sediments.

2,4-Dichlorophenol (2,4-DCP) was anaerobically degraded in freshwater lake sediments. From observed intermediates in incubated sediment samples and from enrichment cultures, the following sequence of transformations was postulated. 2,4-DCP is dechlorinated to 4-chlorophenol (4-CP), 4-CP is dechlorinated to phenol, phenol is carboxylated to benzoate, and benzoate is degraded via acetate to methane and CO2; at least five different organisms are involved sequentially. The rate-limiting step was the transformation of 4-CP to phenol. Sediment-free enrichment cultures were obtained which catalyzed only the dechlorination of 2,4-DCP, the carboxylation of phenol, and the degradation of benzoate, respectively. Whereas the dechlorination of 2,4-DCP was not inhibited by H2, the dechlorination of 4-CP, and the transformation of phenol and benzoate were. Low concentrations of 4-CP inhibited phenol and benzoate degradation. Transformation rates and maximum concentrations allowing degradation were determined in both freshly collected sediments and in adapted samples: at 31 degrees C, which was the optimal temperature for the dechlorination, the average adaptation time for 2,4-DCP, 4-CP, phenol, and benzoate transformations were 7, 37, 11 and 2 days, respectively. The maximal observed transformation rates for these compounds in acclimated sediments were 300, 78, 2, 130, and 2,080 micromol/liter(-1)/day(-1), respectively. The highest concentrations which still allowed the transformation of the compound in acclimated sediments were 3.1 m/M 2,4-DCP, 3.1 mM 4-CP, 13 mM phenol, and greater than 52 mM benzoate. The corresponding values were lower for sediments which had not been adapted for the transformation steps.     

Evidence for osmotic regulation of hydrolytic enzyme production in germinating barley seeds

α-Amylase levels in intact seeds of barley (Hordeum vulgare L. cv. Himalaya) reach a maximum at 3 to 4 days of germination while gibberellin levels continue to increase beyond 6 days of germination. In contrast to its effect on half seeds, gibberellic acid does not increase the total amount of α-amylase produced in germinating seeds. The inability of gibberellic acid to stimulate α-amylase production is not related to its availability; rather, evidence suggests that a factor(s) in whole seeds prevents further enhancement of α-amylase formation and accumulation. Hydrolysis products accumulate in the subaleurone space of the endosperm of germinating seeds up to concentrations of 570 milliosmolar. Chromatography of these hydrolysis products indicate the presence of maltose and glucose. Calculations based on reducing sugar determinations show that glucose accounts for as much as 57% of the solutes present in the endosperm fluid. Both maltose and glucose in the range of 0.2 to 0.4 M effectively inhibit the production of α-amylase by isolated barley aleurone layers. This inhibition is quantitatively similar to that brought about by solutions of polyethylene glycol and mannitol. On the basis of these data we propose that hydrolysis products which accumulate in the starchy endosperm of germinating seeds function to regulate the production of hydrolytic enzymes by the aleurone layer.     

Tetrachloroethylene as electron acceptor for the anaerobic degradation of benzoate

Abstract Teltrachloroethylene (PCE) was biotransformed by reductive dehalgenation under anoxic conditions with benzoate as the electron donor. The experiments were carried out under batch culture conditions with biomass from an anoxic fixed bed reactor fed with benzoate and PCE. Inhibition of methanogenesis by bromoethane-sulfonic acid (BES) resulted in a complete inhibition of benzoate degradation. Benzoate, however, was decomposed in the presence of BES if PCE was added to the cultures. With 2.8 mmol/1 PCE, that was transformed to 1.4 mmol/1 cis-1,2-dichloroethylene (DCE) and 3.8 mmol/1 chloride, 2 mmol/1 benzoate were degraded to about 3.2 mmol/1 acetate. The elimination of benzoate was directly proportional to DCE accumulation, ranging between 1:0.5 and 1:1.     

Influence of hydrogen-consuming bacteria on cellulose degradation by anaerobic fungi.

The presence of methanogens Methanobacterium arboriphilus, Methanobacterium bryantii, or Methanobrevibacter smithii increased the level of cellulose fermentation by 5 to 10% in cultures of several genera of anaerobic fungi. When Neocallimastix sp. strain L2 was grown in coculture with methanogens the rate of cellulose fermentation also increased relative to that for pure cultures of the fungus. Methanogens caused a shift in the fermentation products to more acetate and less lactate, succinate, and ethanol. Formate transfer in cocultures of anaerobic fungi and M. smithii did not result in further stimulation of cellulolysis above the level caused by H2 transfer. When Selenomonas ruminatium was used as a H2-consuming organism in coculture with Neocallimastix sp. strain L2, both the rate and level of cellulolysis increased. The observed influence of the presence of methanogens is interpreted to indicate a shift of electrons from the formation of electron sink carbon products to H2 via reduced pyridine nucleotides, favoring the production of additional acetate and probably ATP. It is not known how S. ruminantium exerts its influence. It might result from a lowered production of electron sink products by the fungus, from consumption of electron sink products or H2 by S. ruminantium, or from competition for free sugars which in pure culture could exert an inhibiting effect on cellulolysis.     

Metabolic by-products of anaerobic toluene degradation by sulfate-reducing enrichment cultures.

Two dead-end metabolites of anaerobic toluene transformation, benzylsuccinic acid and benzylfumaric acid, accumulated in sulfate-reducing enrichment cultures that were fed toluene as the sole carbon source. Stable isotope-labeled toluene and gas chromatography-mass spectrometry were used to confirm that the compounds resulted from toluene metabolism. The two metabolites constituted less than 10% of the toluene carbon (over 80% was mineralized to carbon dioxide, according to a previous study). This study demonstrates that the novel nonproductive pathway proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for a denitrifying pure culture applies to disparate anaerobic bacteria.     

Metabolic by-products of anaerobic toluene degradation by sulfate-reducing enrichment cultures.

Two dead-end metabolites of anaerobic toluene transformation, benzylsuccinic acid and benzylfumaric acid, accumulated in sulfate-reducing enrichment cultures that were fed toluene as the sole carbon source. Stable isotope-labeled toluene and gas chromatography-mass spectrometry were used to confirm that the compounds resulted from toluene metabolism. The two metabolites constituted less than 10% of the toluene carbon (over 80% was mineralized to carbon dioxide, according to a previous study). This study demonstrates that the novel nonproductive pathway proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for a denitrifying pure culture applies to disparate anaerobic bacteria.