Search

The biodegradability of three aliphatic adipic acid diesters and a 1,3-butylene glycol adipic acid polyester was determined in acclimated, activated sludge systems. Rapid primary biodegradation from 67 to 99+% was observed at 3- and 13-mg/liter feed levels for di-n-hexyl adipate, di(2-ethylhexyl) adipate, and di(heptyl, nonyl) adipate in 24 h. When acclimated, activated sludge microorganisms were employed as the seed for two carbon dioxide evolution procedures, greater than 75% of the theoretical carbon dioxide was evolved for the three diesters and the polyester in a 35-day test period. The essentially complete biodegradation observed in these studies suggests that these esters would not persist when exposed to similar mixed microbial populations in the environment.

An anaerobic population of bacteria became acclimated to catechol and phenol in 32 and 18 days, respectively. Evidence from carbon balance measurements indicates that the aromatic ring is cleaved and that the products are stoichiometrically fermentable to methane and carbon dioxide.

The Y-ATP (energy efficiency) of intraperiplasmic growth of Bdellovibrio bacteriovorus was determined from the distribution of radioactivity of the substrate organism ([U-14C]Escherichia coli) btween CO2 and bdellovibrio cells at the end of growth. A "best" Y-ATP value of 18.5 was obtained from single growth cycle experiments and an average value of 25.9 from multicycle experiments. Both values are much higher than the usual value of 10.5 for bacteria growing in rich media. The bases for the unusual energy efficiency for growth of B. bacteriovorus are discussed.

The fate of 14C-labeled 2,4,6-trinitrotoluene (TNT) in an activated-sludge system was investigated. No [14C]TNT could be detected in the contents of an aerated reactor after 3 to 5 days of incubation. No significant 14CO2 was formed, and the radioactivity was about equally divided between the floc and the supernatant. The radioactive carbon present in the microflora was mainly associated with the lipid and protein components, but the characteristic constituents of these compounds (e.g., fatty acids and amino acids) were not radioactive. The major part of the 14C present in the lipid and protein fractions was found in precipitates that formed in both fractions. The solubility properties and infrared spectra of these precipitates suggested that they are macromolecular structures of the polyamide type formed by the reaction of TNT biotransformation products with lipids, fatty acids, and protein constituents of the microbial flora. This hypothesis is further supported by the correspondence of the infrared spectrum of the lipid precipitate with that of a model compound synthesized from TNT transformation products and lipid precursors. The resistance of these macromolecules to further biodegradation was paralleled by the reported resistance to microbial attack of polyamides containing similar linkages.

The primary and ultimate biodegradability of phthalic acid, monobutyl phthalate, and five structurally diverse phthalic acid ester plasticizers in river water and activated sludge samples were determined via ultraviolet spectrophotometry, gas chromatography, and CO2 evolution. The compounds studied underwent rapid primary biodegradation in both unacclimated river water and acclimated activated sludge. When activated sludge acclimated to phthalic acid esters was used as the inoculum for the CO2 evolution procedure, greater than 85% of the total theoretical CO2 was evolved. These studies demonstrate that the phthalic acid ester plasticizers and intermediate degradation products readily undergo ultimate degradation in different mixed microbial systems at concentrations ranging from 1 to 83 mg/liter.

A simple method for measuring gas production by microorganisms by using a pressure transducer to sense pressure buildup was developed and tested with members of the coliform group. The test system consisted of a 5.0 lb/in(2) pressure transducer and a pressure equalizer valve attached to the metal cap of a test tube (20 by 150 mm); gas pressure was recorded on a strip-chart recorder. Gas pressure response curves consisted of (i) a lag period with no marked increase in pressure, (ii) a rapid pressure buildup period, and (iii) a leveling-off period. A linear relationship was established between inoculum size and length of the lag period. Cultures shaken at 200 oscillations/min showed a marked increase in rate of gas release over stationary cultures. Cell concentrations at the time of rapid buildup in pressure were 10(8)/ml. Mean maximum pressure recordings, lb/in(2) per 10 ml of broth, were: Enterobacter aerogenes, 3.70; Citrobacter intermedium, 2.70; and Escherichia coli, 2.10. Mean CO(2) concentrations, ppm of headspace gas, for E. coli were: (i) 2,000 at time of inoculation, (ii) 25,000 at time of rapid buildup in pressure, and (iii) 150,000 at maximum pressure. These results indicate the potential application of the pressure transducer method for rapidly detecting coliforms and other gas-producing microorganisms in clinical samples and in sterility testing of foods.

Microorganisms present in Atlantic Ocean sediment samples collected at a depth of 4,940 m were found to be capable of utilizing hydrocarbons under both ambient and in situ pressures. The rate of utilization under in situ pressure (500 atm) and ambient temperature (20 C) was found to be significantly less compared with hydrocarbon utilization examined under conditions of ambient temperature (20 C) and pressure (1 atm).

In the analysis of an ethanol-CO(2) enrichment of bacteria from an anaerobic sewage digestor, a strain tentatively identified as Desulfovibrio vulgaris and an H(2)-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 H(2) from ethanol and acetate, H(2), and, presumably, CO(2) from lactate; but growth was slight and little of the energy source was catabolized unless the organism was combined with an H(2)-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 H(2) 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.

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.

The experiments were confined to the effects of the addition of different sources of carbon (glucose, wheat straw, and sawdust) on the microbial activities in soils: loamy sand, loam and saline clay were used. The parameters used for this purpose were total bacterial counts, dehydrogenase test, CO2 evolution, and oxidation of organic carbon. Salinity of soils had deleterious effects on all the parameters used. Addition of organic matter resulted in marked increases in bacterial counts, formazan production, and CO2 evolution. Glucose showed the most rapid effect and highest levels. Wheat straw yielded the highest results and sawdust always showed the least effect.

A method, based on quantitating 14CO2 produced from [14C]hexadecane, has been developed for estimating the rate of hexadecane decomposition in seawater of Tokyo Bay during the summer stagnation period. The rate of hexadecane decomposition was from 0.1 to 1.3 mug/h per liter of seawater at the surface layer in the polluted gyre of the inner part of Tokyo Bay during the summer of 1974. A similar horizontal distribution pattern was seen for the density of hexadecane-decomposing bacteria.

The biodegradability of seven different crude oils was found to be highly dependent on their composition and on incubation temperature. At 20 C lighter oils had greater abiotic losses and were more susceptible to biodegradation than heavier oils. These light crude oils, however, possessed toxic volatile components which evaporated only slowly and inhibited microbial degradation of these oils at 10 C. No volatile toxic fraction was associated with the heavier oils tested. Rates of oil mineralization for the heavier oils were significantly lower at 20 C than for the lighter ones. Similar relative degradation rates were found with a mixed microbial community, using CO2 evolution as the measure, and with a Pseudomonas isolate from the Arctic, using O2 consumption as the measure. The paraffinic, aromatic, and asphaltic fractions were subject to biodegradation. Some preference was shown for paraffin degradation, especially at low temperatures. Branched paraffins, such as pristane, were degraded at both 10 and 20 C. At best, a 20% residue still remained after 42 days of incubation. Oil residues generally had a lower relative percentage of paraffins and higher percentage of asphaltics than fresh or weathered oil.

Methods are described for the detection of low numbers of bacteria by monitoring (14)CO(2) evolved from (14)C-labeled substrates. Cell suspensions are filtered with membrane filters, and the filter is then moistened with 0.1 ml of labeled medium in a small, closed apparatus. Evolved (14)CO(2) is collected with Ba(OH)(2)-moistened filter pads and assayed with conventional radioactivity counting equipment. The kinetics of (14)CO(2) evolution are shown for several species of bacteria. Fewer than 100 colony-forming units of most species tested were detected in 2 h or less. Bacteria were inoculated into blood and the mixture was treated to lyse the blood cells. The suspension ws filtered and the filter was placed in a small volume of labeled medium. The evolved (14)CO(2) was trapped and counted. A key development in the methodology was finding that an aqueous solution of Rhyozyme and Triton X-100 produced lysis of blood but was not detrimental to bacteria.

A method is presented for measuring growth of bacteria under high hydrostatic pressure in subsamples taken without pressure change in the incubation vessel. Subsamples may be withdrawn rapidly (5 s) and are not subjected to shear forces. Vice versa, nutrient media, labeled substrates, etc., may be introduced into the culture while under pressure. Chemical fixation of subsamples for electron microscopy or adenosine 5'-triphosphate determinations under pressure is also possible without affecting the growing culture. Data are given of growth experiments demonstrating the feasibility of the method. Problems of oxygen depletion are discussed.

A flask is described for use in conjunction with a gas chromatograph and a spectrophotometer to follow methane and oxygen uptake, carbon dioxide production, and cell growth concurrently in methane-oxidizing cultures.

Two blood culturing systems were compared in parallel-a semi-automated radiometric method, BACTEC1, and the conventional method used in our laboratory. BACTEC contains radioactive 14C labelled substrates and monitors the level of 14CO2 evolved by actively metabolizing bacteria. BACTEC was as efficient as the conventional method in detecting positive cultures and indicated that it was superior in detecting positive cultures earlier than the conventional system.

Dimethoate and malathion added to soil at 10 and 100 microgram/g caused an initial stimulation of CO2 production. Total counts of bacterial propagules were increased. All insecticide applications increased bacteria producing phospholipases from week 1 until week 4 after the application; bacteria then returned to the original levels.

Twenty-eight soil fungi, 49 soil bacteria, and 10 actinomycetes were tested as to their ability to degrade the insecticide endosulfan. Using 14C-labeled material, the qualitative as well as the quantitative formation of metabolities, as well as of 14CO2, could be followed. Sixteen fungi, 15 bacteria, and 3 actinomycetes were found capable of metabolizing more than 30% of the applied endosulfan. The major metabolities detected were endosulfate, formed by oxidation of the sulfite group, and endodiol, formed by hydrolysis of the ester bond. The majority of highly active fungi formed endosulfate as the major metabolite, whereas the majority of active bacteria formed endodiol. In addition to endosulfate and endodiol, individual cultures contained small quantities of endohydroxyether and two unidentified products. The very small quantities of 14CO2 evolved from cultures indicated that an extensive mineralization of the carbon skeleton of endosulfan did not occur.

Various processes for the production of carbon dioxide by microorganisms are presented. It is postulated that a 'microniche' developed in a reducing environment; a symbiotic relationship between alga-like organisms and bacterium-like organisms in the 'microniche' governed the production of carbon dioxide resulting in the establishment of the steady state carbon dioxide system in the sea.

Two different mechanisms were responsible for the disappearance of styrene in enrichment cultures: (i) a mixed population of microorganisms, capable of utilizing styrene as a sole carbon source, oxidized this substrate to phenylethanol and phenylacetic acid; (ii) the culture also mediated polymerization of the monomer to low-molecular-weight styrene oligomers. This chemical reaction probably occurred as the result of microbial degradation of butylcatechol, an antioxidant polymerization inhibitor present in commercial styrene. The resultant polymer material was subsequently metabolized. In soil incubation studies, 14CO2 evolution from applied [8-14C] styrene was used to estimate microbial degradation. Approximately 90 percent of the labeled carbon was evolved from a 0.2 percent addition, and about 75 percent was lost from the 0.5 percent application over a 16-week period.

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.

Three investigations were carried out. The first determined the relationships between dehydrogenase acitivty throughout the course of composting wheat straw and the changes in both organic carbon and total nitrogen. The second investigation tested the validity of the first investigation results. The third dealt with the effect of the degree of ripening of a compost, expressed by age and C/N ratio, on its dehydrogenase activity, and production of CO2. The results revealed continual decreases in the organic carbon, coincident with increases in total N and dehydrogenase activity. Noticeable chemical and biological changes occurred in the first and third month. Levels of dehydrogenase were highest in rice straw and lowest in cotton stalk compost, while the wheat straw compost occupied an intermediate position. Aging of the compost resulted in increases in the dehydrogenase figures and decreases in the amounts of CO2 produced.

An acetate-fermenting strain of Methanosarcina was isolated from an acetate enrichment culture inoculated with anaerobic sludge from a waste treatment digestor. In pure culture, this organism fermented acetate in the absence of added hydrogen at rates comparable in magnitude to those found in digestor systems. This rate was significantly higher than previously obtained for pure cultures of this genus. Mineral components of yeast extract were highly stimulatory for cultures growing on methanol. Comparable stimulation was not observed for cultures growing on acetate. Labeling studies indicated that acetate was converted to methane and CO2 as predicted by previous studies on mixed cultures. Total oxidation or reduction of acetate was not the mechanism of conversion of acetate to methane by the pure culture. The ability of this strain to form colonies or to produce methane from acetate was apparently influenced by the choice of substrate and conditions used for growing the inoculum.

A method of recording C-mineralization using an infrared-CO2-analyser is explained. The procedure is based on a closed circuit system containing the incubationvessels. In that circuit gas is streeming until CO2-concentration is constant. Using the observed concentration-value produced CO2 quantity can be determined.

Under anaerobic conditions, resting cell suspensions of Coprococcus sp. Pe15 degraded 1 molecule of phloroglucinol to 2 molecules of acetic acid and 2 molecules of carbon dioxide. The organism metabolized the flavonoids rhamnetin and quercetin anaerobically in 20% rumen fluid medium but failed to grow under similar conditions at the expense of any of 39 other aromatic or flavonoid compounds tested.

The ferric chelate of ethylenediaminetetraacetic acid (EDTA) was biologically degraded by a mixed population of microorganisms present in an aerated lagoon receiving this chemical in its feed. As determined radiorespirometrically, 28% of the acetate-2-C and 30% of the ethylene position of the ammonium ferric chelate of [14C]EDTA was recovered as 14CO2 after 5 days. In a separate experiment using gas liquid chromatography and the sodium ferric chelate, as much as 89% disappearance of EDTA (0.1% wt/vol) was observed during a similar time period. Optimum 14CO2 evolution was observed at a pH value between 7 and 8 and at room temperature. Degradation of NH4Fe-[2-14C]EDTA was stimulated by the addition of either unlabeled NaFe-EDTA, nitrilotriacetic acid or ethylenediamine, and inhibited by the addition of a variety of different sugars and amino acids. Consistent with the biological nature of this degradation, little or no 14CO2 evolution was observed after heat treatment of the microorganisms at 100 C for 10 min, or after the addition of antibiotics to the incubation mixtures. Gas-liquid chromatography and mass spectral analyses were performed to demonstrate EDTA disappearance and to identify possible intermediates of EDTA degradation.

A technique using selective inhibitors was used to estimate the relative contributions of bacterial and fungal populations to the respiration of six soils and one litter sample. The ratios of bacterial to fungal respiration in the four agricultural soils, given in percentage of the total microbial activity, ranged from 10/90 to 35/65, with the average ratio being about 30/70. In the forest soils, the ratios were 20/80 and 30/70, and in a beech litter sample, the ratio was 40/60. The fungi clearly dominated in all samples. The ratios were not found to be pH related. The difficulties which had previously limited the use of selective inhibitors for in situ soil ecological investigations, such as insufficient inhibitor specificity, inhibitor inactivation or degradation, and errors of measurement caused by elimination of competitor populations, were either resolved or methodologically avoided in the experiments. Inhibitor selectivity was demonstrated using both mixed and pure cultures of microorganisms from each soil. Through the use of experiments with short incubation periods (6-8 h), problems with population shifts and inhibitor degradation were eliminated.

Cell suspensions of acetate-grown Methanosarcina barkeri mediate the conversion of CO and H2O to CO2 and H2. The reaction is coupled with the phosphorylation of ADP. Evidence is presented that CO oxidation by the cells is associated with the transient acidification of the suspension medium. Up to 2 mol vectorial protons were measured/mol CO oxidized when the transmembrane electrical gradient was kept low by the addition of valinomycin (20 nmol/mg protein) and KCl (200 mM) or of KSCN (50 mM). No transient acidification was observed in the presence of the protonophore tetrachlorosalicylanilide which stimulated rather than inhibited CO oxidation. Proton extrusion remained unaltered when the proton-translocating ATPase was specifically inhibited by dicyclohexylcarbodiimide. The latter finding indicates that proton translocation is associated with CO conversion to CO2 and H2 rather than with ATP hydrolysis in the cells. The data substantiate that the coupling of CO oxidation with ADP phosphorylation in M. barkeri occurs via a chemiosmotic mechanism.

Soil respiration (CO2-evolution) was measured at different intervals during mineralization of organic materials in an Egyptian clay-loam soil. The rate of CO2 output was found to decline by time, and a linear negative correlation was found between the logarithm of time and the logarithm of the rate of CO2 output. Applying the linear regression equation it was possible to describe this relationship mathematically. A significant positive correlation was also found between the rate of CO2 output and the total plate count of bacteria and the ATP content of the soil.

Two pathways are involved in the fermentation of glutamate to acetate, butyrate, carbon dioxide, and ammonia-the methylaspartate and the hydroxyglutarate pathways which are used by Clostridium tetanomorphum and Peptococcus aerogenes, respectively. Although these pathways give rise to the same products, they are easily distinguished by different labeling patterns of the butyrate when [4-(14)C]glutamate is used as substrate. Schmidt degradation of the radioactive butyrate from C. tetanomorphum yielded equally labeled propionate and carbon dioxide, whereas nearly all the radioactivity of the butyrate from P. aerogenes was recovered in the corresponding propionate. This procedure was used as a test for the pathway of glutamate fermentation by 15 strains (9 species) of anaerobic bacteria. The labeling patterns of the butyrate indicate that glutamate is fermented via the methylaspartate pathway by C. tetani, C. cochlearium, and C. saccarobutyricum, and via the hydroxyglutarate pathway by Acidaminococcus fermentans, C. microsporum, Fusobacterium nucleatum, and F. fusiformis. Enzymes specific for each pathway were assayed in crude extracts of the above organisms. 3-Methylaspartase was found only in clostridia which use the methylaspartate pathway, including Clostridium SB4 and C. sticklandii, which probably degrade glutamate to acetate and carbon dioxide by using a second amino acid as hydrogen acceptor. High levels of 2-hydroxyglutarate dehydrogenase were found exclusively in organisms that use the hydroxyglutarate pathway. The data indicate that only two pathways are involved in the fermentation of glutamate by the bacteria analyzed. The methylaspartate pathway appears to be used only by species of Clostridium, whereas the hydroxyglutarate pathway is used by representatives of several genera.

Microthrix parvicella, cultivated in a medium with Tween 80 and Casamino acids, utilized only the oleic acid moiety of Tween 80 as carbon and energy source. The cell yield from Tween 80 was about 0.32 g dry weight of cells per g of Tween 80 consumed. As only the oleic acid moiety of Tween 80 was utilized, the cell yield from oleic acid was 1.3 g dry weight of cells per g oleic acid consumed. The amount of carbon produced as CO2 was less than 30% of the oleic acid-carbon and this low value was in agreement with the high cell yield. In batch culture M. parvicella stored large amounts of lipid material during the early growth phase. The fatty acids of the lipid globules were similar to the fatty acids supplied as carbon source. The percentage composition of the biomass changed to give C/N percentage ratios of about 15 during the early growth phase due to the high concentration of internal lipids and the low concentration of protein. The growth rate in batch culture was about 0.016 h-1 but was affected by the concentration of Casamino acids in the medium.

A relatively simple shake-flask system for determining CO2 evolution was developed to assess the ultimate biodegradability by soil and sewage micro-organisms of chemicals which enter the environment. Linear alkylbenzene sulfonates (LAS) were used as model compounds to evaluate the method and were found to undergo substantial biodegradation in this dilute system. At the 30 mg/liter test concentration, higher-molecular-weight LAS compounds were biodegraded at a slower rate and to a lesser extent than lower-molecular-weight LAS, an effect which was eliminated or greatly reduced upon incremental addition of the LAS to the test medium during the first week of incubation. LA35S was used to demonstrate rapid LAS desulfonation, and 14CO2 evolution studies with (14C) benzene ring-labeled LAS indicated concomitant biodegradation of the entire LAS molecule as well as the LAS aromatic component. The test can be employed to examine numerous compounds at the same time and is readily adapted to studies of the effect of variation in temperature and oxygen concentration on biodegradation.

A modified spectrophotometric method was developed to estimate ioxynil and bromoxynil residues. The method when compared with a 14C-tracer method was less sensitive but allowed rapid and accurate estimation of the herbicides. A clay loam soil with high organic matter content, which degraded ioxynil completely to CO2, also degraded bromoxynil completely. Bromoxynil degradation proceeded at a faster rate than that of ioxynil. The half-life of degradation was estimated to be 7 days for bromoxynil and 9-10 days for ioxynil. However, soil microorganisms which degraded ioxynil either completely to CO2 or partially did not seem to completely degrade bromoxynil. Degradation products from bromoxynil were detected on thin-layer chromatograms of extracts from pure cultures containing an exogenous carbon source. Complete degradation of bromoxynil and ioxynil in soil could be due to the action of different microorganisms.

The comparative rates of microbial degradation of 14C-lignin-labeled lignocelluloses and 14C-Kraft lignins were investigated using selected soil and water samples as sources of microorganisms. Natural lignocelluloses containing 14C primarily in their lignin components were prepared by feeding plants uniformly labeled L-[14C]phenylalanine through their cut stems. 14C-Kraft lignins were prepared by pulping lignin-labeled lignocelluloses. Rates of lignin biodegradation were determined by monitoring 14CO2 evolution from incubation mixtures over incubation periods of up to 1000 h. Observed rates of lignin degradation were slow in all cases. Kraft lignins appeared more resistant to microbial attack than natural lignins, even though they were decomposed more rapidly during the first 100-200 h of incubation. Similar degradation patterns were observed in both soil and water. Individual samples, however, varied greatly in their overall rates of degradation of either lignin type. A Kraft-lignin preparation was separated into a variety of molecular weight fractions by column chromatography on LH-20 Sephadex and the biodegradability of the different molecular weight fractions determined. The lower molecular weight fractions of the Kraft lignin were decomposed at a significantly faster rate by the microflora of soil than were the fractions of higher molecular weight.

Measuring the potential soil respiration according to the incubator method allows the physiological performance of the celluloytic microflora to be determined. Laboratory tests were carried out to analyse catabolic processes after soil samples had been enriched with native cellulose + fungides. As the temporary capacity of mineralization primarily induced by the substrate invariably gives rise to a substrate-specific "change in position" in the microbial biocenosis, the measured values cannot furnish a parameter for microbiological comparisons of the soil content. Preliminary analyses provided information on the processes of depolymerisation of various C-sources and characterized the celluloysis as well as the order of respiration of its monomer. The rising respiration curve markedly drops off after 12 days, because with the CO2 development at its maximum, total and partial respiration alternate, a phenomenon that was verified in soils incubated with glucose 14C (Keefer and Mortensen, 1963; Macura, 1963; Simonart and Mayaudon, 1958). The test results obtained with fungicide-treated soils (dithiocarbamine acid derivatives) allow the conclusion that the fungicides exercise a varying influence: in the cellulolysis the activity of the cellulase systems is reduced depending on the active substance and concentration, but what is surprising is that respiration is stimulated; this applies to amounts of pesticides being 0.75 and 1.5 times the practical quantity applied. Nor have the "dissociation tests" with cellobiose, which is supposed to be the final link of celluloysis, shown any inhibition of the 1.4--glucodimerase or high respiration values comparable with the glucose metabolism. Tests with cellulases (Cx with dimerase function, Messrs. Roehm and Haas, Darmstadt), which are at present being carried out, are expected to support these observations experimentally. Investigations intended to clarify whether an additional administration of Mn2+ and Fe3+ in the presence of Maneb and Ferbam permits interference with possible adsorption and exchange processes have not yet yielded any generally valid results. The results of the open-field investigations also tend to indicate that the mentioned dithio-carbamates determine the magnitude and the extent of the physiological and CO2-ecological processes to a varying degree; it can be seen that a remarkable "capacity fo compensation" is discernible in the succession of the investigated phases of soil-physiological processes which we tried to clarify.

The Hungate anaerobic technique was used with a standard procedure for bacterial mutagenesis employing N-methyl-N-nitro-N'-nitrosoguanidine to obtain mutants of an obligate anaerobe. Three mutant strains were derived from a Coprococcus sp., strain Pe15, a rumen anaerobe capable of growing on phloroglucinol. The mutants did not grow on phloroglucinol but did degrade the compound in anaerobic washed-cell suspensions, producing the same end products in approximately the same proportions as the wild type. It was concluded that the mutants were blocked in a unique step or steps necessary for carbon skeleton or energy synthesis from phloroglucinol and not in formation of an enzyme involved in the pathway of phloroglucinol degradation.

The influence of salt mixtures consisting of Ca(H2PO4)2, trace elements, CaSO4, CaCO3, Na2CO3, NaCl and K2SO4 in different combinations on the nitrifying power, evolution of carbon dioxide and the total number of bacteria was studied in arid soils (sandy and alluvial) and semi-humid ones (chernozem and rendzina). Salt mixtures comprising of either mono-calcium phosphate or sodium chloride showed highly inhibiting action on the studied microbial activities in sandy, alluvial and chernozem soils, whilst mono-calcium phosphate stimulated the heterotrophs of rendzina. Trace elements stimulated the autotrophs of the clay soils. The other different salts mixtures acted according to the salt combination and nature of the microbial activity.

The metabolism of a saltwater leachate of 14C-labeled Spartina alterniflora was examined in laboratory systems using mixed, salt marsh microbial communities and, by addition of appropriate antibiotics, communities with bacteria or eukaryotes inhibited. Label uptake was more rapid in the systems with bacteria alone and with the mixed microbial community than with fungi alone. Mineralization of the added label was more extensive in the mixed and bacterial systems, whereas the fungi appear more efficient at converting the label into particulate biomass. Particulate biomass production efficiencies ranged from a high of 0.82 for the fungal system to 0.21 in the mixed community, with the bacterial system giving an intermediate value of 0.54. The presence of protozoa and microcrustaceans in the mixed system appears to account for an increase in the mineralization of the label assimilated. Additional experiments with whole labeled Spartina, a leachate from Spartina, and Spartina after leaching revealed that the seawater-soluble portions of the plants were attacked most rapidly by the microbial community.

A most-probable-number method using 14C-labeled substrates is described for the enumeration of aquatic populations of heterotrophic microorganisms. Natural populations of microorganisms are inoculated into dilution replicates prepared from the natural water from which the organisms originated. The natural water is supplemented with a 14C-labeled compound added so as to approximate a true environmental concentration. 14CO2 evolved by individual replicates is trapped in NaOH and counted by liquid scintillation techniques for use in scoring replicates as positive or negative. Positives (14CO2 evolution) are easily distinguished from negatives (no 14CO2 evolution). The results from a variety of environments using the 14CO2 procedure agreed well with previously described methods, in most instances. The 14C-most-probable-number method described here reduces handling procedures over previously described most-probable-number procedures using 14C-labeled substrates. It also appears to have advantages over other enumeration methods in its attempt to approximate natural conditions more closely.

In field experiments wheat in the phase of shooting was sprayed with solutions of chlorocholinechloride (CCC) and urea, CCC and ammonium salt MCPA (Aminex) or CCC, urea and Aminex. The effect of the treatment on dry weight of overground parts of wheat, number of bacteria, production of carbon dioxide, urease activity and content of ammonium in the rhizosphere soil was investigated. In all cases evolution of carbon dioxide in the rhizosphere soil was higher than that in the control soil. Highest numbers of bacteria were found in the rhizosphere soil of plants treated with urea, the herbicide and their mixtures. Content of ammonium was higher in the control soil than in the rhizosphere soils, the urease activity was highest in the rhizosphere soil of plants treated with the solution of the herbicide and with the combination of the herbicide with urea.

Methane was oxidized to carbon dioxide in the absence of oxygen by water samples from Lake Mendota, Madison, Wis. The anaerobic oxidation of methane did not result in the assimilation of carbon from methane into material precipitable by cold 10% trichloracetic acid. Only samples taken at the suface of the sediment of Lake Mendota were capable of catalyzine the anaerobic oxidation of methane. The rate of methane oxidation in the presence of oxygen was highest in samples taken from near the thermocline. Of the radioactive methane oxidized, 30 to 60% was assimilated into material precipitable by cold 10% trichloroacetic acid during aerobic incubation of the samples. These data support the conclusion that two distinct groups of methane-oxidizing organisms occur in stratifield lakes. Enrichments with acetate and methane as the sole sources of carbon and energy and sulfate as the electron acceptor resulted in the growth of bacteria that oxidize methane. Sulfate, acetate, and methane were all required for growth of enrichments. Acetate was not oxidized to carbon dioxide but was assimilated by cells. Methane was not assimilated but was oxidized to carbon dioxide in the absence of air.

The influence of calcium-cyanamide upon the microbiological activity was tested in pot experiments under controlled conditions in two Portuguese soils (sandy and loamy) after the addition of rice or wheat straw (rice straw 0.275% N, wheat straw 0.307% N). The amount of straw was equalled to 100 dz/ha, the application of calcium-cyanamide to 25, 50 and 100 kg N/ha. In the containers treated with straw the total amount of microorganisms (Koch-method) was higher in sandy than in loamy soil after 30 days, but after 70 days it was higher in loamy soil. The content of active nitrogen (NH4 + NO3) increased, when calcium-cyanamide was added, but decreased after the application of straw. After 70 days sandy soil again showed an increase of active nitrogen. Straw increased the rates of CO2-production considerably, wheat straw was superior to rice straw. Calcium-cyanamide increased the CO2-production more in sandy than in loamy soil or German loess, which was also used for this experiment. Only in the case of rice straw higher doses of calcium-cyanamide had a positive effect. After 70 days the CO2-production rose only when rice straw was applied. The dehydrogenase-activity was increased in both soils, but a superiority of wheat straw occurred in sandy soil only. The microbiological activity in the pots with straw was higher in sandy than in loamy soil, the addition of calcium-cyanamide accelerated it. Doses of 25-50 kg N/ha are sufficient generally. The period of the formation of insoluble organic N-compounds, usually connected with the application of organic matter with a wide N:C-ratio, seems to be reduced by the addition of calcium-cyanamide.