Anomalies in mineralization of low concentrations of organic compounds in lake water and sewage

The rates of mineralization of nitrilotriacetic acid (NTA), 2,4-dichlorophenoxyacetic acid (2,4-D), p-nitrophenol, aniline, and isopropyl N-phenylcarbamate (IPC) at one or more concentrations ranging from 100 pg/ml to 1.0 microgram/ml were proportional to chemical concentrations in samples of three lakes. The rates at 100 pg of NTA, 2,4-D, p-nitrophenol, and aniline per ml in samples of one or more lakes were less than predicted, assuming the rates were linearly related to the concentration. Neither NTA nor 2,4-dichlorophenol at 2.0 ng/ml was mineralized in some lake waters, but higher levels of the two chemicals were converted to CO2 in samples of the same waters. In samples from two lakes, little or no mineralization of IPC or 2,4-D occurred at 1.0 microgram/ml, but 10 ng/ml or lower levels of the herbicides were mineralized. The mineralization in sewage of 1.0 microgram of NTA per ml was biphasic; about 20% of the substrate was mineralized in 20 h, and mineralization was only reinitiated after a period of 130 h. The biphasic transformation was not a result of the accumulation of organic products, and it was still evident if protozoan activity was inhibited. NTA also underwent a biphasic mineralization in lake waters, and the biphasic pattern was not altered by additions of growth factors and inorganic nutrients. From 40 to 60% of the carbon of aniline added to lake water at levels of 100 pg/ml to 1.0 microgram/ml was mineralized, but more than 90% of the carbon of NTA, 2,4-D, or p-nitrophenol added to lake water at 10 ng/ml or 1.0 microgram/ml was mineralized.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anomalies in mineralization of low concentrations of organic compounds in lake water and sewage.

The rates of mineralization of nitrilotriacetic acid (NTA), 2,4-dichlorophenoxyacetic acid (2,4-D), p-nitrophenol, aniline, and isopropyl N-phenylcarbamate (IPC) at one or more concentrations ranging from 100 pg/ml to 1.0 microgram/ml were proportional to chemical concentrations in samples of three lakes. The rates at 100 pg of NTA, 2,4-D, p-nitrophenol, and aniline per ml in samples of one or more lakes were less than predicted, assuming the rates were linearly related to the concentration. Neither NTA nor 2,4-dichlorophenol at 2.0 ng/ml was mineralized in some lake waters, but higher levels of the two chemicals were converted to CO2 in samples of the same waters. In samples from two lakes, little or no mineralization of IPC or 2,4-D occurred at 1.0 microgram/ml, but 10 ng/ml or lower levels of the herbicides were mineralized. The mineralization in sewage of 1.0 microgram of NTA per ml was biphasic; about 20% of the substrate was mineralized in 20 h, and mineralization was only reinitiated after a period of 130 h. The biphasic transformation was not a result of the accumulation of organic products, and it was still evident if protozoan activity was inhibited. NTA also underwent a biphasic mineralization in lake waters, and the biphasic pattern was not altered by additions of growth factors and inorganic nutrients. From 40 to 60% of the carbon of aniline added to lake water at levels of 100 pg/ml to 1.0 microgram/ml was mineralized, but more than 90% of the carbon of NTA, 2,4-D, or p-nitrophenol added to lake water at 10 ng/ml or 1.0 microgram/ml was mineralized.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Sorption on Mineralization of Low Concentrations of Aromatic Compounds in Lake Water Samples

Montmorillonite-benzylamine complexes were formed immediately upon addition of 20 pg to 20 μg of amine per ml of suspensions containing the clay. The extent of amine sorbed was a linear function of equilibrium amine concentration in lake water. Increases in the clay concentration decreased the percentage of the organic compound that was mineralized at amine levels of 20 pg to 200 ng, but not at 20 μg/ml. A larger percentage of the chemical was released from the complex during mineralization in the presence of high clay concentrations than in the presence of low clay concentrations. The rates of desorption and mineralization increased linearly with benzylamine levels up to 200 ng/ml. Montmorillonite did not enhance mineralization rates at amine levels of 200 ng/ml or lower, but it was stimulatory at 20 μg/ml. Except at high amine and clay concentrations, mineralization was more rapid than desorption during the early periods of decomposition when the amine concentration in solution was relatively high. However, relative to the microbial demand, desorption was more rapid during later periods of decomposition when the amine level in solution was very low. Mineralization of benzoate was not usually affected by montmorillonite, kaolinite, or glass beads. More than 90% of the carbon from benzylamine and benzoate was often mineralized when the substrate concentration was 250 ng/ml or less. After incubation of the chemical in lake water, none of the radioactivity from benzylamine was in the particulate fraction containing natural sediment and microbial cells. The data indicate that clay may have a significant effect on the microbial decomposition of low concentrations of certain organic compounds.     

Kinetics and Extent of Mineralization of Organic Chemicals at Trace Levels in Freshwater and Sewage

A sensitive and rapid method was developed to measure the mineralization of ¹⁴C-labeled organic compounds at picogram-per-milliliter or lower levels in samples of natural waters and sewage. Mineralization was considered to be equivalent to the loss of radioactivity from solutions. From 93 to 98% of benzoate, benzylamine, aniline, phenol, and 2,4-dichlorophenoxyacetate at one or more concentrations below 300 ng/ml was mineralized in samples of lake waters and sewage, indicating little or no incorporation of carbon into microbial cells. Assimilation of ¹⁴C by the cells mineralizing benzylamine in lake water was not detected. Mineralization in lake waters was linear with time for aniline at 5.7 pg to 500 ng/ml, benzylamine at 310 ng/ml, phenol at 102 fg to 10 mg/ml, 2,4-dichlorophenoxyacetate at 1.5 pg/ml, and di-(2-ethylhexyl) phthalate at 21 pg to 200 ng/ml, but it was exponential at several p-nitrophenol concentrations. The rate of mineralization of 50 and 500 ng of aniline per ml and 200 pg and 2.0 ng of the phthalate per ml increased with time in lake waters. The phthalate and 2,4-dichlorophenoxyacetate were mineralized in samples from a eutrophic but not an oligotrophic lake. Addition to eutrophic lake water of a benzoate-utilizing bacterium did not increase the rate of benzoate mineralization at 34 and 350 pg/ml but did so at 5 and 50 ng/ml. Glucose and phenol reduced the percentage of p-nitrophenol mineralized at p-nitrophenol concentrations of 200 ng/ml but not at 22.6 pg/ml and inhibited the rates of mineralization at both concentrations. These results show that the kinetics of mineralization, the capacity of the aquatic community to assimilate carbon from the substrate or the extent of assimilation, and the sensitivity of the mineralizing populations to organic compounds are different at trace levels than at higher concentrations of organic compounds.     

Rates of Mineralization of Trace Concentrations of Aromatic Compounds in Lake Water and Sewage Samples

The rates of mineralization of phenol, benzoate, benzylamine, p-nitrophenol, and di(2-ethylhexyl) phthalate added to lake water at concentrations ranging from a few picograms to nanograms per milliliter were directly proportional to chemical concentration. The rates were still linear at levels of <1 pg of phenol or p-nitrophenol per ml, but it was less than the predicted value at 1.53 pg of 2,4-dichlorophenoxyacetate per ml. Mineralization of 2,4-dichlorophenoxyacetate was not detected in samples of lake water containing 200 ng of the chemical per ml. The slope of a plot of the rate of phenol mineralization in samples of three lakes as a function of its initial concentration was lower at levels of 1 to 100 μg/ml than at higher concentrations. In lake water and sewage supplemented with <60 ng of ¹⁴C-labeled benzoate or phenylacetate per ml, 95 to 99% of the radioactivity disappeared from solution, indicating that the microflora assimilated little or none of the carbon. The extent of mineralization of some compounds in samples of two lakes and sewage was least in the water with the lowest nutrient levels. No mineralization of 2,4-dichlorophenoxyacetate and the phthalate ester was observed in samples of an oligotrophic lake. These data suggest that mineralization of some chemicals at concentrations of <1 μg/ml is the result of activities of organisms different from those functioning at higher concentrations or of organisms that metabolize the chemicals at low concentrations but assimilate little or none of the substrate carbon.     

Cometabolism of low concentrations of propachlor, alachlor, and cycloate in sewage and lake water.

Low concentrations of propachlor (2-chloro-N-isopropylacetanilide) and alachlor [2-chloro-2',6'-diethyl-N-(methoxymethyl)acetanilide] were not mineralized, cycloate (S-ethyl-N-ethylthiocyclohexanecarbamate) was slowly or not mineralized, and aniline and cyclohexylamine were readily mineralized in sewage and lake water. Propachlor, alachlor, and cycloate were extensively metabolized, but the products were organic. Little conversion of propachlor and alachlor was evident in sterilized sewage or lake water. The cometabolism of propachlor was essentially linear with time in lake water and was well fit by zero-order kinetics in short periods and by first-order kinetics in longer periods in sewage. The rate of cometabolism in sewage was directly proportional to propachlor concentration at levels from 63 pg/ml to more than 100 ng/ml. Glucose but not aniline increased the yield of products formed during propachlor cometabolism in sewage. No microorganism able to use propachlor as a sole source of carbon and energy was isolated, but bacteria isolated from sewage and lake water metabolized this chemical. During the metabolism of this herbicide by two of the bacteria, none of the carbon was assimilated. Our data indicate that cometabolism of these pesticides takes place at concentrations of synthetic compounds that commonly occur in natural waters.     

Cometabolism of low concentrations of propachlor, alachlor, and cycloate in sewage and lake water

Low concentrations of propachlor (2-chloro-N-isopropylacetanilide) and alachlor [2-chloro-2',6'-diethyl-N-(methoxymethyl)acetanilide] were not mineralized, cycloate (S-ethyl-N-ethylthiocyclohexanecarbamate) was slowly or not mineralized, and aniline and cyclohexylamine were readily mineralized in sewage and lake water. Propachlor, alachlor, and cycloate were extensively metabolized, but the products were organic. Little conversion of propachlor and alachlor was evident in sterilized sewage or lake water. The cometabolism of propachlor was essentially linear with time in lake water and was well fit by zero-order kinetics in short periods and by first-order kinetics in longer periods in sewage. The rate of cometabolism in sewage was directly proportional to propachlor concentration at levels from 63 pg/ml to more than 100 ng/ml. Glucose but not aniline increased the yield of products formed during propachlor cometabolism in sewage. No microorganism able to use propachlor as a sole source of carbon and energy was isolated, but bacteria isolated from sewage and lake water metabolized this chemical. During the metabolism of this herbicide by two of the bacteria, none of the carbon was assimilated. Our data indicate that cometabolism of these pesticides takes place at concentrations of synthetic compounds that commonly occur in natural waters.     

Microbial Methanogenesis and Acetate Metabolism in a Meromictic Lake

Methanogenesis and the anaerobic metabolism of acetate were examined in the sediment and water column of Knaack Lake, a small biogenic meromictic lake located in central Wisconsin. The lake was sharply stratified during the summer and was anaerobic below a depth of 3 m. Large concentrations (4,000 μmol/liter) of dissolved methane were detected in the bottom waters. A methane concentration maximum occurred at 4 m above the sediment. The production of ¹⁴CH₄ from ¹⁴C-labeled HCOOH, HCO₃⁻, and CH₃OH and [2-¹⁴C]acetate demonstrated microbial methanogenesis in the water column of the lake. The maximum rate of methanogenesis calculated from reduction of H¹⁴CO₃⁻ by endogenous electron donors in the surface sediment (depth, 22 m) was 7.6 nmol/h per 10 ml and in the water column (depth, 21 m) was 0.6 nmol/h per 10 ml. The methyl group of acetate was simultaneously metabolized to CH₄ and CO₂ in the anaerobic portions of the lake. Acetate oxidation was greatest in surface waters and decreased with water depth. Acetate was metabolized primarily to methane in the sediments and water immediately above the sediment. Sulfide inhibition studies and temperature activity profiles demonstrated that acetate metabolism was performed by several microbial populations. Sulfide additions (less than 5 μg/ml) to water from 21.5 m stimulated methanogenesis from acetate, but inhibited CO₂ production. Sulfate addition (1 mM) had no significant effect on acetate metabolism in water from 21.5 m, whereas nitrate additions (10 to 14,000 μg/liter) completely inhibited methanogenesis and stimulated CO₂ formation.     

Carbon- and Nitrogen-to-Volume Ratios of Bacterioplankton Grown under Different Nutritional Conditions

Carbon- and nitrogen-to-volume (C/V and N/V) ratios were determined for freshwater bacterial assemblages grown in lake water filtrate or in water enriched with nutrients (aqueous extract of lake seston, glucose, arginine, phosphate, or ammonium). Biovolume was measured by epifluorescence microphotography, and carbon and nitrogen biomasses were measured with a CHN analyzer. Despite large variations of nutritional conditions (i.e., the composition and concentration of the dissolved organic carbon) and different mean cell sizes of the bacterial assemblage (0.17 to 1.8 μm³ per cell), the C/V, N/V, and carbon-to-nitrogen weight ratios varied little (C/V ratio, 0.14 pg of C per μm³ [standard deviation, 0.057; n = 15]; N/V ratio, 0.027 pg of N per μm³ [standard deviation; 0.011, n = 15]; carbon-to-nitrogen weight ratio, 5.6 [standard deviation, 2.2, n = 15]). An average C/V ratio of 0.12 pg of C per μm³ that was derived from natural and cultured bacterial assemblages is proposed as an appropriate conversion factor for estimation of the biomass of freshwater bacteria.     

Measurement of Proteolysis in Natural Waters

Microbiological proteolysis in Lake Champlain water was measured in situ and in vitro by the spectrophotometric measurement of the rate of release of soluble color from an insoluble azure dye derivative of hide powder. Water samples sterilized by microfiltration were never proteolytic. In situ proteolysis was found to be very dependent upon water temperature (1 to 23°C). No measurable activity was observed below 4°C. The in vitro proteolysis rate at 20°C was found to be 2.3 times the rate at 15°C and 6 times the rate at 10°C. Water taken from beneath the ice-covered lake throughout the winter and tested in the laboratory at 20°C was found to show an increasing proteolytic potential during the winter months. The highest activity was obtained as the ice broke up in early spring. Microbiological proteolysis in water from Burlington Harbor was often four times that found in center lake water. In most experiments proteolysis was inhibited completely by 2 μg of Cu²⁺ and inhibited 67% by 0.75 μg/ml. Proteolysis was markedly stimulated by 20 to 40 μg of Casitone or Casamino Acids per ml. The predominant bacteria growing in the proteolysis flasks were species of Pseudomonas and Flavobacterium. Pure cultures of Pseudomonas required traces of Casitone, Casamino Acids, or yeast extract for proteolysis of hide powder azure, whereas those of Flavobacterium did not. The requirement could not be met by a mixture of 21 amino acids and eight vitamins.     

Effect of Concentration of Organic Chemicals on Their Biodegradation by Natural Microbial Communities

The effect of concentration on the biodegradation of synthetic organic chemicals by natural microbial communities was investigated by adding individual ¹⁴C-labeled organic compounds to stream water at various initial concentrations and measuring the formation of ¹⁴CO₂. The rate of degradation of p-chlorobenzoate and chloroacetate at initial concentrations of 47 pg/ml to 47 μg/ml fell markedly with lower initial concentrations, although half or more of the compound was converted to CO₂ in 8 days or less. On the other hand, little mineralization of 2,4-dichlorophenoxyacetate and 1-naphthyl-N-methylcarbamate, or the naphthol formed from the latter, occurred when these compounds were present at initial concentrations of 2 to 3 ng/ml or less, although 60% or more of the chemical initially present at higher concentrations was converted to CO₂ in 6 days. It is concluded that laboratory tests of biodegradation involving chemical concentrations greater than those in nature may not correctly assess the rate of biodegradation in natural ecosystems and that low substrate concentration may be important in limiting biodegradation in natural waters.     

Selecting inocula for the biodegradation of organic compounds at low concentrations

The inability of many organisms to degrade pollutants at low concentrations is a problem when selecting inocula for bioremediation of sites with these low concentrations. Thus, a study was conducted to determine the effect of low concentrations of p-nitrophenol (PNP) on growth of four PNP-degrading bacteria and their abilities to metabolize low concentrations of the compound in culture and samples from an oligotrophic lake. PNP did not increase the growth rates of Flavobacterium sp. M4, Pseudomonas sp. K, Flavobacterium sp. M1, and Pseudomonas sp. SP3 at concentrations of less than 2, 4, 10, and 100 ng/ml, respectively, when it was the sole added carbon source in culture, but it stimulated multiplication at higher concentrations. In liquid culture with the nitro compound as sole added carbon source, the four bacteria extensively mineralized PNP at 50 and 100 ng/ml, and three of the four degraded much of the substrate at 25 ng/ml. Pseudomonas sp. SP3 mineralized more than 20% but the two Flavobacterium strains converted less than 10% of the substrate to C02 at 10 ng/ml, and none of the three mineralized more than 5% at 1 and 5 ng PNP/ml. Under conditions where more than 99% of the radioactivity from ¹⁴C-PNP added at 1 ng/ml remained in solution, two of the isolates formed organic products. Pseudomonas sp. K had no activity at 1, 5, and 10 ng/ml. In contrast, when each of the bacteria was separately inoculated into samples of water from an oligotrophic lake and from a well in which PNP was not biodegraded, the bacteria were able to mineralize as little as 1 ng PNP/ml. The addition to a salts solution of 10 ng of glucose per ml resulted in mineralization of PNP at concentrations too low to be mineralized when the nitro compound was the sole source of added carbon. Bacteria may thus be able to mineralize substrates in natural waters at concentrations below those suggested by tests conducted in culture media, possibly because of the availability of other carbon sources for the bacteria.Offprint requests to: M. Alexander.     

Phosphorus enhancement of mineralization of low concentrations of p-nitrophenol by Flavobacterium sp. in lake water

Abstract A study was conducted to determine whether the supply of inorganic phosphorus in lake water was sufficient for the mineralization of p -nitrophenol (PNP) by a Flavobacterium sp. The bacterium grew following its inoculation into sterile lake water amended with only 20 or 200 ng PNP per ml, but depending on when the water sample was taken, either PNP was not mineralized or it was mineralized slowly. PNP at both concentrations was rapidly mineralized if 10 mM P was added. The degradation of 200 ng PNP per ml was most rapid if the lake water was amended with 10 mM P, it proceeded more slowly with 1 mM P and the initiation of mineralization was delayed to equal extents if the sterile water received 0.1 mM, 0.01 mM or no P. The possible significance of these findings to biodegradation in natural waters is discussed.     

Genotoxicity of food preservative sodium sorbate in human lymphocytes in vitro

The genotoxic effects of antimicrobial food additive sodium sorbate (SS) was assessed by using chromosome aberrations (CAs), sister-chromatid exchanges (SCEs), and micronucleus (MN) in cultured human lymphocytes and comet assay in isolated human lymphocytes. Lymphocytes were treated with four concentrations (100, 200, 400 and 800 μg/ml) of SS as well as a negative (sterile distilled water) and a positive control (Mitomycin-C: MMC for cultured lymphocytes and H₂O₂ for isolated lymphocytes). The result of this study indicated that SS increased the frequency of CAs at both 24 and 48 h period compared to control. When gaps were included, this increase was significant at 200, 400 and 800 μg/ml concentrations at 24 h and, at all concentrations at 48 h treatment time. When gaps were excluded, this increase was significant at only 800 μg/ml concentration at both 24 and 48 h treatments. In addition, SS increased SCEs/cell and MN frequency at 400 and 800 μg/ml concentrations at both 24 and 48 h compared to negative control. Furthermore, this additive caused DNA damage at all concentrations in isolated human lymphocytes after 1 h in vitro exposure. The present results show that SS is genotoxic to the human peripheral blood lymphocytes in vitro at the highest concentrations.     

Nematostatic Activity of Oxamyl and N,N-Dimethyl-1-cyanoformamide (DMCF) on Meloidogyne incognita Juveniles

The nematostatic activity of oxamyl, methyl-N'',N''-dimethy]-N-hydroxy-l-thiooxamimidate (oxamyl-oxime) and N,N-dimethyl-l-cyanoformamide (DMCF) was studied by immersing 10 Meloidogyne incognita second-stage juveniles into aqueous solutions of various concentrations of each chemical. At concentrations of 500 to 8,000 μg/ml, oxamyl quickly immobilized immersed juveniles. In all other concentrations studied (down to 4 μg/ml), oxamyl stopped or reduced movement of juveniles within 24 hours. DMCF also quickly immobilized juveniles at concentrations of 4,000 and 8,000 μg/ml and reduced movement at 2,000 μg/ml. Lower concentrations had no observed effect on movement. In solutions of the oxime from 2,000 to 8,000 μg/ml, some reduction of movement was observed, but most juveniles maintained some motion over a period of 24 hours. Juveniles were transferred to water from 4,000 μg/ml solutions of oxamyl and DMCF after various intervals of time in order to determine the effect of duration of exposure to the chemicals on the ability of the immobilized juveniles to recover normal motion. Some recovery was observed even after 24 hours of exposure to DMCF, but none after exposure to oxamyl for longer than 40 minutes.     

Growth of phenol-mineralizing microorganisms in fresh water.

A method was developed to enumerate the procaryotic and eucaryotic phenol-mineralizing microorganisms present in samples of fresh water. Sixty-five percent or greater mineralization of [U-14C]phenol was considered a positive tube (contained phenol-mineralizing microorganisms) in the most-probable-number technique. Replicate most-probable-number tubes contained no microbial inhibitors, streptomycin and tetracycline, or cyclohexamide and nystatin plus 200 pg to 100 micrograms of phenol per ml. Phenol mineralization rates were obtained by measuring the amount of exogenous phenol that disappeared from solution over time in the presence or absence of the microbial inhibitors. Initially, less than 100 phenol-mineralizing bacteria per ml and 1 phenol-mineralizing fungus per ml were present at both 200 pg and 100 micrograms of phenol per ml. Phenol mineralization rates were 6.3 times greater for the mineralizing bacteria than for the fungi at 200 pg of phenol per ml. Phenol concentrations above 10 micrograms/ml were inhibitory to the microorganisms capable of mineralizing phenol. The phenol mineralizers grew in the water samples in the absence of phenol, indicating that there were sufficient indigenous nutrients in the lake water to support growth. There was no difference in the growth rate of these microorganisms in the presence or absence of 1 ng of phenol per ml, whereas the growth rate was more rapid at 1 microgram of phenol per ml than in its absence. There was a correlation between microbial growth and the amount of phenol mineralized at 1 microgram but not at 1 ng of phenol per ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth of phenol-mineralizing microorganisms in fresh water

A method was developed to enumerate the procaryotic and eucaryotic phenol-mineralizing microorganisms present in samples of fresh water. Sixty-five percent or greater mineralization of [U-14C]phenol was considered a positive tube (contained phenol-mineralizing microorganisms) in the most-probable-number technique. Replicate most-probable-number tubes contained no microbial inhibitors, streptomycin and tetracycline, or cyclohexamide and nystatin plus 200 pg to 100 micrograms of phenol per ml. Phenol mineralization rates were obtained by measuring the amount of exogenous phenol that disappeared from solution over time in the presence or absence of the microbial inhibitors. Initially, less than 100 phenol-mineralizing bacteria per ml and 1 phenol-mineralizing fungus per ml were present at both 200 pg and 100 micrograms of phenol per ml. Phenol mineralization rates were 6.3 times greater for the mineralizing bacteria than for the fungi at 200 pg of phenol per ml. Phenol concentrations above 10 micrograms/ml were inhibitory to the microorganisms capable of mineralizing phenol. The phenol mineralizers grew in the water samples in the absence of phenol, indicating that there were sufficient indigenous nutrients in the lake water to support growth. There was no difference in the growth rate of these microorganisms in the presence or absence of 1 ng of phenol per ml, whereas the growth rate was more rapid at 1 microgram of phenol per ml than in its absence. There was a correlation between microbial growth and the amount of phenol mineralized at 1 microgram but not at 1 ng of phenol per ml.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Water Extracts of Carob Pods, Tannic Acid, and Their Derivatives on the Morphology and Growth of Microorganisms

The effect of aqueous extracts of carob (Ceratonia siliqua) pods, gallotannic acid, gallic acid, and catechol on several microorganisms was studied. Carob pod extract and tannic acid showed a strong antimicrobial activity toward some cellulolytic bacteria. On the basis of tannin content, to which antimicrobial effect was related, carob pod extracts inhibited Cellvibrio fulvus and Clostridium cellulosolvens at 15 μg/ml, Sporocytophaga myxococcoides at 45 μg/ml, and Bacillus subtilis at 75 μg/ml. The inhibiting concentrations for tannic acid were found to be 12, 10, 45, and 30 μg/ml, respectively. Gallic acid and catechol were much less effective. Tannic acid and the tannin fraction of carob extract exerted both bacteriostatic and bactericidal effects on C. fulvus. Respiration of C. fulvus in the presence of bactericidal concentrations of tannic acid or tannin fraction of carob extract was inhibited less than 30%. A partial formation of “protoplasts” by C. fulvus was obtained after 2 hr of incubation in a growth medium to which 20% sucrose, 0.15% MgSO₄·7H₂O, and 10 to 50 μg/ml of tannic acid or 500μg/ml of penicillin, or both, had been added. Tannic acid and the tannin fraction of carob extract protected C. fulvus from metabolic lysis in sucrose solution. Although the growth of other microorganisms tested was only slightly affected, the morphology of some of them was drastically changed in the presence of subinhibitory concentrations of carob pod extracts of tannic acid. It is suggested that the site of action of tannins on sensitive microorganisms is primarily the cell envelope.     

Effect of Sulfide on Nitrogen Fixation in a Stream Sediment-Water System

Nitrogen fixation (C₂H₂ reduction) in a sediment-water system was studied under anaerobic incubation conditions. Sodium sulfide at low concentrations stimulated activity, with a twofold increase in C₂H₄ production occurring in the presence of 8 μmol of S²⁻ per ml of stream water. Sodium sulfide at concentrations of 16 μmol of S²⁻ per ml or greater inhibited nitrogen fixation, with 64 μmol of S²⁻ per ml being completely inhibitory. Sulfide at levels of 16 μmol/ml or above inhibited CO₂ production, and the degree of inhibition increased with increasing concentration of sulfide. Titanium (III) citrate (used to modify Eh levels) stimulated both nitrogen fixation and CO₂ production, but could not duplicate, at any concentration tested, the twofold increase in nitrogen fixation caused by 8 μmol of S²⁻ per ml. Sulfide additions caused pH changes in the sediment, and when the sediment was adjusted and maintained at pH 7.0 all concentrations of sulfide inhibited nitrogen fixation activity. From considerations of the redox equilibria of H₂, H₂S, and other sulfur species at various pH values, it appeared that H₂S was the toxic entity and that HS⁻ was less toxic. The observed stimulation of activity was apparently due to a pH change coupled with the concurrent production of HS⁻ from H₂S.     

Comparative study of genotoxicity and antimutagenicity of methanolic extracts from Teucrium chamaedrys and Teucrium montanum in human lymphocytes using micronucleus assay

Since Teucrium chamaedrys and Teucrium montanum are the most popular plants used in the treatment of many diseases, we evaluated genotoxic potential of their methanolic extracts on cultured human peripheral blood lymphocytes (PBLs) using cytokinesis-block micronucleus (MN) assay. Cultures were treated with four concentrations of both plants (125, 250, 500 and 1,000 μg/ml), both separately and in combination with mitomycin C (MMC). The results revealed that extract of T. chamaedrys administered at the tested concentrations did not significantly affect the mean MN frequency in comparison to untreated cells. Methanolic extract of T. montanum increased the mean MN frequency in PBL at the tested concentrations, but significantly only at the concentration of 1,000 μg/ml. In all tested concentrations, the extract of T. chamaedrys significantly reduced the MMC-induced MN frequency, in a dose dependent manner (r = − 0.687, p < 0.01). The extract of T. montanum decreased the MMC-induced MN frequency at the tested concentrations, but statistically only at 125 μg/ml. Both extracts administered alone did not significantly affect the nuclear division index (NDI) at the tested concentrations. In the combined treatments with MMC, the extract obtained from T. chamaedrys in the concentrations of 500 and 1,000 μg/ml significantly decreased NDI values in comparison to MMC-treated cells alone, while the extract of T. montanum significantly decreased NDI at all tested concentrations. Both extracts nonsignificantly decreased NDI at all tested concentrations in comparison to untreated cells. Our results suggest the important function of T. chamaedrys extract in cancer therapy, this methanolic extract may prevent genotoxic effects of chemotherapy in PBLs.