F. Kunc

Continuous flow method in soil microbiology

The continuous flow method, applied to the study of microbiological processes in soil samples, was used to study the biological immobilization of mineral forms of nitrogen and phosphorus. By means of this method, physical-chemical adsorption of mineral elements was differentiated from biological immobilization over a given period. Biological immobilization of nitrogen and phosphorus was related to the metabolic activity of the soil microflora. It was found that the amount of biological immobilization of nitrogen and phosphorus was closely associated with glucose decomposition. A correlation was found between the immobilization of nitrate nitrogen and carbon dioxide evolution during glucose decomposition and the amount of glucose utilized. The ratio of the amount of glucose carbon assimilated by the soil microflora to the amount of nitrogen immobilized depended on the C∶N ratio in the added solution.AbstractНепрерывный проточный метод, применяемый при изучении микробиологических процессов в образцах почв, был использован для исследований биологической иммобилизации минеральных форм азота и фосфора. Этим методом было возможно отделить во времени физико-химическую сорбцию минеральных элементов от биологической иммобилизации. Биологическая иммобилизация азота и фосфора находилась в тесной связи с активностью метаболизма почвенной микрофлоры. Было установлено, что количество биологически иммобилизированных азота и фосфора тесно связано с количеством глюкозы, потребляемой микрофлорой почвы. Была обнаружена корреляция между иммобилизацией азота нитратов и продукцией углекислоты при разложении глюкозы—и количеством использованной глюкозы. Отношение углерода глюкозы, ассимилируемого микрофлорой почвы, к количествы иммобилизированного азота зависело от соотношения C∶N в добавляемом растворе.

Decomposition of vanillin by soil microorganisms

In chernozem soil, vanillin was decomposed via vanillic and protocatechuic acid before the aromatic ring opened. The rate curves of oxygen consumption for the oxidation of vanillin were seen to have more than one maximum. During incubation of the soil with vanillin, the number of bacteria increased, especially those capable of utilizing vanillin as the sole carbon source. Of the 21 such strains isolated, 15 were identified asPseudomonas sp., five asCellulomonas sp. and one asAchromobacter sp. It was found that the course of the oxidation of vanillin varied at different p.H values and in different strains was found that the course of the oxidation of vanillin varied at different p.H values and in different strains of bacteria. In some cases, the phase of the oxidation of vanillin to vanillic acid was clearly differentiated from the subsequent decomposition of vanillic acid.

Degradation of 3-chlorobenzoate in soil by pseudomonads carrying biodegradative plasmids.

Degradation of continuously added 3-chlorobenzoate (3-CB) was studied in samples of chernozem soil. Soil columns were inoculated withPseudomonas putida growing on 3-CB and carrying the biodegradation plasmid and withPseudomonas aeruginosa incapable of growth on 3-CB and carrying the inserted biodegradation plasmid pBS 2 determining ortho-cleavage of the aromatic ring. While the 3-CB degradation was observed in both inoculated variants, the native microflora of the soil under study was incapable to degrade 3-CB. Among pseudomonads isolated from inoculated soil at different stages of cultivation and growth on 3-CB, some had the taxonomic features ofP. putida as well as those differing in 1 –5 characteristics. The study of the activities of the enzymes cleaving the aromatic ring revealed the presence of pyrocatechol 1,2-dioxygenase in the isolated strains only, as estimated by means of benzoate and 3-CB as substrates.

Decomposition of the herbicide bromoxynil in soil and in bacterial cultures

It was found in field, and laboratory experiments that of 50 ppm of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile added to grey forest soil 20-80% were still detected after three months). Bromoxynil did not influence (except for a short-termed stimulation of the number of bacteria) the amount and composition of the basic groups of soil microorganisms. In enrichment cultures of soil microorganisms metabolie products of bromoxynil decomposition (3,5-dibromo-4-hydroxybenzamide and 3,5-dibromo-4-hydroxybenzoic acid) were detected and a stimulating effect of cosubstratos on its decomposition was demonstrated. Bromoxynil concentration, aeration conditions and the presence of cosubstrates (ribose in particular) influenced the rate and degree of the decomposition process inPsevdomonas putida. In addition to the degradation products mentioned above, production of methoxylated and partially dehalogenated aromatic compounds was detected.

Decomposition of root exudates in soil

The conversion of synthetic root exudates, i.e. of a mixture of amino acids, organic acids and sugars, added to soil in a single dose or continuously, was studied. After the addition of a single dose, the root exudates were gradually mineralized and after 76 hours, 85% of carbon had been released in the form of carbon dioxide. The extent and rate of mineralization was not influenced by the simultaneous addition of ammonium phosphate. The continuous addition of substrate formed a model artificial rhizosphere. In the steady state, 93% of the carbon in the added substrate was mineralized to carbon dioxide. The conversion of organic acids, sugars and amino acids and the mineralization of nitrogen was studied simultaneously by chromatography. In soil continuously enriched with root exudates, phenomena similar to the rhizosphere effect in nature were observed both in the numbers of microorganisms and in the relative incidence of the nutritional groups of bacteria.

Degradation of the herbicide bromoxynil in Pseudomonas putida.

Biological conversion of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) was studied in a batch culture ofPseudomonas putida by using HPLC. The process had a cometabolic character and proceeded only in the presence of another, simultaneously metabolizable, carbon and energy source. The intensity of degradation correlated with the growth rate, the degradation stopping when the cosubstrate becomes exhausted or the pH value of the medium falls below 6.5. In a medium with glucose, no lag phase longer than one day was observed concerning growth, sugar and herbicide consumption and formation of metabolic herbicide derivatives (3,5-dibromo-4-hydroxybenzamide and 3,5-dibromo-4-hydroxybenzoic acid). In a medium with ribose, the initial lag of the above processes took 2 d. No formation of other degradation products was detected. Growth inhibition was proportional to the concentration of bromoxynil.

Oxidation of aromatic compounds in soil

The oxidation ofp-hydroxybenzoic acid, quinic acid, vanillin and coumarin in soil was studied. With vanillin, and particularly with coumarin, the lag phase for oxygen consumption was longer and the rate of oxygen consumption attained more than one peak. In soil preincubated with the relevant substrate, the second dose of the same substrate was oxidized more rapidly. If the soil was preincubated with glucose, the lag phase was also shortened and oxygen consumption was raised with all aromatic substrates.

Decomposition of glucose continuously added to soil

The continuous flow method was used to study the decomposition of uniformly tagged glucose in soil with different inorganic nitrogen and phosphorus levels. It was found that the amount of glucose carbon mineralized to carbon dioxide was higher if nitrogen and phosphorus were added together with the glucose. Some of the labelled carbon escaped from the soil and the amount of leached-out carbon was in inverse proportion to the amount of nitrogen and phosphorus in the soil. The level of mineral nutrient elements stimulated the rate of glucose mineralization in the initial phase of the continuous process. The rate of glucose mineralization in the steady state was stimulated in soil continuously enriched with glucose together with nitrogen and phosphorus. The quantitative relationship between the assimilation and oxidation of glucose carbon depended on the nitrogen and phosphorus concentration and was in inverse proportion to the mineral element level. The continuous addition of glucose stimulated decomposition of the native soil organic matter. The resultant priming effect was balanced, however, by the retention of glucose carbon in the soil, with the result that the carbon balance remained positive. The rate of glucose oxidation, the amount of carbon retained in the soil and the priming effect of glucose were strongly influenced by the flow rate.AbstractНепрерывный проточный метог был использован для изучения разложения в почве единообразно меченой глюкозы при различных уровнях неорганического азота и фосфора. Было установлено, что количество глюкозы, меченой14C и окисляемой до двуокиси углерода, находилось в прямой зависимости от содержания азота и фосфора. Значительная часть14C уходила из почвы, и количество бымываемого14C было обратно пропорционально содержанию в почве азота и фосфора. Согержание элементов минерального питания влияло на скорость минерализации глюкозы в начальной фазе непрерывного процесса, а отчасти также на скорость минерализации глюкозы в состоянии равновесия. Отношение между ассимиляцией и окислением глюкозы-14C зависело от содержания азота и фосфора и понижалось при повышении содержания минеральных элементов. Непрерывное поступление глюкозы стимулировало разложение нативного органического вещества почвы. Но результирующий priming effect перекрывался задержкой14C в почве, так что углеродныи баланс оставался положительным. Скорость протока существенно влияла на скорость окисления глюкозы, количество14C, задерживающегося в почве, и на priming effect глюкозы.

Mineralization of 2,4-dichlorophenoxyacetic acid in soil simultaneously enriched with saccharides

Detoxication of 2,4-dichlorophenoxyacetic acid (2,4-D) in samples of chernozem soil was determined by a biological test and the time course of production of14CO2 a product of microbial degradation of 2-14C-2,4-D, was measured during 38-d incubation at 28°C in the dark. Enrichment of the soil with glucose (1000 ppm), two exocellular bacterial glucan and glucomannan polysaccharides (750 ppm), or a mixture of glucose with (NH4)2SO4 (C:N=5∶1) brought about acceleration of both detoxication and mineralization of 2,4-D (50 ppm) added simultaneously with the saccharides. Mineralization of the saccharides always preceded the degradation of the herbicide. The lag phase of 2,4-D mineralization, did not exceed 3 d. In samples with saccharides the doubling time of the mineralization activity in the exponential phase of the process was substantially shortened and the mineralization of 2,4-D was accelerated even when the soil was inoculated with a suspension of soil in which microbial 2,4-D decomposers had accumulated. The extent, of mineralization was not affected by the presence of saccharides (about 1/3 of the introduced radioactive carbon was transformed into14CO2). All saccharides had a similar effect which reflected an increase in the overall bacterial count and in the relative abundance of bacterial 2,4-D decomposers. The role of other mechanisms such as co-metabolism in the stimulation of the degradation process is discussed.

Effect of glucose on the decomposition of organic materials added to soil

The effect of glucose on the decomposition of plant material in soil was studied. Soil samples were enriched with different fractions of labelled alfalfa: the soluble dialyzable fraction, the soluble nondialyzable fraction, cellulose-lignin, lucerne meal. The added substances were decomposed for 42 days. One soil sample in every experimental variant was then percolated with water and another with 200 ml. 0.5% glucose for another 21 days. It was found that the effect of glucose on decomposition of the plant material depended on the latter’s composition and degree of decomposition. It was small in soils pre-enriched with soluble alfalfa fractions, decomposition of which was largely completed within the given time. Glucose markedly inhibited the release of radioactive carbon from the celluloselignin fraction and stimulated the formation of radioactive carbon dioxide from alfalfa meal.AbstractИсследовалось действие глюкозы на разложение растительного материала в почве. Образцы почвы обогащали различными фракциями мененой люцерны: растворимая диализующаяся фракция, растворимая недиализующаяся фракция, целлюлоза-лигнин, мука из люцерны. Прибавленные материалы разлагались в течение 42 дней, после чего по 1 образцу почвы каждого варианта проливали водой, а второй образец— 200 мл 0,5% глюкозы в течение еще 21 дня. Оказалось, что действие глюкозы на разложение растительного материала зависит от его состава и от степени разложения: это действие было незначительным в почве, предварительно обогащенной растворимыми фракциями люцерны, разложение которых в указанные сроки уже было в значительной степени закончено. Действие глюкозы заметно понижало освобождение радиоактивного углерода из фракции целлюлоза-лигнин и, наоборот, стимулировало образование из люцерны двуокиси углерода.