C. van der Drift

Differential analyses of glyoxylate derivatives

Abstract A differential glyoxylate analysis is given that enables differentiation between allantoin, allantoate, ureidoglycolate, and glyoxylate. The optimal conditions for glyoxylic acid phenylhydrazone formation are studied. At higher phenylhydrazine concentrations the rate of this reaction is limited by the dehydration of glyoxylate. The latter reaction is catalyzed by the presence of bivalent cations and phosphate. The conditions for selective quantitative hydrolysis of the glyoxylate derivatives are described and based upon these data six different methods of analysis are given. The conditions of optimal color formation are studied and some novel properties of the product formed are given. The analysis presented is more accurate, sensitive, and selective than former methods.

Microbial cycling of volatile organic sulfur compounds

Abstract. Microbial cycling of volatile organic sulfur compounds (VOSCs), especially dimethyl sulfide (DMS) and methanethiol (MT), is intensively studied because these compounds play an important role in the processes of global warming, acid precipitation, and the global sulfur cycle. VOSC concentrations in freshwater sediments are low due to the balance between the formation and degradation of these compounds. These reactions occur for the greater part at the oxic/anoxic interphase of sediment and water column. In contrast to marine ecosystems, where dimethylsulfoniopropionate is the main precursor of MT and DMS, in freshwater ecosystems, VOSCs are formed mainly by methylation of sulfide and to a lesser extent from the degradation of S-containing amino acids. One of the major routes for DMS and MT formation through sulfide methylation is anaerobic O-demethylation of methoxylated aromatic compounds. Inhibition studies have revealed that the major part of the endogenously produced MT and DMS is degraded anaerobically by methanogens. The major bacterial groups involved in formation and consumption of VOSCs are described.

Bacteria in the Intestinal Tract of Different Species of Arthropods

A bstractThe number of bacteria in the intestine of 12 species of arthropods, belonging to 7 different orders, was determined to obtain information about the significance of intestinal bacteria for the digestion of food. Therefore, a simple and effective method for direct counts of 4′, 6-diamidino-2-phenylindole (DAPI) stained bacteria from the gastrointestinal tract of arthropods was developed. The intestinal bacteria could be released from the gut wall by ultrasonic treatment in the presence of sodium tetrapyrophosphate (PPi). The bacterial counts ranged between 0.2 and 3.6 × 109 (ml gut)−1 in the foregut, 0.2 and 28 × 109 (ml gut)−1 in the midgut, and 0.1 and 190 × 109 (ml gut)−1 in the hindgut. The foregut and hindgut of Hylotrupes bajules larvae were devoid of bacteria; the whole intestinal tract of Eurycanta calcarata and Schistocerca gragaria contained only low numbers of bacteria. The population of bacteria in some parts of the intestinal tract of several arthropods were high enough to suggest a potential contribution to digestive processes.

Nitrogen assimilating enzymes in the white button mushroom Agaricus bisporus

Agaricus bisporus has the enzymic potential to assimilate ammonia by the activities of glutamine synthetase (EC 6.3.1.2), NADp-dependent glutamate dehydrogenase (EC 1.4.1.2) and NADP-dependent glutamate dehydrogenase (EC 1.4.1.4). It also contains glutamate synthase (EC 1.4.7.1) and a number of transaminating activities like glutamate-oxaloacetate transaminase (EC 2.6.1.1), glutamate-pyruvate transaminase (EC 2.6.1.2) and alanine-glyoxylate transaminase (EC 2.6.1.44). A. bisporus showed good growth in a defined buffered medium on glucose as a carbon source and a number of organic nitrogen compounds or ammonia as a nitrogen source. No growth was observed using nitrate as a nitrogen source. A. bisporus was not able to use organic nitrogen containing substances as a sole nitrogen and carbon source. Specific activities of the ammonia assimilating enzymes showed some variation when mycelia were cultivated on different nitrogen sources. Highest specific activities for glutamine synthetase, NAD-dependent glutamate dehydrogenase and NADP-dependent glutamate dehydrogenase were found when mycelia were grown on glutamate as a nitrogen source. Lowest values were found when the mycelia were grown on ammonia or glutamine. The specific activities of the ammonia assimilating enzymes showed no variation during maturation of the sporophores.

Inhibition of propionate degradation by acetate in methanogenic fluidized bed reactors

SummaryThe degradation of acetate, propionate and butyrate was monitored during start-up of five lab-scale methanogenic fluidized bed reactors on an artificially prepared waste water. The acetate concentration in the reactor content was found to influence the degradation of propionate but not of butyrate. In general, at acetate levels over 200 mg/l the degradation of propionate was below 60%, whereas the degradation was complete at acetate levels under 100 mg/l. The rationale of the inhibition of propionate degradation by acetate is discussed.

Acetate, methanol and carbon dioxide as substrates for growth of Methanosarcina barkeri

Methanosarcina barkeri grows in defined media with acetate, methanol or carbon dioxide as carbon sources. Methanol is used for methanogenesis at a 5 times higher rate as compared with the other substrates. M. barkeri can use the substrates simultaneously, but due to acidification or alkalification of the medium during growth on methanol or acetate, respectively, growth and methanogenesis may stop before the substrates are exhausted. Growth and methanogenesis on methanol or acetate are inhibited by the presence of an excess of H2; the inhibition is abolished by the addition of carbon dioxide, which probably serves as an essential source of cell carbon, in the absence of which methano-genesis ceases.

Early stages in biofilm development in methanogenic fluidized-bed reactors.

SummaryBiofilm development in methanogenic fluidized-bed reactors with sand as the carrier was studied on a laboratory scale. The microorganisms present in consecutive layers of the biofilm of mature sludge granules were preliminarily characterized on the basis of their morphology, element composition and adhesion capacity and were compared to bacteria which take part in the initial colonization of sand. The early phase of biofilm development was monitored with reactors receiving waste-waters containing different mixtures of volatile fatty acids and inoculated with fluidized-bed reactor effluent for different lengths of time. The results obtained indicate that facultative anaerobic bacteria abundantly present in the outermost biofilm layers of mature sludge granules are probably the main primary colonizers of the sand. Methanothrix spp. or other methanogens were rarely observed among the primary colonizers. The course of biofilm formation was comparable under the various start-up conditions employed including variations in waste-water composition, inoculation and anaerobicity. However, omission of waste-water and thus of substrate resulted in rapid wash-out of the attached biomass.

Purification and some properties of hydroxypyruvate isomerase of Bacillus fastidiosus

Hydroxypyruvate isomerase of Bacillus fastidiosus is a novel enzyme (Braun, W. and Kaltwasser, H. (1979) Arch. Microbiol. 121, 129–134) which catalyzes the reversible conversion of tartronate semialdehyde into hydroxypyruvate. The enzyme was purified to homogeneity. The native molecule had a molecular weight of 265 000–280 000 and was composed of six subunits with a molecular weight of 45 000. The enzyme showed optimal activity at pH 6.6–7.4 and 57°C. Hydroxypyruvate isomerase is stable on heating for 10 min at 67°C. The enzyme appeared to be specific for tartronate semialdehyde and hydroxypyruvate and no cofactors were involved in the reaction. The equilibrium constant K=[tartronate semialdehyde][hydroxypyruvate] was found to be 2.5 at pH 7.1, and 30°C.

Degradation of structural polysaccharides by the plant cell-wall degrading enzyme system from anaerobic fungi : An application study

Abstract The degradation capacity of extracellular enzyme preparations from two anaerobic fungi, i.e., Piromyces sp. strain E2 and Neocallimastix patriciarum strain N2, toward several types of lignocellulosic substrates was studied. The enzymes were capable of effectively converting up to 2% (w/v) microcrystalline cellulose (Avicel) to glucose, thereby indicating the presence of all catalytic activities (endoglucanases, exoglucanases, and β-glucosidases) needed. Performance of these enzymes surpassed that of a combined preparation of the commercial enzymes Celluclast and Novozym in batch degradation of 2% (w/v) Avicel. The enzymes from anaerobic fungi are very stable at 40°C. After one week of incubation at this temperature in the absence of substrate, about 50% of activity towards Avicel was retained. Although the performance of the enzyme system was lowered when increasing initial glucose concentrations were applied, still over 30% of sugar release was observed at an initial monomer concentration of 200 m m . Various natural lignocellulosic materials were ground and tested as substrates. A negative correlation was found between the amount of lignin in the substrate and the release of soluble sugars.

Purification and characterization of NADP-dependent glutamate dehydrogenase from the commercial mushroom Agaricus bisporus

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent glutamate dehydrogenase (NADP-GDH) of Agaricus bisporus, a key enzyme in ammonia assimilation, was purified to apparent electrophoretic homogeneity with 27% recovery of the initial activity. The molecular weight of the native enzyme was 330 kDa. The enzyme is probably a hexamer, composed of identical subunits of 48 kDa. The isoelectric point of the enzyme was found at pH 4.8. The N-terminus appeared to be blocked. The enzyme was specific for NADP(H). The Km-values were 2.1, 3.2, 0.074, 27.0, and 0.117mM for ammonia, 2-oxoglutarate, NADPH, L-glutamate, and NADP respectively. The pH optima for the amination and deamination reactions were found to be 7.6 and 9.0, respectively. The temperature optimum was 33°C. The effect of several metabolites on the enzymes activity was tested. Pyruvate, oxaloacetate, ADP, and ATP showed some inhibitory effect. Divalent cations slightly stimulated the aminating reaction. Antibodies raised against the purified enzyme were able to precipitate NADP-GDH activity from a cell-free extract in an anticatalytic immunoprecipitation test. Analysis of a Western blot showed the antibodies to be specific for NADP-GDH.