The mechanism of aggregate formation by Selenomonas ruminantium

Summary The mechanism of granule formation by Selenomonas ruminantium was investigated. A basic protein has been isolated from the lysate of S. ruminantium which triggers cluster formation (small aggregates of 20–100 cells) of suspended cells. Evidence is presented that these basic proteins were of ribosomal origin. It is suggested that ribosomes are released into the culture broth by lysis and that the associated basic proteins are subsequently dissociated by high monovalent cation concentrations. It was found that these positively charged basic proteins interact with the negatively charged lipopolysaccharide of the organism to form the clusters. Adding lysate to suspended cells, followed by lowering of the pH from 5.8 to 4.5 also induced clustering. At dilution rates exceeding the maximum growth rate clusters were retained in anaerobic gas-lift reactors and grew into granules (1–3 mm). It is postulated that granules evolve from clusters. Within the clusters, lysis and a low pH are induced due to diffusion limitations. As a consequence dividing cells are entrapped within the clusters, resulting in growth.     

Dilution rates influence ammonia-assimilating enzyme activities and cell parameters of Selenomonas ruminantium strain D in continuous (glucose-limited) culture

Aims: The objective of this study was to examine the effect of dilution rates (Ds, varying from 0·05 to 0·42 h^?1) in glucose‐limited continuous culture on cell yield, cell composition, fermentation pattern and ammonia assimilation enzymes of Selenomonas ruminantium strain D. Methods and Results: All glucose‐limited continuous culture experiments were conducted under anaerobic conditions. Except for protein, all cell constituents including carbohydrates, RNA and DNA yielded significant cubic responses to Ds with the highest values at Ds of either 0·10 or 0·20 h^?1. At Ds higher than 0·2 h^?1, fermentation acid pattern shifted primarily from propionate and acetate to lactate production. Succinate also accumulated at the higher Ds (0·30 and 0·42 h^?1). Glucose was most efficiently utilized by S. ruminantium D at 0·20 h^?1 after which decreases in glucose and ATP yields were observed. Under energy limiting conditions, glutamine synthetase (GS) and glutamate dehydrogenase (GDH) appeared to be the major enzymes involved in nitrogen assimilation suggesting that other potential ammonia incorporating enzymes were of little importance in ammonia assimilation in S. ruminantium D. GS exhibited lower activities than GDH at all Ds, which indicates that the bacterial growth rate is not a primary regulator of their activities. Conclusions: Studied dilution rates influenced cell composition, fermentation pattern and nitrogen assimilation of S. ruminantium strain D grown in glucose‐limited continuous culture. Significance and Impact of the Study: Selenomonas ruminantium D is an ecologically and evolutionary important bacterium in ruminants and is present under most rumen dietary conditions. Characterizing the growth physiology and ammonia assimilation enzymes of S. ruminantium D during glucose limitation at Ds, which simulate the liquid turnover rates in rumen, will provide a better understanding of how this micro‐organism responds to differing growth conditions.     

Anaerobic degradation of phenylacetic acid by mixed and pure cultures

Summary A phenylacetic acid-degrading mixed culture was enriched from effluent of an anaerobic reactor for the treatment of waste water from cellulose bleaching. From this consortium a phenylacetic acid-degrading pure culture, strain DSU3, was isolated and, due to its typical morphology and substrate spectrum, tentatively classified as a Desulfosarcina sp. It could grow on and degrade phenylacetic acid, cyclohexane carboxylate, cyclohexylacetate, benzoate, fumaric acid and several volatile fatty acids, while phenol, o-hydroxybenzoate, p-hydroxybenzoate and glucose were not utilized. Production of mandelic acid from phenylacetic acid by the enrichment culture and utilization of benzoate, an intermediate of the mandelic acid pathway, by strain DSU3 may presumably indicate degradation of phenylacetic acid via the mandelic acid pathway.     

Aggregate-formation by Clostridium butyricum

Summary Clostridium butyricum was grown in a glucose-limited chemostat culture at a dilution rate of 0.1 h^–1 at pH 6.0. With 0.9% w/v input glucose in the medium the cells were found to grow in suspension and glucose was fermented completely to acetate and butyrate. An increase in the input concentration of glucose resulted in increased concentrations of end-products, but not all extra glucose was consumed. It could be demonstrated that this was due to a lowering of the maximal growth rate by elevated levels of butyric acid. However, prolonged growth in the presence of high glucose concentrations led to an increase in biomass. This was caused by the selection of a variant that was less sensitive to butyrate. This variant was able to form aggregates in an anaerobic gas-lift reactor at high dilution rates. Inoculation of these aggregates in a conventional chemostat culture with high glucose input resulted in an aggregated culture that remained stable for at least 6 months, and in which all glucose was consumed. Whether the organisms grew in suspension or in aggregates was found to be determined by the concentration of butyrate. The isolation of aggregate-forming variants from chemostat cultures leads to a very simple and new type of immobilization technique.Offprint requests to: G. R. Zoutberg     

Characterization ofClostridium bifermentans and its biotransformation of 2,4,6-trinitrotoluene (TNT) and 1,3,5-triaza-1,3,5-trinitrocyclohexane (RDX)

Summary We have determined that an organism able to degrade both RDX and TNT in a pure culture is a strain ofClostridium bifermentans. The consortium from which this organism is derived also degrades these compounds, and we suspect thatC. bifermentans is also the responsible organism within that consortium. The bioconversion of RDX and TNT occurs under anaerobic conditions both in the consortium and in pure culture without the need of an added reductant. The presence of co-metabolites speeded these biotransformations.     

Synergism between different species of proteolytic rumen bacteria

Proteolytic strains ofButyrivibrio alactacidigens, Butyrivibrio fibrisolvens, Selenomonas ruminantium, andStreptococcus bovis grew markedly better in medium containing casein as sole nitrogen source when they were inoculated in pairs rather than singly, when growth was poor or nil. The improved growth ofSelenomonas ruminantium with other species was a consequence of a faster rate of proteolysis by the mixed culture. The cooperativity withStreptococcus bovis was mediated by the extracellular activity ofSelenomonas ruminantium. Synergistic growth betweenBu. alactacidigens and other bacteria was not due to cooperativity between proteolytic activities but probably to nutritional interdependence between the species. Cooperative proteolysis also occurred in mixtures ofSelenomonas ruminantium andBacteroides ruminicola, a proteolytic isolate capable of growth on casein in monoculture.     

Application of Pseudomurein Endoisopeptidase to Fluorescence In Situ Hybridization of Methanogens within the Family Methanobacteriaceae

In situ detection of methanogens within the family Methanobacteriaceae is sometimes known to be unsuccessful due to the difficulty in permeability of oligonucleotide probes. Pseudomurein endoisopeptidase (Pei), a lytic enzyme that specifically acts on their cell walls, was applied prior to 16S rRNA-targeting fluorescence in situ hybridization (FISH). For this purpose, pure cultured methanogens within this family, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanosphaera stadtmanae, and Methanothermobacter thermautotrophicus together with a Methanothermobacter thermautotrophicus-containing syntrophic acetate-oxidizing coculture, endosymbiotic Methanobrevibacter methanogens within an anaerobic ciliate, and an upflow anaerobic sludge blanket (UASB) granule were examined. Even without the Pei treatment, Methanobacterium bryantii and Methanothermobacter thermautotrophicus cells are relatively well hybridized with oligonucleotide probes. However, almost none of the cells of Methanobrevibacter ruminantium, Methanosphaera stadtmanae, cocultured Methanothermobacter thermautotrophicus, and the endosymbiotic methanogens and the cells within UASB granule were hybridized. Pei treatment was able to increase the probe hybridization ratio in every specimen, particularly in the specimen that had shown little hybridization. Interestingly, the hybridizing signal intensity of Methanothermobacter thermautotrophicus cells in coculture with an acetate-oxidizing H₂-producing syntroph was significantly improved by Pei pretreatment, whereas the probe was well hybridized with the cells of pure culture of the same strain. We found that the difference is attributed to the differences in cell wall thicknesses between the two culture conditions. These results indicate that Pei treatment is effective for FISH analysis of methanogens that show impermeability to the probe.     

Isolation of Selenomonas ruminantium from an aquatic ecosystem

A crescentic Gram-negative rod-shaped bacterium motile by a laterally inserted tuft of flagella was isolated from a boggy ditch water habitat. Cells occurred usually singly or in pairs, but sometimes short chains, long helical cells or spheroplasts with flagella still attached were observed. Its metabolism was obligate fermentative. The fermentation of glucose yielded mainly acetate and propionate. It grew with a generation time of 1 h 50 min. The DNA base ratio was found to be 51.6 mol % G+C. The characteristics of this organism indicated that it belongs to the genus Selenomonas closely similar to and by its main characteristics identical with the rumen bacterium Selenomonas ruminantium. The differing characteristics — production of catalase and lower temperature optimum (25°C) — interpretable as the result of adaptation to the specific environmental conditions may justify classification of the isolate into a new subspecies of S. ruminantium named Selenomonas ruminantium subsp. psychrocatalagenes. Additional information on the DNA base composition in strains of Selenomonas ruminantium (GA 192 and HD 1) was obtained.     

Kinetic study of the anaerobic degradation of toluene by a mixed culture

Toluene was anaerobically degraded by an enriched mixed culture under methanogenic conditions. The mixed culture was originally developed from cow-dung and sludge from a laboratory reactor, in which benzene was anerobically degraded by sulphate-reducing bacteria. First the mixed culture was enriched on toluene over a year with and without the use of sulphate in the medium. For the evaluation of growth-kinetic and maintenance parameters, namely μ_max, K_s, k_d and Y, the anaerobic degradation of toluene was carried out in batch as well as in continous reactors systems. The gas volume and the methane content in the produced gas was somewhat lover than the theoretical value expected, indicating an incomplete degradation of some of the complex intermediates of the toluene degradation pathway. However, the mixed culture was able to transform 41.3% of the toluene carbon into methane.     

d(−)lactic acid production by suspended and aggregated continuous cultures of Bacillus laevolacticus

Summary A new method of d(–)lactic acid production based on the aggregated form of growth of Bacillus laevolacticus in continuous culture in an anaerobic gaslift reactor is presented. With glucose as the substrate a bacterial dry weight of 25 g·1^–1 and a lactic acid production rate of 13 g·1^–1·h^–1 was attained. In conventional glucose-limited chemostat cultures elevated levels of lactic acid stimulated specific lactic acid production while the formation rates of other end-products remained unaffected. In glucose-limited aggregated cultures lactic acid positively influenced the aggregation of cells, improving the volumetric production rate. It is concluded that lactic acid itself is a positive effector in the optimisation of lactic acid production with aggregated cultures of B. laevolacticus.Offprint requests to: J. P. de Boer     

Anaerobic degradation of chlorophenol by an enrichment culture

Summary An anaerobic mixed culture from sewage sludge was enriched in a yeast extract and peptone-containing medium; it was able to degrade 2-cholorophenol completely to methane and CO₂. Degradation rates of 2-chlorophenol of up to 0.18 g/l per day were observed in suspended cultures without biomass retention and of 0.375 g/l per day in cultures immobilized on Liapor clay beads. Attempts to isolate the dechlorinating organism failed. The mixed culture was reduced to three morphologically distinctive microorganisms using a medium with limited amounts of yeast extract and peptone and n-butyrate as a co-substrate. Under these conditions the phenol-degrading bacterium was lost and phenol accumulated in the medium. No growth and no dehalogenation of 2-chlorophenol was obtained when yeast extract and peptone were omitted completely. Besides serving as a source of supplementary components, yeast extract and peptone were apparently required as the main source of carbon, wereas reducing equivalents for reductive dehalogenation were obtained by oxidation of n-butyrate. A spirochaete-like organism was presumably the dechlorinating bacterium. The mixed culture lost its dehalogenation capability if this organism was lost. n-Butyrate could be replaced by n-valerate, hexanoate, heptanoate, octanoate, pelargonic acid, n-decanoic acid or palmitate as co-substrates for dehalogenation of either 2-chlorophenol, 2-bromophenol or complete dechlorination of 2,6-dichlorophenol, whereas from 2,4-dichlorophenol only the substituent in the ortho-position could be eliminated.Dedicated to Professor O. Kandler on the occassion of his 70th birthdayOffprint requests to: J. Winter     

Studies of insufficient mixing in bioreactors: Effects of limiting oxygen concentrations and short term oxygen starvation on Penicillium chrysogenum

The effect of oxygen limitation on the respiration rate of Penicillium chrysogenum was studied. The results show that measurements of critical oxygen tensions within a process that on morphological or on physical grounds exhibits an inhomogenous structure are not likely to resemble the Monod model.In order to study the effects of short term oxygen starvation on the respiratory capacity of Penicillium chrysogenum, a two compartment fermenter was constructed. This fermenter consists of one well mixed aerobic part (CSTR) and one minor anaerobic part (CPFR). In the latter the circulation time as well as the volume can be varied. After passage of the whole cell culture volume through the anaerobic part, irreversible inhibition of the respiration was observed. This was caused by a circulation time of 5 and 10 min in the plug flow reactor and with a volume of 6% of the stirred tank reactor volume. However, circulation times of 1 and 2 min with an anaerobic zone of 1% of the stirred tank reactor volume did not give any irreversible effects on the respiratory capacity.This was compared with the results of the previously established model ln(1 — I _OUR//100)^–1 = kt [1]. The I _OUR is the percentage irreversible inhibition of the respiration, t is the anaerobic circulation time and k is a constant. The two compartment fermenter results agree with the earlier model at circulation times of 5 and 10 min, but not with the shorter times, and this suggests that a lag phase exists in the inactivation kinetics.     

Effect of tea saponin on methanogenesis,microbial community structure and expression of mcrA gene,in cultures of rumen micro‐organisms

Aims: To determine the in‐vitro effect and mode of action of tea saponin on the rumen microbial community and methane production. Methods and Results: Saponin extracted from tea seeds was added to (1) an in‐vitro fermentation inoculated with rumen fluid and (2) a pure culture of Methanobrevibacter ruminantium. Methane production and expression of the methyl coenzyme‐M reductase subunit A (mcrA) were monitored in both cultures. Abundance of methanogens, protozoa, rumen fungi and cellulolytic bacteria were quantified using real‐time PCR, and bacterial diversity was observed using denaturing gradient gel electrophoresis. Addition of tea saponin significantly reduced methane production and mcrA gene expression in the ruminal fermentation but not with the pure culture of M. ruminantium. The abundance of protozoa and fungi were significantly decreased 50% and 79% respectively but methanogen numbers were not affected, and Fibrobacter succinogenes increased by 41%. Bacterial diversity was similar in cultures with or without tea saponin. Conclusions: Tea saponin appeared to reduce methane production by inhibiting protozoa and presumably lowering methanogenic activity of protozoal‐associated methanogens. Significance and Impact of the Study: Tea saponin may be useful as a supplement to indirectly inhibit methane production in ruminants without a deleterious effect on rumen function.     

Formate dependent nitrate and nitrite reduction to ammonia by Citrobacter freundii and competition with denitrifying bacteria

Citrobacter freundii, Paracoccus denitrificans and Pseudomonas stutzeri were grown either singly or in mixed culture in anaerobic nitrate or nitrite limited chemostats with formate and/or succinate as electron donors and carbon sources. C. freundii reduced nitrate or nitrite stoichiometrically to ammonia. Maximum molar growth yields for nitrate (nitrite) were 15.3 (9.9) g/mol for C. freundii on formate with succinate as carbon source, 15.3 (9.5) g/mol for Ps. stutzeri on succinate and 32.3 (20.4) g/mol for Pa. denitrificans on succinate. The almost identical growth yields indicate that the ATP output of the anaerobic processes in the nitrate (nitrite) ammonifying organism and Ps. stutzeri are nearly the same. In mixed cultures with either Ps. stutzeri or Pa. denitrificans, C. freundii was the best competitor for nitrate. These results show that in anaerobic environments C. freundii may compete successfully with denitrifying organisms.     

Anaerobic biodegradation of triphenylmethane dyes in a hybrid UASFB reactor for wastewater remediation

Anaerobic digestions have been proved more successful than aerobic systems for the degradation and destruction of dye-containing wastewaters. The performance of a hybrid up flow anaerobic sludge-filter bed (UASFB) reactor was tested with a synthetic wastewater containing Crystal violet (CV) as a carbon source and sodium acetate as a co-substrate. Continuous feeding of the reactor started with an initial OLR of 0.9 g COD/l-d and then it was increased step wise to 4 g COD l⁻¹ d⁻¹, while maintaining constant HRT (24 h). The optimum pH value and temperature for decolorization of crystal violet by this mixed culture species under anaerobic conditions were found to be 8–9 and 30–35°C respectively. N,N-dimethylaminophenol and N,N-bis (dimethylamino) benzophenone (Michler’s Ketone) were detected as the degradative metabolites of Crystal Violet. Subsequently, N,N-dimethylaminophenol was further degraded to aniline in the reactor whereas Michler’s ketone was not degraded under anaerobic conditions. The UASFB bioreactor was able to remove the CV completely up to a loading rate of 100 mg CV l⁻¹d⁻¹.     

Specific and sensitive detection of Alcaligenes species from an agricultural environment

A quantitative real‐time PCR assay to specifically detect and quantify the genus Alcaligenes in samples from the agricultural environment, such as vegetables and farming soils, was developed. The minimum detection sensitivity was 106 fg of pure culture DNA, corresponding to DNA extracted from two cells of Alcaligenes faecalis. To evaluate the detection limit of A. faecalis, serially diluted genomic DNA from this organism was mixed with DNA extracted from soil and vegetables and then a standard curve was constructed. It was found that Alcaligenes species are present in the plant phytosphere at levels 10²–10⁴ times lower than those in soil. The approach presented here will be useful for tracking or quantifying species of the genus Alcaligenes in the agricultural environment.     

Spatial distribution of major bacterial species and different volatile fatty acids in a two-phase anaerobic biofilm reactor with PVA gel beads as bio-carrier

Abstract

Conventional completely mixed anaerobic treatment systems limit the chances of the different species of bacteria to spatially group together according to their mutual cooperation and as a result, show a lower efficiency and vulnerability towards shock situations. It is interesting to know about the stratification of the different bacterial species participating in the degradation process and the intermediates that they produce. In this study, we established and optimized a two-phase anaerobic packed bed biofilm reactor system (AnPBR) with porous PVA gel beads used as bio-carriers and ran the reactor system in a steady state to observe the VFAs produced along with the microbial diversity of the predominant species at different stages of the reactor system. We observed that acetate and butyrate were the predominant intermediate VFAs while concentrations of other VFAs such that propionic acid were low. Acetobacterium and Clostridium were found to be the most abundant bacterial species in acidogenic reactor while methanogenic reactor was highly enriched with Methanobacterium and Methanosarcina. Apart from the above, syntrophic populations such as Syntrophobactor wolinii were also observed to be dominant in both the reactors – especially towards the end of acidogenic reactor and the initial part of the methanogenic reactor.     

Molecular analysis of a sulphate-reducing consortium used to treat metal-containing effluents

A sulphate-reducing consortium used in a bioprocess to remove toxic metals from solution as insoluble sulphides, was characterised using molecular (PCR-based) and traditional culturing techniques. After prolonged cultivation under anoxic biofilm-forming conditions, the mixed culture contained a low diversity of sulphate-reducing bacteria, dominated by one strain closely related to Desulfomicrobium norvegicum, identified by three independent PCR-based analyses. The genetic targets used were the 16S rRNA gene, the 16S-23S rRNA gene intergenic spacer region and the disulfite reductase (dsr) gene, which is conserved amongst all known sulphate-reducing bacteria. This organism was also isolated by conventional anaerobic techniques, confirming its presence in the mixed culture. A surprising diversity of other non-sulphate-reducing facultative and obligate anaerobes were detected, supporting a model of the symbiotic/commensal nature of carbon and energy fluxes in such a mixed culture while suggesting the physiological capacity for a wide range of biotransformations by this stable microbial consortium.     

Power production in MFCs inoculated with Shewanella oneidensis MR‐1 or mixed cultures

Power densities and oxidation–reduction potentials (ORPs) of MFCs containing a pure culture of Shewanella oneidensis MR‐1 were compared to mixed cultures (wastewater inoculum) in cube shaped, 1‐, 2‐, and 3‐bottle batch‐fed MFC reactor configurations. The reactor architecture influenced the relative power produced by the different inocula, with the mixed culture generating 68–480% more power than MR‐1 in each MFC configuration. The mixed culture produced the maximum power density of 858 ± 9 mW m^?2 in the cubic MFC, while MR‐1 produced 148 ± 20 mW m^?2. The higher power by the mixed culture was primarily a result of lower internal resistances than those produced by the pure culture. Power was a direct function of ohmic resistance for the mixed culture, but not for strain MR‐1. ORP of the anode compartment varied with reactor configuration and inoculum, and it was always negative during maximum power production but it did not vary in proportion to power output. The ORP varied primarily at the end of the cycle when substrate was depleted, with a change from a reductive environment during maximum power production (approximately ?175 mV for mixed and approximately ?210 mV for MR‐1 in cubic MFCs), to an oxidative environment at the end of the batch cycle (～250 mV for mixed and ～300 mV for MR‐1). Mixed cultures produced more power than MR‐1 MFCs even though their redox potential was less negative. These results demonstrate that differences between power densities produced by pure and mixed cultures depend on the MFC architecture. Biotechnol. Bioeng. 2010; 105: 489–498. © 2009 Wiley Periodicals, Inc.     

Spectroscopic properties of complexes of acridine orange with glycosaminoglycans II. Aggregated complexes-evidence for long-range order.

Aggregated complexes of acridine orange with dermatan and chondroitin sulfates have been studied in aqueous solution by absorption and circular dichroism spectroscopy. Aggregation was found to be favored at high-dye and glycosaminoglycan concentrations, and in solutions where anionic sites of the glycosaminoglycan are effectively complexed with dye. The aggregates can be removed from solution by centrifugation at 27,000 × g for 1 hr or by filtration through a membrane containing pores of 0.1 μm diameter. The aggregated complexes exhibit large-magnitude-ellipticity circular dichroism bands. In addition, the circular dichroism spectrum observed for a solution containing aggregated acridine orange/chondroitin 4-sulfate complexes is nearly a mirror image of that obtained for aggregated acridine orange/dermatan sulfate complexes. Cooperative alterations (sharp transitions) in the circular dichroism ellipticities of the aggregates occur at elevated temperatures, and result in spectroscopically distinct aggregates upon cooling. The circular dichroism properties and temperature effects are attributed to a supramolecular ordering of acridine orange/glycosaminoglycan complexes within the aggregates, which can be reorganized to a more stable form at high temperatures. Mixed aggregates, containing two different glycosaminoglycans, can be formed. The circular dichroism properties of the mixed aggregates also indicate the existence of long-range order in the arrangement of the complexes. Mixed aggregates containing dermatan sulfate and either chondroitin 4-sulfate or chondroitin 6-sulfate resemble pure dermatan sulfate aggregates in circular dichroism characteristics.