Biomedical subjects
Publications and source records attributed to W Verstraete.
Ca2+ -ATPase enzymes facilitate active trans-membrane transport of Ca2+ in micro-organisms. This study investigates the hypothesis that active calcium metabolism under conditions of alkaline stress is a key element of microbial carbonate precipitation, and that the latter plays an integral part in survival of bacteria under alkaline conditions.
In Flanders, it is not possible to centralise the infrastructure for handling all wastewater. Decentralised treatment should be considered an essential part of an overall regional policy. Decentralised wastewater treatment can be small-scale (20 to 2,200 Inhabitant Equivalent (IE)) or individual (less than 20 IE). This paper deals with the operation, performances, technological aspects and costs of Individual Wastewater Treatment Plants (IWTPs) in Flanders. Different treatment systems are described and tentatively evaluated. The total costs (capital and operation) can be of the same order of those of centralised systems, i.e. 100 EURO/IE.year. In terms of overall performance, most of the systems tested in Flanders, i.e. aerobic biological filter, activated sludge system, submerged aerated filter, biorotor and percolation reed bed, appear capable to perform well in principle. However, there remain problems to be solved. The effluent quality differs 'in situ' considerably, depending on the care and attention given by the owner. Hence, the legislator should not only focus on certification of installations as such, but foremost give attention to regular validation of the performance at the household. In this respect, there is an urgent need for the design and the logistic management of proper performance control systems.
The effects of a long-term herbicide treatment on the microbial community of an agricultural soil were investigated. Therefor, molecular techniques were used to evaluate the structure and diversity of the soil microbial community. Eubacterial and group-specific primers for methanotrophs type I and II were used to amplify 16S rRNA gene fragments from total soil DNA. These fragments were subsequently separated by denaturing gradient gel electrophoresis (DGGE). The structure of the methanotrophic community was affected by the herbicides as the patterns obtained from a herbicide treated soil (conventional) clustered separately from the control soil (biological). It seems that group-specific PCR followed by DGGE is a very powerful and sensitive technique to differentiate fields, which have received a herbicide treatment from those who did not. The diversity of the methanotrophic community was quantified by calculating the Shannon-Weaver index of biodiversity. The type I methanotrophs showed a significant decreased biodiversity due to the herbicide treatment but the diversity of the methanotrophs type II was slightly higher in the herbicide treated soil.
The homogeneity of the microbial community structure of a sediment landfill was examined by a culture-independent method and compared with physico-chemical parameters, i.e. organic matter, CaCO3 content, pH, and texture. Total genomic DNA was extracted from samples derived from different places and depths. After amplification with two different primer sets of partial bacterial 16S rRNA genes, the products were separated by denaturing gradient gel electrophoresis (DGGE). The DGGE fingerprints of different sediment samples taken in regular patterns at the same depth were similar, which indicates a spatial homogeneity in the numerically dominant bacterial populations in a landfill over 10,000 m2 in size. In a vertical column of approx. 10 m, only some differences in a few bands of the bacterial community structure were observed between samples taken from different depths. This DNA homogeneity coincided with a similar homogeneity of the physico-chemical parameters in the landfill at this site. Nevertheless, the DGGE technique revealed small differences in less prominent bacteria and was capable of separating the upper and lower samples of one column into two clusters. It therefore seems more sensitive than the physico-chemical approach for characterising the homogeneity of an environmental habitat.
Bacterial bile salt hydrolysis is considered a risk factor for the development of colon cancer because of the risk of forming harmful secondary bile salts after an initial deconjugation step. In this study, the influence of enhanced bacterial bile salt transformation by the bile salt hydrolase-active Lactobacillus reuteri was studied in batch culture using the microbial suspension of the Simulator of the Human Intestinal Microbial Ecosystem; (SHIME), which was supplemented with oxgall at 5 g/l or 30 g/l. Changes in the fermentative capacity of the microbial ecosystem and the (geno)toxic properties of the SHIME supernatants were investigated. Increasing concentrations of oxgall inhibited the fermentation. Transient cell toxicity was observed for samples supplemented with 5 g oxgall/l, while samples with 30 g oxgall/l exhibited toxicity. The results of the haemolysis test suggest that the detrimental effects were probably due to the membrane-damaging effects of bile salts. In all cases, the adverse effects could be counteracted by the addition of 7.5 +/- 0.5 log10 CFU L. reuteri/ml. Plausible mechanisms for the protective properties of L. reuteri could involve a precipitation of the deconjugated bile salts and a physical binding of bile salts by the bacterium, thereby making the harmful bile salts less bioavailable.
Lactobacillus acidophilus 74-2, which is used in probiotic products, was administered, with fructo-oligosaccharide in a milk-based product, to the second vessel (duodenum/jejunum) of the SHIME reactor, an in vitro simulation of the human intestinal microbial ecology. The main focus of this study was to monitor the changes of the population density of selected bacterial species in the intestine and the changes of metabolic activities during the supplementation of L. acidophilus and fructooligosaccharide in the SHIME reactor. Interestingly, the addition of L. acidophilus 74-2 with fructooligosaccharide gave rise to an increase of bifidobacteria. Moreover, major positive changes occurred in the production of volatile fatty acids: a strong upward trend was observed especially in the case of butyric acid and propionic acid. Furthermore a noticeable increase of beta-galactosidase activity was monitored, while the activity of beta-glucuronidase, generally considered undesirable, declined.
The E. coli cell method for nitrate measurement consists of two-steps: nitrate reduction by the E. coli cell usually under anaerobic conditions and subsequently nitrite measurement with the Griess reaction. It was found that the E. coli DSM 498k wildtype cell can reduce nitrate to nitrite under aerobic conditions. Therefore, the E. coli method for nitrate measurement was adapted to be performed under aerobic conditions in a microtiter plate. The adapted method is simpler than the original E. coli method and other nitrate methods such as those with inorganic reductants and with purified enzymes. Furthermore, it was found that for the Griess reaction the pH values of samples after addition of the Griess reagent A should be lower than 1.8 for a stable absorbance at 540 nm to be reached. It is important to add the two Griess reagents separately and to read the absorbance twice consecutively in a microtiter plate. The adapted E. coli method was successfully applied to measure the traces of nitrate in MRS and other medium components by measuring the standard curve of a dilution of each individual medium component. It was found that many organic medium components contain traces of nitrate, while none of them contain detectable nitrite. Among these, the extract of meat and yeast extract contain relatively high amounts of nitrate: 217 mg N/kg and 99 mg N/kg respectively. MRS broth contains nitrate from 0.3 to 0.6 mg N/l depending on the batch numbers of the product. The adapted E. coli can also be used for nitrate measurement in other matrices.
Degradation of branched octylphenol was studied in a bacterial culture of a Sphingomonas sp. strain. Octylphenol is considered to be the most stable degradation intermediate formed from the corresponding nonionic octylphenol polyethoxylates surfactants during biological wastewater treatment. Since octylphenol can exert estrogenic effects in wildlife, a detailed study of its biodegradation is warranted. The aerobic microbiological transformation of octylphenol was examined with and without the addition of the easily assimilable sodium acetate. In both cases the formation of the metabolite 2,4,4-trimethyl-2-pentanol, representing the intact alkyl chain as a tertiary alcohol, was observed. Since the octylphenol degradation rate was not affected by the presence of acetate, this strain did not show any diauxic metabolic behaviour when incubated with octylphenol and sodium acetate as the sources of carbon and energy. As a result of the biotransformation of octylphenol, its estrogenic potency was removed because it is the phenolic moiety that interacts with the estrogen receptors. This feature opens perspectives for the use of this strain in the framework of an adequate treatment of wastewater with high levels of alkylphenol polyethoxylates.
The effect of a continuous supply of a water extract of Moringa oleifera seeds (WEMOS) on the hydrolytic microbial population of biomass grown in mesophilic upflow anaerobic sludge blanket reactors treating domestic wastewater was investigated. The WEMOS-treated sludge had seemingly a wider diversity, with enterobacter and klebsiella as dominant hydrolytic bacteria, compared with the control sludge. Additional tests indicated that various hydrolytic bacteria could degrade WEMOS. It appeared that a continuous supply of WEMOS to an anaerobic digester, treating domestic wastewater, increased the diversity of hydrolytic bacteria and therefore enhanced the biological start-up of the reactor.
After inoculation of the plasmid-free non-aggregative Ralstonia eutropha-like strain AE815 in activated sludge, followed by reisolation on a selective medium, a mutant strain A3 was obtained, which was characterized by an autoaggregative behaviour. Strain A3 had also acquired an IncP1 plasmid, pLME1, co-aggregated with yeast cells when co-cultured, and stained better with Congo red than did the AE815 strain. Contact angle measurements showed that the mutant strain was considerably more hydrophobic than the parent strain AE815, and scanning electron microscopy (SEM) revealed the production of an extracellular substance. A similar hydrophobic mutant (AE176R) could be isolated from the AE815-isogenic R. eutropha-like strain AE176. With the DNA fingerprinting technique repetitive extragenic palindromic-polymerase chain reaction (REP-PCR), no differences between these four strains, AE815, A3, AE176 and AE176R, could be revealed. However, using the amplified fragment length polymorphism (AFLP) DNA fingerprinting technique with three different primer combinations, small but clear reproducible differences between the banding patterns of the autoaggregative mutants and their non-autoaggregative parent strains were observed for each primer set. These studies demonstrate that, upon introduction of a strain in an activated sludge microbial community, minor genetic changes readily occur, which can nevertheless have major consequences for the phenotype of the strain and its aggregation behaviour.
An in vitro model, designated the Simulator of the Human Intestinal Microbial Ecosystem (SHIME), was used to study the effect of a soygerm powder rich in beta-glycosidic phytoestrogenic isoflavones on the fermentation pattern of the colon microbiota and to determine to what extent the latter metabolize the conjugated phytoestrogens. Initially, an inoculum prepared from human feces was introduced into the reactor vessels and stabilized over 3 wk using a culture medium. This stabilization period was followed by a 2-wk control period during which the microbiota were monitored. The microbiota were then subjected to a 2-wk treatment period by adding 2.5 g/d soygerm powder to the culture medium. The addition resulted into an overall increase of bacterial marker populations (Enterobacteriaceae:, coliforms, Lactobacillus: sp., Staphylococcus: sp. and Clostridium: sp.), with a significant increase of the Lactobacillus: sp. population. The short-chain fatty acid (SCFA) concentration increased approximately 30% during the supplementation period; this was due mainly to a significant increase of acetic and propionic acids. Gas analysis revealed that the methane concentration increased significantly. Ammonium and sulfide concentrations were not influenced by soygerm supplementation. Use of an electronic nose apparatus indicated that odor concentrations decreased significantly during the treatment period. The beta-glycosidic bonds of the phytoestrogenic isoflavones were cleaved under the conditions prevailing in the large intestine. The increased bacterial fermentation after addition of the soygerm powder was paralleled by substantial metabolism of the free isoflavones (genistein, daidzein and glycitein), resulting in recovery of only 12-17% of the supplemented isoflavones.
Soil treated with linuron for more than 10 years showed high biodegradation activity towards methoxy-methyl urea herbicides. Untreated control soil samples taken from the same location did not express any linuron degradation activity, even after 40 days of incubation. Hence, the occurrence in the field of a microbiota having the capacity to degrade a specific herbicide was related to the long-term treatment of the soil. The enrichment culture isolated from treated soil showed specific degradation activity towards methoxy-methyl urea herbicides, such as linuron and metobromuron, while dimethyl urea herbicides, such as diuron, chlorotoluron, and isoproturon, were not transformed. The putative metabolic intermediates of linuron and metobromuron, the aniline derivatives 3, 4-dichloroaniline and 4-bromoaniline, were also degraded. The temperature of incubation drastically affected degradation of the aniline derivatives. Whereas linuron was transformed at 28 and 37 degrees C, 3,4-dichloroaniline was transformed only at 28 degrees C. Monitoring the enrichment process by reverse transcription-PCR and denaturing gradient gel electrophoresis (DGGE) showed that a mixture of bacterial species under adequate physiological conditions was required to completely transform linuron. This research indicates that for biodegradation of linuron, several years of adaptation have led to selection of a bacterial consortium capable of completely transforming linuron. Moreover, several of the putative species appear to be difficult to culture since they were detectable by DGGE but were not culturable on agar plates.
In this study Vibrio proteolyticus CW8T2 has been identified as a virulent pathogen for Artemia spp. Its infection route has been visualized with transmission electron microscopy. The pathogen affected microvilli and gut epithelial cells, disrupted epithelial cell junctions, and reached the body cavity, where it devastated cells and tissues. In vivo antagonism tests showed that preemptive colonization of the culture water with nine selected bacterial strains protected Artemia juveniles against the pathogenic effects. Two categories of the selected strains could be distinguished: (i) strains providing total protection, as no mortality occurred 2 days after the experimental infection with V. proteolyticus CW8T2, with strain LVS8 as a representative, and (ii) strains providing partial protection, as significant but not total mortality was observed, with strain LVS2 as a representative. The growth of V. proteolyticus CW8T2 in the culture medium was slowed down in the presence of strains LVS2 and LVS8, but growth suppression was distinctly higher with LVS8 than with LVS2. It was striking that the strains that gave only partial protection against the pathogen in the in vivo antagonism test showed also a restricted capability to colonize the Artemia compared to the strains providing total protection. The in vivo antagonism tests and the filtrate experiments showed that probably no extracellular bacterial compounds were involved in the protective action but that the living cells were required to protect Artemia against V. proteolyticus CW8T2.
A strain identified as Comamonas testosteroni I2 was isolated from activated sludge and found to be able to mineralize 3-chloroaniline (3-CA). During the mineralization, a yellow intermediate accumulated temporarily, due to the distal meta-cleavage of chlorocatechol. This strain was tested for its ability to clean wastewater containing 3-CA upon inoculation into activated sludge. To monitor its survival, the strain was chromosomally marked with the gfp gene and designated I2gfp. After inoculation into a lab-scale semicontinuous activated-sludge (SCAS) system, the inoculated strain maintained itself in the sludge for at least 45 days and was present in the sludge flocs. After an initial adaptation period of 6 days, complete degradation of 3-CA was obtained during 2 weeks, while no degradation at all occurred in the noninoculated control reactor. Upon further operation of the SCAS system, only 50% 3-CA removal was observed. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes revealed a dynamic change in the microbial community structure of the activated sludge. The DGGE patterns of the noninoculated and the inoculated reactors evolved after 7 days to different clusters, which suggests an effect of strain inoculation on the microbial community structure. The results indicate that bioaugmentation, even with a strain originating from that ecosystem and able to effectively grow on a selective substrate, is not permanent and will probably require regular resupplementation.
Transfer of the 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil was investigated as a means of bioaugmentation. When the soil was amended with nutrients, plasmid transfer and enhanced degradation of 2,4-D were observed. These findings were most striking in the B horizon, where the indigenous bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) during at least 22 days but where inoculation with either of the two plasmid donors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon and subsequent formation of transconjugants (10(5) CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated soil. However, donor inoculation in the nonamended B-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at all was observed in noninoculated soil during 89 days. With plasmid pEMT1, this enhanced degradation seemed to be due only to transconjugants (10(5) CFU/g of soil), since the donor was already undetectable when degradation started. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial community structure of the nonamended B-horizon soils. The new 16S rRNA gene fragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D-degrading transconjugant colonies isolated on agar plates. This result indicates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates that bioaugmentation can constitute an effective strategy for cleanup of soils which are poor in nutrients and microbial activity, such as those of the B horizon.
There is an urgent need in aquaculture to develop microbial control strategies, since disease outbreaks are recognized as important constraints to aquaculture production and trade and since the development of antibiotic resistance has become a matter of growing concern. One of the alternatives to antimicrobials in disease control could be the use of probiotic bacteria as microbial control agents. This review describes the state of the art of probiotic research in the culture of fish, crustaceans, mollusks, and live food, with an evaluation of the results obtained so far. A new definition of probiotics, also applicable to aquatic environments, is proposed, and a detailed description is given of their possible modes of action, i.e., production of compounds that are inhibitory toward pathogens, competition with harmful microorganisms for nutrients and energy, competition with deleterious species for adhesion sites, enhancement of the immune response of the animal, improvement of water quality, and interaction with phytoplankton. A rationale is proposed for the multistep and multidisciplinary process required for the development of effective and safe probiotics for commercial application in aquaculture. Finally, directions for further research are discussed.
Sustainable wastewater treatment requires that household wastewater is collected and treated separately from industrial wastewater and rainwater run-offs. This separate treatment is, however, still inadequate, as more than 70% of the nutrients and much of the chemical oxygen demand (COD) and potential pathogens of a domestic sewage system are confined to the few litres of black water (faeces, urine and toilet water). Whilst grey water can easily be filter treated and re-used for secondary household purposes, black water requires more intensive treatment due to its high COD and microbial (pathogens) content. Recently developed vacuum/dry toilets produce a nutrient rich semi-solid waste stream, which, with proper treatment, offers the possibility of nutrient, carbon, water and energy recovery. This study investigates the terrestrial applicability of Life Support System (LSS) concepts as a framework for future domestic waste management. The possibilities of treating black water together with other types of human-generated solid waste (biowastes/mixed wastes) in an anaerobic reactor system at thermophilic conditions, as well as some post treatment alternatives for product recovery and re-use, are considered. Energy can partially be recovered in the form of biogas produced during anaerobic digestion. The system is investigated in the form of theoretical mass balances, together with an assessment of the current feasibility of this technology and other post-treatment alternatives.
The on-line estimation of the maximum specific growth rate of autotrophic biomass is addressed in this article. A general nitrification process model, which is valid for any realistic flow pattern, is used to develop the estimation algorithm. Depending on the measurements available, two estimation equations are derived. While both require measuring the nitrification activity of the activated sludge, one requires the additional measurement of the nitrifiable nitrogen concentrations at the two ends of the bioreactor, and the other requires the nitrate nitrogen concentrations at the same locations. The algorithm also requires some stoichiometric and kinetic parameters. However, sensitivity analysis shows that the estimate is insensitive to the parameters other than the autotrophic decay rate. Compared to the existing algorithms, the algorithm developed in this article does not rely on the assumption of ideal flow pattern in the plant and does not require an error-prone estimate of the autotrophic biomass concentration. Experimental and simulation studies show that the algorithm performs well and is robust to influent variations and accidental sludge losses.