Description of filamentous bacteria present in industrial activated sludge WWTPs by conventional and molecular methods.

Conventional cultivation methods and molecular approaches were utilised to describe the filamentous bacterial population of industrial activated sludge WWTPs. In total 43 strains were isolated by micromanipulation and were affiliated with 12 different species, comprising two new species and a new genus. In particular, a new species of Microthrix, a new genus of a filamentous Alphaproteobacteria morphologically similar to Nostocoida limicola, and a new filamentous species closely related to the opportunistic pathogen Propionibacterium propionicum were obtained. Despite the high number of isolates, the cultivation approach was unable to describe the filamentous bacteria most common in industrial WWTP. A culture-independent approach, termed the cell sorting/RT-PCR method, was therefore applied to identify fastidious or non-culturable filamentous microrganisms from different industrial plants. By this method the relevant filaments were micromanipulated and their 16S rDNA genes were amplified by RT-PCR. This approach was highly efficient. In total 31 16S rRNA sequences were obtained and 16 of them were used for the design of new specific oligonucleotide probes that highlighted dominant filaments in industrial activated sludge plants.

Compact optical sensor for real-time monitoring of bacterial growth for space applications.

During long-duration manned space missions, complex chemical and biological processes need to be managed accurately for recycling human wastes and to produce human consumables. As a result, there is increasing interest in how the characteristics of microbes are influenced by microgravity. Compact optical instrumentation allows for real-time and non-invasive measurement of bacterial growth parameters during flight experiments. In close collaboration, the National Aerospace Laboratory of the Netherlands (NLR) and Bioclear Environmental Biotechnology developed and tested an on-line optical biomass sensor successfully. The sensor concept is based on a turbidity measurement technique operating in the VIS-blue part of the light spectrum with use of blue LED sources. A diagnostic tool has been developed using com-pact spectrometers and optical fibers to characterize bacterial cultures. As a result a few sensor applications operating at different colors and sensor layouts are discussed in the paper.

Molecular monitoring of bulking sludge in industrial wastewater treatment plants.

Fluorescent In Situ Hybridisation (FISH) was used to monitor the presence of filamentous microorganisms in industrial wastewater treatment plants (WWTPs). Monitoring with a restricted set of FISH probes in WWTPs from potato industry showed growth and decline of Thiothrix populations that could be linked to operational procedures. In a follow up project new FISH probes were developed for filamentous bacteria in industrial WWTPs and 70 WWTPs were analysed for presence of these filaments. Several newly described species of filamentous bacteria appear to be common and dominant in industrial WWTPs. Monitoring of a WWTP from textile industry showed growth and decline of one of these organisms when operational conditions in the plant were varied. The present paper demonstrates that bulking sludge in industrial wastewater treatment plants can effectively be monitored using a combination of standard chemical analyses and the FISH technique.

Lactobacillus diolivorans sp. nov., a 1,2-propanediol-degrading bacterium isolated from aerobically stable maize silage.

Inoculation of maize silage with Lactobacillus buchneri (5 x 10(5) c.f.u. g(-1) of maize silage) prior to ensiling results in the formation of aerobically stable silage. After 9 months, lactic acid bacterium counts are approximately 10(10) c.f.u. g(-1) in these treated silages. An important subpopulation (5.9 x 10(7) c.f.u. g(-1)) is able to degrade 1,2-propanediol, a fermentation product of L. buchneri, under anoxic conditions to 1-propanol and propionic acid. From this group of 1,2-propanediol-fermenting, facultatively anaerobic, heterofermentative lactobacilli, two rod-shaped isolates were purified and characterized. Comparative 16S rDNA sequence analysis revealed that the newly isolated bacteria have identical 16S rDNA sequences and belong phylogenetically to the L. buchneri group. DNA-DNA hybridizations, whole-cell protein fingerprinting and examination of phenotypic properties indicated that these two isolates represent a novel species, for which the name Lactobacillus diolivorans sp. nov. is proposed. The type strain is LMG 19667T (= DSM 14421T).

Anaerobic conversion of lactic acid to acetic acid and 1, 2-propanediol by Lactobacillus buchneri.

The degradation of lactic acid under anoxic conditions was studied in several strains of Lactobacillus buchneri and in close relatives such as Lactobacillus parabuchneri, Lactobacillus kefir, and Lactobacillus hilgardii. Of these lactobacilli, L. buchneri and L. parabuchneri were able to degrade lactic acid under anoxic conditions, without requiring an external electron acceptor. Each mole of lactic acid was converted into approximately 0.5 mol of acetic acid, 0.5 mol of 1,2-propanediol, and traces of ethanol. Based on stoichiometry studies and the high levels of NAD-linked 1, 2-propanediol-dependent oxidoreductase (530 to 790 nmol min(-1) mg of protein(-1)), a novel pathway for anaerobic lactic acid degradation is proposed. The anaerobic degradation of lactic acid by L. buchneri does not support cell growth and is pH dependent. Acidic conditions are needed to induce the lactic-acid-degrading capacity of the cells and to maintain the lactic-acid-degrading activity. At a pH above 5.8 hardly any lactic acid degradation was observed. The exact function of anaerobic lactic acid degradation by L. buchneri is not certain, but some results indicate that it plays a role in maintaining cell viability.

Characterization of 3-chlorobenzoate degrading aerobic bacteria isolated under various environmental conditions.

The rates of bacterial growth in nature are often restricted by low concentrations of oxygen or carbon substrates. In the present study the metabolic properties of 24 isolates that had been isolated using various concentrations of 3-chlorobenzoate, benzoate and oxygen as well as using continuous culture at high and low growth rates were determined to investigate the effects of these parameters on the metabolism of monoaromatic compounds. Bacteria were enriched from different sampling sites and subsequently isolated. In batch culture this was done both under low oxygen (2% O(2)) and air-saturated concentrations. Chemostat enrichments were performed under either oxygen or 3-chlorobenzoate limiting conditions. Bacteria metabolizing aromatics with gentisate or protocatechuate as intermediates (gp bacteria) as well as bacteria metabolizing aromatic compounds via catechols (cat bacteria) were isolated from batch cultures when either benzoate or 3CBA were used as C sources, regardless of the enrichment conditions applied. In contrast, enrichments performed in chemostats at low dilution rates resulted in gp-type organisms only, whereas at high dilution rates cat-type organisms were enriched, irrespective of the oxygen and 3-chlorobenzoate concentration used during enrichment. It is noteworthy that the gp-type of bacteria possessed relatively low µ(max) values on 3CBA and benzoate along with relatively high substrate and oxygen affinities for these compounds. This is in contrast with cat-type of bacteria, which seemed to be characterized by high maximum specific growth rates on the aromatic substrates and relatively high apparent half saturation constants. In contrast, bacteria degrading chlorobenzoate via gentisate or protocatechuate may possibly be better adapted to conditions leading to growth at reduced rates such as low oxygen and low substrate concentrations.

Degradation of 3-chlorobenzoate under low-oxygen conditions in pure and mixed cultures of the anoxygenic photoheterotroph Rhodopseudomonas palustris DCP3 and an aerobic Alcaligenes species.

The presence or absence of molecular oxygen has been shown to play a crucial role in the degradability of haloaromatic compounds. In the present study, it was shown that anaerobic phototrophic 3-chlorobenzoate (3CBA) metabolism by Rhodopseudomonas palustris DCP3 is oxygen tolerant up to a concentration of 3 microM O2. Simultaneous oxidation of an additional carbon source permitted light-dependent anaerobic 3CBA degradation at oxygen input levels which, in the absence of such an additional compound, would result in inhibition of light-dependent dehalogenation. Experiments under the same experimental conditions with strain DCP3 in coculture with an aerobic 3CBA-utilizing heterotroph, Alcaligenes sp. strain L6, revealed that light-dependent dehalogenation of 3CBA did not occur. Under both oxygen limitation (O2 < 0.1 microM) and low oxygen concentrations (3 microM O2), all the 3CBA was metabolized by the aerobic heterotroph. These data suggest that biodegradation of (halo)aromatics by photoheterotrophic bacteria such as R. palustris DCP3 may be restricted to anoxic photic environments.

Isolation of Alcaligenes sp. strain L6 at low oxygen concentrations and degradation of 3-chlorobenzoate via a pathway not involving (chloro)catechols.

Isolations of 3-chlorobenzoate (3CBA)-degrading aerobic bacteria under reduced O2 partial pressures yielded organisms which metabolized 3CBA via the gentisate or the protocatechuate pathway rather than via the catechol route. The 3CBA metabolism of one of these isolates, L6, which was identified as an Alcaligenes species, was studied in more detail. Resting-cell suspensions of L6 pregrown on 3CBA oxidized all known aromatic intermediates of both the gentisate and the protocatechuate pathways. Neither growth on nor respiration of catechol could be detected. Chloride production from 3CBA by L6 was strictly oxygen dependent. Cell-free extracts of 3CBA-grown L6 cells exhibited no catechol dioxygenase activity but possessed protocatechuate 3,4-dioxygenase, gentisate dioxygenase, and maleylpyruvate isomerase activities instead. In continuous culture with 3CBA as the sole growth substrate, strain L6 demonstrated an increased oxygen affinity with decreasing steady-state oxygen concentrations.