Deuterated styrene ([2 H 8 ]styrene) was used as a tracer in combination with phospholipid fatty acid (PLFA) analysis for characterization of styrene-degrading microbial populations of biofilters used for treatment of waste gases. Deuterated fatty acids were detected and quantified by gas chromatography-mass spectrometry. The method was evaluated with pure cultures of styrene-degrading bacteria and defined mixed cultures of styrene degraders and non-styrene-degrading organisms. Incubation of styrene degraders for 3 days with [ 2 H 8 ]styrene led to fatty acids consisting of up to 90% deuterated molecules. Mixed-culture experiments showed that specific labeling of styrene-degrading strains and only weak labeling of fatty acids of non-styrenedegrading organisms occurred after incubation with [ 2 H 8 ]styrene for up to 7 days. Analysis of actively degrading filter material from an experimental biofilter and a full-scale biofilter by this method showed that there were differences in the patterns of labeled fatty acids. For the experimental biofilter the fatty acids with largest amounts of labeled molecules were palmitic acid (16:0), 9,10-methylenehexadecanoic acid (17:0 cyclo9-10), and vaccenic acid (18:1 cis11). These lipid markers indicated that styrene was degraded by organisms with a Pseudomonas-like fatty acid profile. In contrast, the most intensively labeled fatty acids of the full-scale biofilter sample were palmitic acid and cis-11-hexadecenoic acid (16:1 cis11), indicating that an unknown styrene-degrading taxon was present. Iso-, anteiso-, and 10-methyl-branched fatty acids showed no or weak labeling. Therefore, we found no indication that styrene was degraded by organisms with methyl-branched fatty fatty acids, such as Xanthomonas, Bacillus, Streptomyces, or Gordonia spp.Extraction and analysis of chemotaxonomically important lipid markers from environmental samples constitute a wellestablished method for characterizing microbial communities, detecting community changes through time, or obtaining information about the metabolic status of a community (39). Recently, phospholipid fatty acid (PLFA) analyses were combined with carbon isotope labeling techniques to link degradation activities with specific microbial populations (2, 12, 25). 14 C tracers were successfully used for this approach (28). However, the main disadvantage of the procedure was the low efficiency of separation of the radiolabeled fatty acid methyl esters (FAMEs) caused by the discontinuous collection of fractions prior to scintillation counting. This was avoided by using substrates labeled with the stable 13 C isotope, which facilitated continuous detection of FAMEs by gas chromatography and on-line-combustion isotope ratio mass spectrometry (27).We studied the use of a deuterated substrate as an alternative to 13 C isotopes for characterization of actively degrading microbial populations in complex communities. The use of deuterated substrates with subsequent gas chromatographymass spectrometry analysis of the products is a well-establ...

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Sulfate reducing bacteria (SRB) are widely used for attenuating heavy metal pollution by means of sulfide generation. Due to their low metal tolerance, several SRB species depend on associated bacteria in mixed cultures to cope with metal-induced stress. Yet the identity of the SRB protecting bacteria is largely unknown. We aimed to identify these associated bacteria and their potential role in two highly metal-resistant mixed SRB cultures by comparing bacterial community composition and SRB activity between these cultures and two sensitive ones. The SRB composition in the resistant and sensitive consortia was similar. However, whereas the SRB in the sensitive cultures were strongly inhibited by a mixture of copper, zinc, and iron, no influence of these metals was detected on SRB growth and activity in the resistant cultures. In the latter, a Gram-positive population mostly assigned to Clostridium spp. initiated heavy metal bioremoval based on sulfide generation from components of the medium (mainly sulfite) but not from sulfate. After metal levels were lowered by the Clostridium spp. populations, SRB started sulfate reduction and raised the pH of the medium. The combination of sulfite reducing Clostridium spp. with SRB may improve green technologies for removal of heavy metals.

The bacteriologic treatment efficiency of vertical and horizontal subsurface flow constructed wetlands (SFCWs) was analysed in two multistage wastewater treatment systems by culture dependent and independent methods. When assessed with standard cultivation procedures, bacteria removal efficiency of the vertical and horizontal SFCWs was similar. However, microscopic enumerations of the wastewater bacteria after DNA staining revealed a completely different removal pattern: bacteria removal efficiency of the horizontal SFCWs was in general low and erratic, whereas the vertical SFCWs displayed high bacteria removal rates. The discrepancies in the results obtained by bacteria enumeration and cultivation was due to a strong decrease in bacterial culturability after treatment by the horizontal SFCWs, leading to overestimation of the real bacterial concentrations in these effluents. Additionally, a PCR based approach for the detection of the enteropathogenic bacteria Campylobacter jejuni and Yersinia enterocolitica was tested in the wastewater samples. The methods were specific and reproducible in the analysed samples and could be carried out within 12 h, proving very adequate as an alternative to cultivation. This work recommends a review of the current standard methodology for wastewater quality surveillance, as well as of the design of SFCW.