R. Maarit Niemi

Extraction and purification of DNA in rhizosphere soil samples for PCR-DGGE analysis of bacterial consortia.

Application of DNA fingerprinting methods enables the detection of diverse members of soil bacterial consortia, even including those bacteria not yet cultivated. However, extraction and purification of DNA from soil samples without bias is difficult. We compared five different DNA isolation methods and three purification methods for rhizosphere soil samples. Purified DNA extracts were amplified in PCR using universal bacterial primers and the PCR products were analysed with denaturing gradient gel electrophoresis (DGGE) for the visualisation of DNA bands representing dominant bacterial species. Both the isolation and purification methods affected the apparent bacterial community structure of the samples.

Rhizobia isolated from root nodules of tropical leguminous trees characterized using DNA-DNA dot-blot hybridisation and rep-PCR genomic fingerprinting.

Summary Fifty-one fast growing rhizobial strains isolated from root nodules of Acacia Senegal and Prosopis chilensis in Sudan and Kenya were divided into DNA homology groups using non-radioactive DNA-DNA dot-blot hybridisation. Rhizobium leguminosarum, R. galegae, R. tropici, Mesorhizobium loti, Sinorhizobium fredii, S. meliloti used in numerical taxonomy were included in the hybridisation experiments as reference strains and, at a later stage S. saheli and S. terangae. Scores given to the intensities of dots detected in the hybridisation experiments were used in principal component analysis, which clustered the majority of the tree rhizobia in two separate DNA-homology groups. The 51 strains were also analysed by genomic fingerprinting using the repetitive sequence-based polymerase chain reaction (rep-PCR) method with REP, ERIC, BOX and GTG5 primers. The resulting genomic fingerprints were compared with strains representing 15 rhizobial species. The relationship of 17 Sudanese strains to established sinorhizobial species was examined using the optical renaturation rates method and the G+C content of nine strains was determined. Results from dot-blot hybridisation and rep-PCR experiments were found to be in close agreement with each other and with the results obtained from spectrophotometric reassociation analysis. We suggest that rep-PCR fingerprinting can be used as a first and dot-blot hybridisation as a second rapid and dependable genomic screening method to classify new rhizobial isolates of unknown taxonomic status and to choose the representative strains for the more laborious DNA-DNA reassociation experiments.

Previously uncultured β-Proteobacteria dominate in biologically active granular activated carbon (BAC) filters.

Bacteria colonizing BAC filters used in drinking water purification from lake water were characterized by morphology, physiological tests, whole cell protein profiles and PLFA (phospholipid fatty acid) composition, and identified by partial 16S rRNA gene sequencing. Epifluorescence revealed prothecate bacteria to dominate in BAC. The majority of the isolates belonged to order Burkholderiales of beta-Proteobacteria, a few to Comamonadaceae but the majority to an undescribed family and the related sequences belonged mainly to uncultured bacteria. Among the less common alpha-Proteobacteria the genus Sphingomonas and the genera Afipia, Bosea or Bradyrhizobium of the Bradyrhizobiaceae family were detected. The majority of cultured bacteria persisting in the BAC biofilter were Burkholderiales, which according to ecological information are efficient in the mineralisation of dissolved organic matter in BAC. The biotechnical potential of the previously uncultured dominant bacteria warrants to be further studied.

The impact of crop plant cultivation and peat amendment on soil microbial activity and structure

AbstractA rapid means for restoring soil fertility could be addition of peat to the plough layer. The impact of cultivation of eight different crops (the joint impact of plant and the management tailored for each plant), with and without soil amendment by peat treatment on soil microbiological, physical and chemical properties was assessed for two consecutive growing seasons. As a measure of the functional diversity of soil microbial community we estimated the activity of several different extracellular soil enzymes using the ZymProfiler® test kit. ATP content was measured to yield information on the amount of the active microbial biomass, and phospholipid fatty acid (PLFA) profiles were analysed to reveal the microbial community structure. The enzyme activity patterns of the soil samples indicated several differences due to the different crops and years but ATP content and PLFA profiles were rather stable. However, microbial biomass as total amount of PLFAs depended on the plant and peat treatment and ATP content varied between the years. The effects of the peat treatments were less clearly indicated by the biological parameters one or two years after the amendment, as only arylsulphatase and β-xylosidase activities were affected in both the years. Soil moisture, affecting enzyme activities, depended on the year and crop plant and peat addition increased it. Abbreviations: AMC – 7-amino-4-methylcoumarin; AP – aminopeptidase; ATP – adenosine triphosphate; Cmic– microbial biomass carbon; DNA – deoxyribonucleic acid; EC – electrical conductivity; FAME – fatty acid methyl ester; fw – fresh weight; MUF – 4-methylumbelliferyl; na – not added; Nmic– microbial biomass nitrogen; PDE – phosphodiesterase; PLFA – phospholipid fatty acid; PME – phosphomonoesterase; SOM – soil organic matter

Acetate and ethanol as potential enhancers of low temperature denitrification in soil contaminated by fur farms: a pilot-scale study.

Ethanol and acetate were examined as potential candidates to enhance denitrification at low temperature in soils contaminated by fur farms. Five pilot-scale sand and gravel columns with a top layer of soil from a fur farm were set up and fed with nitrate-containing water (influent concentration of 100 and 200 mg L(-1)) for 459 days at 6+/-2 degrees C. Two of the columns also received acetate and two other ethanol while one received no additional C-substrate. During the experiment, various C:N-ratios were tested to find the most optimal concentration of the added C-substrates, and effluent concentrations of nitrate, nitrite, and TOC were monitored. At the end of the experiments, soils in the columns were unpacked and the soils were used to measure a pattern of enzyme activities and the rates of denitrification in microcosms. The fur farm contaminated soil appeared to harbour a good intrinsic potential for denitrification, which could be greatly enhanced by the introduction of ethanol or acetate. Consequently, in the C-substrate-fed columns, 95-100% of the influent nitrate was removed after an acclimatization period of some weeks. Ethanol with C:N-ratio of ca. 6 at the nitrate level 200 mg L(-1) proved to be the most promising candidate to be used in field trials.

Enumeration of intestinal enterococci and interfering organisms with Slanetz‐Bartley agar, KF streptococcus agar and the MUST method

The recovery of intestinal species of enterococci and streptococci and potentially interfering nonfaecal species was measured on KF streptococcus agar, Slanetz‐Bartley agar and in a medium based on 4‐methylumbelliferyl‐β‐d‐glucoside (MUST method) using pure cultures. Both of the solid media yielded high recoveries of the target species. Their selectivity was better at elevated incubation temperature but nonfaecal Enterococcus and Staphylococcus species were not eliminated even at the elevated temperature. The MUST method tended to give slightly lower recoveries than the agar cultivation methods with some target species at 44°C but recoveries were better at 41°C.

Confirmation of Escherichia coli and its distinction from Klebsiella species by gas and indole formation at 44 and 44.5°C

Aims: In the enumeration of coliform bacteria, confirmation of Escherichia coli has been based upon gas and indole production at the elevated incubation temperature. The test for gas production has recently been questioned. The aim of this study was to investigate the impact of gas production test on the reliability of confirmation of E. coli.

Virulence genes of Aeromonas isolates, bacterial endotoxins and cyanobacterial toxins from recreational water samples associated with human health symptoms

Exposure to cyanobacterial water blooms has been associated with various kinds of adverse health effects. In addition to cyanobacteria and their toxins, the bacteria associated with cyanobacteria could also be the etiological agents. We isolated Aeromonas strains (n = 176) from water samples (n = 38) taken from sites where cyanobacteria were suspected to have caused human health symptoms, of which fever and gastrointestinal symptoms were the most common. The isolates were screened by PCR for six virulence gene types (12 genes). The majority (90%) of the strains contained at least one of the virulence genes. Most common amplification products were those of genes (act/aerA/hlyA) that encode cytotoxic enterotoxin and haemolytic products. The genes encoding cytotonic enterotoxins (ast and alt), phospholipase (lip/pla/lipH3/alp-1), elastase (ahyB) and flagellin subunits (flaA/flaB) were also present in 5-37% of the Aeromonas strains. Analysed toxins (cyanobacterial hepatotoxins and neurotoxins, and bacterial endotoxins) were not detectable or were present in only low concentrations in the majority of the samples. The results indicated that the toxins were unlikely to be the main cause of the reported adverse health effects, whereas more attention should be paid to bacteria associated with cyanobacteria as a source of health effects.