Analysis of bacteria contaminating ultrapure water in industrial systems

Bacterial populations inhabiting ultrapure water (UPW) systems were investigated. The analyzed UPW systems included pilot scale, bench scale, and full size UPW plants employed in the semiconductor and other industries. Bacteria present in the polishing loop of the UPW systems were enumerated by both plate counts and epifluorescence microscopy. Assessment of bacterial presence in UPW by epifluorescence microscopy (cyanotolyl tetrazolium chloride [CTC] and DAPI [4',6'-diamidino-2-phenylindole] staining) showed significantly higher numbers (10 to 100 times more bacterial cells were detected) than that determined by plate counts. A considerable proportion of the bacteria present in UPW (50 to 90%) were cells that did not give a positive signal with CTC stain. Bacteria isolated from the UPW systems were mostly gram negative, and several groups seem to be indigenous for all of the UPW production systems studied. These included Ralstonia pickettii, Bradyrhizobium sp., Pseudomonas saccharophilia, and Stenotrophomonas strains. These bacteria constituted a significant part of the total number of isolated strains (>or=20%). Two sets of primers specific to R. pickettii and Bradyrhizobium sp. were designed and successfully used for the detection of the corresponding bacteria in the concentrated UPW samples. Unexpectedly, nifH gene sequences were found in Bradyrhizobium sp. and some P. saccharophilia strains isolated from UPW. The widespread use of nitrogen gas in UPW plants may be associated with the presence of nitrogen-fixing genes in these bacteria.     

Analysis of Bacteria Contaminating Ultrapure Water in Industrial Systems

Bacterial populations inhabiting ultrapure water (UPW) systems were investigated. The analyzed UPW systems included pilot scale, bench scale, and full size UPW plants employed in the semiconductor and other industries. Bacteria present in the polishing loop of the UPW systems were enumerated by both plate counts and epifluorescence microscopy. Assessment of bacterial presence in UPW by epifluorescence microscopy (cyanotolyl tetrazolium chloride [CTC] and DAPI [4′,6′-diamidino-2-phenylindole] staining) showed significantly higher numbers (10 to 100 times more bacterial cells were detected) than that determined by plate counts. A considerable proportion of the bacteria present in UPW (50 to 90%) were cells that did not give a positive signal with CTC stain. Bacteria isolated from the UPW systems were mostly gram negative, and several groups seem to be indigenous for all of the UPW production systems studied. These included Ralstonia pickettii, Bradyrhizobium sp., Pseudomonas saccharophilia, and Stenotrophomonas strains. These bacteria constituted a significant part of the total number of isolated strains (≥20%). Two sets of primers specific to R. pickettii and Bradyrhizobium sp. were designed and successfully used for the detection of the corresponding bacteria in the concentrated UPW samples. Unexpectedly, nifH gene sequences were found in Bradyrhizobium sp. and some P. saccharophilia strains isolated from UPW. The widespread use of nitrogen gas in UPW plants may be associated with the presence of nitrogen-fixing genes in these bacteria.     

Selective Growth Inhibition of Sphaerotilus natans and Beggiatoa sp. by Nucleosides

9-beta-d-Arabinofuranosyladenine (ara-A) has been found to specifically inhibit the growth of Sphaerotilus natans and Beggiatoa sp. at a low concentration (0.78 mug/ml). The nucleoside had no antimicrobial activity against various microorganisms other than Candida albicans at 1,000 mug/ml. 3'-Deoxyadenosine, 2'-deoxyadenosine, formycin, and some derivatives of ara-A also showed inhibitory activity against Sphaerotilus natans. The growth of Beggiatoa sp. was also inhibited by 9-beta-arabinofuranosylhypoxanthine, 3'-deoxyadenosine, 2'-deoxyadenosine, formycin, toyocamycin, tubercidin, and some derivatives of ara-A. ara-A was quite stable in water and had no harmful effect on fish at 200 mug/ml. The possible uses of ara-A and some nucleosides in controlling the proliferation of S. natans and Beggiatoa sp. in the environment are discussed.     

The half-saturation coefficient for dissolved oxygen: A dynamic method for its determination and its effect on dual species competition

A simple approach was developed to determine the half-saturation coefficient for dissolved oxygen (K(DO)) for three bacteria by maintaining a constant oxygen concentration in continuous culture, and employing a dynamic method to obtain the specific growth rate (mu) for each species. Measurement of mu at selected dissolved oxygen concentrations (DO) resulted in a typical Monod curve for a plot of mu vs. DO. Values for K(DO) and mu(max) were obtained from the Lineweaver-Burk reciprocal plot. The bacteria studied included representative strains of three microorganisms isolated in pure culture from poorly settling activated sludge: two filamentous microorganisms, Sphaerotilus natans and a second Sphaerotilus sp., and an unidentified floc-forming microorganism. The K(DO) values obtained for Sphaerotilus sp., S. natans, and the floc former were 0.014, 0.033, and 0.073 mg/L, respectively. Dual species competition experiments were conducted in continuous culture under low and high DO conditions. Successful growth competition by these microorganisms under DO-limiting conditions was consistent with experimentally determined K(DO) values. The finding of lower K(DO) values for the two Sphaerotilus species, compared to the floc former, confirmed the hypothesis that these filamentous microorganisms can outgrow floc-forming microorganisms in activated sludge when DO in the aeration basin is low.     

Rapid, semiautomated quantification of bacterial cells in freshwater by using a microfluidic device for on-chip staining and counting

A microfluidic device-based system for the rapid and semiautomated counting of bacteria in freshwater was fabricated and examined. Bacteria in groundwater and in potable water, as well as starved Escherichia coli O157:H7 spiked in pond water, were able to be on-chip stained and enumerated within 1 h using this system.     

Culture dependent methods for enumeration of sulphate reducing bacteria (SRB) in the Oil and Gas industry

The group of anaerobic microorganisms collectively referred to as Sulphate Reducing Bacteria (SRB) is a major concern in the Oil and Gas industry primarily because of this group’s ability to generate substantial amounts of hydrogen sulfide and insoluble ferrous sulfide in the presence of iron. Traditionally, the Oil industry has relied on two recommended standard practices i.e. API RP-38 and NACE TM0194 for the detection and enumeration of culturable sulphate reducing bacteria for routine field monitoring. API RP-38 has now been withdrawn without any replacement. Data generated by nonstandard molecular microbiological methods which are still in the developmental stage cannot be compared with the accepted control levels for SRBs in oil field systems, monitored over the years with viable culture methods. Culture based methodologies are still important tools for the study of SRB, as they help in understanding the physiological characteristics which may be similar or different across phylogenetically similar bacteria. This review article therefore tries to highlight the continued importance of culture dependent methods for detection and enumeration of SRB in Oil field systems and the need for further development of an universal standard culture based method for studying SRB in the Oil and Gas industry.     

Thermogenesis of mesophilic, thermotolerant and thermophilic strains of microorganisms--producers of fungal cell wall--destroying enzymes]

Investigations were carried out to clarify the relationship between thermogenesis and production of yeast wall lyzing enzymes by the mesophilic strain of Bacillus subtilis, thermotolerant strain of Actinomyces sp. II and thermophilic strain of Actinomyces sp. 10. The enzymic lyzing activity was measured in the culture liquid filtrate of those microorganisms. The thermophilic strain of Actinomyces sp. 10 showed the highest enzymic activity. The thermogenetic curves of the cultures had several inflections. The mesophilic culture of Bacillus subtilis whose enzymic lyzing activity was the lowest displayed the highest heat release.     

In vitro modeling of dental water line contamination and decontamination

The contamination of dental unit water lines (DUWL) is an emerging concern in dentistry. The aim of this study was to use an in vitro DUWL to model microbial contamination and evaluate the decontamination efficacy of tetraacetylethylenediamine (TAED) solutions. A DUWL biofilm model used to simulate clinical conditions was used to generate a range of biofilms in DUWL. Three distinct biofilms were generated: (1) biofilm from water, (2) biofilm from a mix of water + contaminating human commensal bacteria, (3) biofilm from water with contaminating oral bacteria added after biofilm formed. The contaminating oral species used were Streptococcus oralis, Enterococcus faecalis and Staphylococcus aureus. Decontamination by simple water flushing or flushing with TAED was evaluated (2, 5 and 10 min intervals). The DUWL tubes were split and samples were plated onto a range of media, incubated and bacteria enumerated. Water flushing did not reduce the number of microorganisms detected. Bacteria were not detected from any of the TAED sampling points for any of the biofilm types tested. Interestingly, if contamination was introduced to new DUWL along with the waterborne species a biofilm was formed containing only the waterborne species. If however, an existing biofilm was present before the introduction of "contaminating" bacteria then these could be detected in the biofilm. This implies that if the DUWL are new or satisfactorily cleaned on a regular basis then the associated cross-contamination aspects are reduced. In conclusion, TAED provides effective control for DUWL biofilms.     

Effect of plant extract on the degradation of nitroaromatic compounds by soil microorganisms

Remediation of soils contaminated by nitroaromatic compounds and nitramines, i.e. explosives, is known as very important, complicated, and rapidly developing area of biotechnology. A search for optimal growth conditions for soil bacteria is of a great importance in order to isolate various xenobiotic degraders. Bacteria consortium A43 was isolated from soils contaminated with explosives. In the presence of carbohydrate and plant extract, an addition of TNT to the solidified minimal medium stimulated the growth of the tested bacteria, as compared to other bacteria consortium isolated from the same soils. Reducing sugars as carbohydrates, and cabbage leaf extract as a plant extract were used in these experiments. Cultivation of the A43 in liquid medium of the same content showed that addition of cabbage leaf extract alone to medium is much more efficient for TNT degradation by growing biomass as compared to addition of carbohydrate alone.     

动静态水环境下不同种植方式对苦草生长的影响

设置扦插法、纱布包裹法、布袋覆土法3种种植方式,研究苦草（Vallisneria natans（Lour.）Hara）在静态水环境下和水体受到持续扰动的动态条件下不同种植方式对植株生长的影响。结果显示,不同种植方式下苦草的生长差异明显。在苦草形态特征方面,布袋覆土法种植方式下苦草的平均株高和叶宽明显高于扦插法和纱布包裹法,但是其分株数和平均根长小于扦插法和纱布包裹法;在苦草的生物量和地下与地上部分之比方面,布袋覆土法种植方式下苦草的地上、地下部分生物量和总生物量明显大于扦插法和纱布包裹法,但是其地下与地上部分生物量之比小于其他2种方法;在苦草叶片叶绿素a含量方面,布袋覆土法明显高于扦插法和纱布包裹法。动静态水环境只对苦草的分株数有显著差异,静态水环境下分株数大于动态水环境,对其他指标无显著影响。研究结果表明动、静态水环境和不同种植方式对苦草的生长具有显著的影响,布袋覆土法种植方式下单株苦草生长最好;静态水环境下生长的苦草株高、叶宽和生物量等指标均优于动态水环境。     

A comparative analysis of microflora during biofilm development on grape seeds exposed to methanol in a biofilter

The attachment of microorganisms onto biotic surfaces to form biofilm structures on the support media of a biofilter has great impact on biodegradation systems. This study examined the composition of the microbial community that developed on grape seeds (GS) used as support media in methanol degradation biofilters. They were analyzed using conventional microbiology techniques and API galleries. Analysis of microbial counts showed that, in GS before methanol exposure, bacteria and filamentous fungi were predominant over yeasts. In contrast, GS exposed to methanol exhibited more bacteria and yeasts than fungi. Most of the Gram-negative bacteria were the Pseudomonas genus, Bacillus staerothermophilus, Bacillus amyloliquefaciens, and Bacillus pumilus. Rhodotorula mucilaginosa was the primary yeast found. The filamentous fungi Aspergillus sp. Cladosporium cladosporioides, Fusarium sp., and Alternaria sp. were also detected. No Gram-positive bacteria growth was found on GS exposed to methanol. Using scanning electron microscopy, biofilm formation on the GS was examined to reveal the presence of both prokaryotic and eukaryotic microorganisms as biomass accumulation was visible on the seeds. Seeds exposed to methanol for 90 days showed a mature biofilm with cuticle and epidermal layer decline, as well as biofilm dissolution into grape seed integuments.     

Rapid enumeration of physiologically active bacteria in purified water used in the pharmaceutical manufacturing process

Physiologically active bacteria in purified water used in the manufacturing process of pharmaceutical products were enumerated in situ. Bacteria with growth potential were enumerated using the micro-colony technique and direct viable counting (DVC), followed by 24 h of incubation in 100-fold diluted SCDB (Soybean Casein Digest Broth) at 30 degrees C. Respiring and esterase-active bacteria were detected by fluorescent staining with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 6-carboxyfluorescein diacetate (6CFDA), respectively. A large number of bacteria in purified water retained physiological activity, while most could not form colonies on conventional media. The techniques applied in this study enabled bacteria to be counted within 24 h so results could be available within one working day. These rapid and convenient techniques should be useful for the systematic monitoring of bacteria in water used for pharmaceutical manufacturing.     

Chlorine resistance patterns of bacteria from two drinking water distribution systems.

The relative chlorine sensitivities of bacteria isolated from chlorinated and unchlorinated drinking water distribution systems were compared by two independent methods. One method measured the toxic effect of free chlorine on bacteria, whereas the other measured the effect of combined chlorine. Bacteria from the chlorinated system were more resistant to both the combined and free forms of chlorine than those from the unchlorinated system, suggesting that there may be selection for more chlorine-tolerant microorganisms in chlorinated waters. Bacteria retained on the surfaces of 2.0-microns Nuclepore membrane filters were significantly more resistant to free chlorine compared to the total microbial population recovered on 0.2-micron membrane filters, presumably because aggregated cells or bacteria attached to suspended particulate matter exhibit more resistance than unassociated microorganisms. In accordance with this hypothesis, scanning electron microscopy of suspended particulate matter from the water samples revealed the presence of attached bacteria. The most resistant microorganisms were able to survive a 2-min exposure to 10 mg of free chlorine per liter. These included gram-positive spore-forming bacilli, actinomycetes, and some micrococci. The most sensitive bacteria were readily killed by chlorine concentrations of 1.0 mg liter-1 or less, and included most gram-positive micrococci, Corynebacterium/Arthrobacter, Klebsiella, Pseudomonas/Alcaligenes, Flavobacterium/Moraxella, and Acinetobacter.     

Determination of total protein content of bacterial cells by SYPRO staining and flow cytometry.

An assay has been developed for measuring protein biomass of marine planktonic bacteria by flow cytometry. The method was calibrated by using five species of Bacteria (an Arcobacter sp., a Cytophaga sp., an Oceanospirillum sp., a Pseudoalteromonas sp., and a Vibrio sp.) recently isolated from seawater samples and grown in culture at different temperatures. The intensity of SYPRO-protein fluorescence of these bacteria strongly correlated with their total protein content, measured by the bicinchoninic acid method to be in the range of 60 to 330 fg of protein cell-1 (r2 = 0.93, n = 34). According to the calibration, the mean biomass of planktonic bacteria from the North Sea in August 1998 was 24 fg of protein cell-1.     

Rapid quantification of bacterial cells in potable water using a simplified microfluidic device

A simplified microfluidic device for quantification of bacteria in potable water was fabricated and examined. Comparisons of counts of Escherichia coli by the microfluidic system and by epifluorescence microscopy closely correlated (r2=0.99). Bacteria in natural mineral water and in purified household tap water were accurately enumerated by using this system within 15 min after fluorescent staining.     

Abundance and function of bacteria in the Southern Ocean.

The very low water temperatures existing in polar oceans that experience seasonal advance and retreat of pack ice do not inhibit the presence of large bacterial populations. Bacteria may contribute significantly to the energy transfers within the Southern Ocean. In the last decades, notable progress has been made in the knowledge of the role of marine bacteria in the Southern Ocean. A short overview of the abundance and function ofAntarctic marine bacteria is given, with respect to metabolic activity. The importance of spatial and temporal variability is described. The ecological function of Antarctic marine bacterioplankton is discussed. Depending on food web structure, bacteria may be either a link in food webs supporting metazoan production, or a sink where bacterial production is metabolised by microorganisms. In the more oligotrophic areas and during certain periods of the year bacterial biomass dominates phytoplankton. The microbial food web is therefore the dominant pathway for carbon and energy flow in Antarctic seawater.     

Characterisation and Comparison of Microbial Populations in Swine Faeces and Manure Storage Pits by 16S rDNA Gene Sequence Analyses

Odour emanating from anaerobic lagoons and swine production facilities has increased the tension among rural neighbors and among urban and rural residents. Storage of swine manure is associated with the production of a variety of odorous compounds, including ammonia, organic acids and alcohols, and sulphides. Although the generation of these chemicals is the result of microbiological activity, little is known about the types of microorganisms responsible for their production. We have initiated an approach to determine and compare the bacterial populations present in both pig faeces and manure storage pits. Total DNA was isolated from both of these ecosystems. DNA sequence analyses of PCR amplified 16S rDNA genes derived from eubacterial sequences were carried out. Similarity analyses of the 16S sequences indicated the presence of primarily low G + C Gram-positive bacteria, such as Clostridium sp., Streptococcus sp., and Lactobacillus sp. in both ecosystems. Many of the sequences were from unidentified microorganisms. These results indicate that the primary eubacteria identified in swine faeces and manure pits are low G + C, Gram-positive bacteria.     

Bacterial interactions in the rhizosphere of seagrass communities in shallow coastal lagoons

Rooted phanerogam communities in the shallow intertidal and subtidal coastal zone represent productive and healthy ecosystems. Inorganic nutrients are assimilated into seagrass biomass. Much of the organic matter resulting from moribund seagrass is rapidly mineralized, principally by bacteria. The microbial community of the rhizosphere is also highly active due to the supply of organic matter released during photosynthesis. This active sediment community plays an important role through carbon, nitrogen and phosphorous cycling in maintaining the stability and productivity of seagrass meadows. Over the last two decades, however, seagrass meadows in European coastal areas have declined due to increasing pollution. As eutrophication advances a trasition occurs from rooted phanerogram dominated communities to planktonic algal blooms and/or cyanobacterial blooms. Such changes represent the decline of a stable, high biodiversity habitat to an unstable one dominated by a few species. These changes of community structure can occur rapidly once the internal nutrient and organic matter control cycles are exceeded. A field investigation was undertaken to establish the spatial distribution of bacterial populations of Zostera noltii colonized and uncolonized sediment in the Bassin d'Arcachon, France. Bacteria were enumerated using both plate count and MPN techniques for different functional groups as well as determining the total bacterial populations present. Nitrogen fixation, ammonification, sulphate reduction rates, as well as alkaline phosphatase activity were also determined. Colonization of the Z. noltii roots and rhizomes was studied by light and scanning electron microscopy. Results confirmed that higher bacterial populations were present in the rhizosphere of Z. noltii compared to uncolonized sediments. Furthermore, electron microscopy identified the rhizome as the main site of colonization for a diverse range of morphological groups of bacteria. Sulphate reducing bacteria were identified as the key group of bacteria involved in N-fixation in the rhizosphere of Z. noltii. The data will be discussed in relation to the role played by the rhizosphere microflora in supplying and mobilising nutrients in Z. noltii.     

Kinetics of SO4−2 reduction under different growth media by sulfate reducing bacteria

Sulfate Reducing Bacteria (SRB) were used to reduce the SO₄ ^–2 concentration in waste water. The growth pattern of SRB was found by varying the concentration of nutrients and the biomass. The specific reaction constant was evaluated in each case.