Incidence of Prosthecate Bacteria in a Polluted Stream

Water samples were collected aseptically several times throughout the year at nine stations on the Red Cedar River, a stream flowing through farmland and receiving effluent from several municipalities in central Michigan. Total prosthecate bacteria were enumerated by both direct and viable counting techniques. By direct techniques, these bacteria accounted for 0.62 to 1.1% of the total microflora during the study. The predominant type of appendaged bacteria was the caulobacters (Caulobacter, Asticcacaulis, and the fusiform caulobacter), which accounted for 64 to 93% of the total prosthecate forms. The others of importance were prosthecomicrobia (< 1 to 24%), including Prosthecomicrobium and Prosthecochloris; hyphomicrobia (< 1 to 15%), including Hyphomicrobium and Rhodomicrobium; and Ancalomicrobium (< 1 to 6%). The viable counts of heterotrophs also indicated that the caulobacters were the most numerous prosthecate bacteria in the stream. They ranged from fewer than 1 per ml to a maximum of almost 4,000 per ml. During the coldest period, when the total viable counts decreased to about 10(4) per ml compared to their summer high of over 10(7) per ml, the caulobacters actually increased in numbers. In December (temperature 0 to 1 C), they comprised from 0.09 to 1.0% of the viable microbial count, and in March (6.0 to 8.0 C) they accounted for 0.14 to 2.8%. The other heterotrophic prosthecate bacteria were generally found at numbers less than 1 per ml, with the exception of the December study when Hyphomicrobium was present in numbers as high as 2,400 per ml. There was no consistent correlation between the frequency of prosthecate bacteria and total coliforms in the stream during the investigation.     

Coliform Bacteria and Nitrogen Fixation in Pulp and Paper Mill Effluent Treatment Systems

The majority of pulp and paper mills now biotreat their combined effluents using activated sludge. On the assumption that their wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N₂ fixation in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase (nifH) gene probing, and bacterial isolations. In situ N₂ fixation was undetectable in all seven bioreactors but was present in six associated primary clarifiers. One primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained nifH, of which >90% were Klebsiella strains. All primary-clarifier coliform bacteria growing on MacConkey agar were identified as klebsiellas, and all those probed contained nifH. In contrast, analysis of 48 random coliform isolates from other mill water system locations showed that only 24 (50%) possessed the nifH gene, and only 13 (27%) showed inducible N₂-fixing activity. Thus, all the pulp and paper mill primary clarifiers tested appeared to be sites of active N₂ fixation (0.87 to 4.90 mg of N liter⁻¹ day⁻¹) and a microbial community strongly biased toward this activity. This may also explain why coliform bacteria, especially klebsiellas, are indigenous in pulp and paper mill water systems.     

Enumeration of High Numbers of Bacteria Using Hydrophobic Grid-Membrane Filters

Printing a wax grid on a conventional membrane filter yields a device functioning as a most probable number apparatus (MPN), used at a single dilution but with a very large number of growth compartments (e.g., 3,650). By restraining the lateral spread and confluence of colonies, the hydrophobic grid-membrane filter (HGMF) allows growth- or colony-forming units (GU) to be resolved at levels far above those which produce an uncountable lawn on a conventional membrane filter. It also eliminates the size variation of normal bacterial colonies. As a result, the HGMF can give more accurate estimates of the concentration of GU. The method by which grid-cell count observations can be used to obtain MPN estimates of the number of GUs is described, and estimates obtained using the MPN method on the HGMF are compared with those resulting from conventional colony count procedures on membrane filters. A linear relation was observed between MPNGU and the number of GUs, at levels up to 30,000 GUs, for pure cultures of bacteria and for samples of natural waters. The HGMF has great potential for reducing the labor required in quantitative microbiology, since it allows, with one filter, enumeration of microorganisms over a very large concentration range and therefore reduces the need to make dilutions.     

Deoxyribonucleic Acid Base Sequence Homologies of Some Budding and Prosthecate Bacteria

The genetic relatedness of a number of budding and prosthecate bacteria was determined by deoxyribonucleic acid (DNA) homology experiments of the direct binding type. Strains of Hyphomicrobium sp. isolated from aquatic habitats were found to have relatedness values ranging from 9 to 70% with strain "EA-617," a subculture of the Hyphomicrobium isolated by Mevius from river water. Strains obtained from soil enrichments had lower values with EA-617, ranging from 3 to 5%. Very little or no homology was detected between the amino acid-utilizing strain Hyphomicrobium neptunium and other Hyphomicrobium strains, although significant homology was observed with the two Hyphomonas strains examined. No homology could be detected between prosthecate bacteria of the genera Rhodomicrobium, Prosthecomicrobium, Ancalomicrobium, or Caulobacter, and Hyphomicrobium strain EA-617 or H. neptunium LE-670. The grouping of Hyphomicrobium strains by their relatedness values agrees well with a grouping according to the base composition of their DNA species. It is concluded that bacteria possessing cellular extensions represent a widely diverse group of organisms.     

Factors Influencing the Occurrence of High Numbers of Iodine-Resistant Bacteria in Iodinated Swimming Pools

It has been shown that, although iodinated swimming-pool waters are usually free from coliform bacteria and enterococci, the total counts frequently become relatively high. Pseudomonas alcaligenes and Alcaligenes faecalis have been shown to account for most of these high counts. It was of interest, therefore, to compare the microbial flora of four alternately chlorinated and iodinated swimming pools. By means of the membrane filter method and suitable selective media, examinations were made for total viable counts, coliform bacteria, enterococci, staphylococci, Streptococcus salivarius, and P. aeruginosa. Colonies also were picked from membrane filters incubated on standard plate count agar and identified. The results showed that, although viable counts were significantly higher during the iodinated periods, the specific types of bacteria determined were either fewer than or the same as in chlorinated periods. During chlorination, the predominant microbial flora consisted of staphylococci and members of the genus Bacillus. During iodination, however, the P. alcaligenes-A. faecalis group accounted for 92 to 99% of the microbial flora. The accumulation of high numbers of these bacteria was shown to be due to their iodine resistance and their ability to grow rapidly in pool water in the absence of free iodine.     

Occurrence of Genes Associated with Enterotoxigenic and Enterohemorrhagic Escherichia coli in Agricultural Waste Lagoons

The prevalence among all Escherichia coli bacteria of the LTIIa toxin gene and STII toxin gene, both associated with enterotoxigenic E. coli, and of three genes (stxI, stxII, and eaeA) associated with enterohemorrhagic E. coli was determined in farm waste disposal systems seasonally for 1 year. Single- and nested-PCR results for the number of E. coli isolates carrying each toxin gene trait were compared with a five-replicate most-probable-number (MPN) method. The STII and LTIIa toxin genes were present continuously at all farms and downstream waters that were tested. Nested-MPN-PCR manifested sensitivity increased over that of single-MPN-PCR by a factor of 32 for LTIIa, 10 for STII, and 2 for the stxI, stxII, and eaeA genes. The geometric mean prevalence of each toxin gene within the E. coli community in waste disposal site waters after nested MPN-PCR was 1:8.5 E. coli isolates (1:8.5 E. coli) for the LTIIa toxin gene and 1:4 E. coli for the STII toxin gene. The geometric mean prevalence for the simultaneous occurrence of toxin genes stxI, stxII, and eaeA, was 1:182 E. coli. These findings based on total population analysis suggest that prevalence rates for these genes are higher than previously reported in studies based on surveys of single isolates. With a population-based approach, the frequency of each toxin gene at the corresponding disposal sites and the endemic nature of diseases on farms can be easily assessed, allowing farmers and public health officials to evaluate the risk of infection to animals or humans.     

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.     

Niche Differentiation of Ammonia-Oxidising Archaea (AOA) and Bacteria (AOB) in Response to Paper and Pulp Mill Effluent

Sediment organic loading has been shown to affect estuarine nitrification and denitrification, resulting in changes to sediment biogeochemistry and nutrient fluxes detrimental to estuarine health. This study examined the effects of organic loading on nutrient fluxes and microbial communities in sediments receiving effluent from a paper and pulp mill (PPM) by applying microcosm studies and molecular microbial ecology techniques. Three sites near the PPM outfall were compared to three control sites, one upstream and two downstream of the outfall. The control sites showed coupled nitrification–denitrification with minimal ammonia release from the sediment. In contrast, the impacted sites were characterised by nitrate uptake and substantial ammonia efflux from the sediments, consistent with a decoupling of nitrification and denitrification. Analysis of gene diversity demonstrated that the composition of nitrifier communities was not significantly different at the impacted sites compared to the control sites; however, analysis of gene abundance indicated that whilst there was no difference in total bacteria, total archaea or ammonia-oxidising archaea (AOA) abundance between the control and impacted sites, there was a significant reduction in ammonia-oxidising bacteria (AOB) at the impacted sites. The results of this study demonstrate an effect of organic loading on estuarine sediment biogeochemistry and highlight an apparent niche differentiation between AOA and AOB.     

Occurrence of Two Resin Acid-Degrading Bacteria and a Gene Encoding Resin Acid Biodegradation in Pulp and Paper Mill Effluent Biotreatment Systems Assayed by PCR

Abstract We examined the distribution of two dehydroabietic acid-degrading bacteria, Pseudomonas abietaniphila BKME-9 and Zoogloea resiniphila DhA-35, in biotreatment systems for pulp and paper mill effluents (PPMEs) using PCR assays. These two bacteria were first isolated from two PPME biotreatment systems and can degrade both dehydroabietic acid (DhA) and other abietane resin acids. We also examined the distribution of a catabolic gene, ditA1, encoding the α subunit of an aromatic ring-hydroxylating dioxygenase involved in DhA degradation by BKME-9. PCR primers specific for the 16S rDNA of BKME-9 and of DhA-35 and specific for ditA1 were used. Among 3 laboratory- and 17 full-scale PPME biotreatment systems, 10 contained phylotype BKME-9, 3 contained phylotype DhA-35, and 11 contained ditA1, indicating the wider distribution of phylotype BKME-9 than of phylotype DhA-35. Both phylotype BKME-9 and ditA1 were detected in the biotreatment system from which BKME-9 was originally isolated in 1994, suggesting the persistance of BKME-9 in that biotreatment system. The detection limit of the PCR assay was one cell per PCR reaction, which corresponds to one BKME-9 cell per 6 × 10⁷ total sludge bacteria. A competitive PCR assay indicated that ditA1 ranged from 51 to 250 copies/mg of dry biomass. BKME-9 appears to contribute to the biodegration of resin acids in some PPME biotreatment systems. Using degenerate PCR primers and touchdown PCR, we obtained from our DhA-degrading strain collection six DNA sequences putatively homologous to that of ditA1. Cluster analysis of these DNA sequences suggests that ditA1 encodes a representative of a novel class of dioxygenase enzymes. Received: 12 February 1999; Accepted: 4 May 1999     

Culture of Five Commonly Used Acid-Producing Bacteria on Banana Pulp

A significant fraction of the bananas and plantains produced in the humid tropics are wasted due to rapid spoilage. The use of acid-producing, anaerobic fermentation may be a safe and economical way to preserve the fruit in both large- and small-scale applications. The abilities of five commonly used, acid-producing bacteria to ferment media containing ripe or green bananas were examined. Three homofermentative bacteria, Lactobacillus bulgaricus, Streptococcus thermophilus, and Streptococcus faecalis, typically reduced the pH of a puree of ripe bananas from 4.8 to about 3.5 to 3.6 in 7 days at 37°C. Action on green bananas was similar, but the drop in pH was less pronounced. Very little gas evolved. Lactobacillus fermentum (formerly Lactobacillus fermenti) and Leuconostoc mesenteroides showed active gas evolution and a significant drop in pH. About twice as much gas evolved from ripe banana medium as from green bananas. The fermented purees were spongy, soft solids floating in a clear liquor. The color was medium to light creamy brown, and the odor was pleasant. Fermented products stored at room temperature retained good appearance and aroma throughout a 1-month storage test.     

Methodology for Estimating Numbers of Free-Living and Attached Bacteria in Estuarine Water

A fundamental problem in estuarine microbiology studies is the accurate determination of the density in the water column of both free-living bacteria and those attached to suspended particulate matter. When a water sample is filtered and the filter is viewed by epifluorescence microscopy, counts can be made of the numbers of bacteria which are seen on the filter background (free-living) and those which appear to lie on sediment particles (both free-living and attached). With only the additional knowledge of the proportion of the filter area covered by particles (a quantity that is straightforwardly determined by stereological point counting), results from geometric probability were used to determine the expected number of bacteria which are hidden by particles and hence to provide an estimation scheme for the true densities of free-living and attached bacteria. Variance equations based on a Taylor series are given, and a partial check of the method is attempted with controlled mixtures of bacteria and sediment. An alternative procedure is also proposed, in which the natural attached/free-living ratio is altered by an intervention experiment, allowing an estimation which is less model dependent but more labor intensive. Both methods are applied to a series of samples from the Tamar estuary, United Kingdom, taken in April 1985. A notable conclusion is that there are always more free-living than attached bacteria in the water column throughout the estuary.     

The Greening of a Pulp and Paper Mill: International Paper's Androscoggin Mill, Jay, Maine

International Paper (IP), the world's largest forest products company, owns the Androscoggin Mill, a large pulp and paper mill in Jay, Maine, in the northeastern United States. This case study describes the transformation of the Androscoggin Mill from an object of public opprobrium and conflict to a show-case for environmental management. In the late 1980s, an 18-month strike had embittered workers and townspeople and left the mill's reputation in tatters. In response to mill environ-mental violations, some of which were considered crimes by state regulators, the town of Jay passed its own environmental ordinance to control mill emissions. Early in the 1990s, new management, including two former corporate-level employ-ees, sought to change the mill's business approach and turn the Androscoggin Mill into IP's best environmental performer. The initial emphasis on establishing and maintaining compliance was expanded to include aggressive pollution prevention efforts that led to cooperative projects with the Maine Department of Environmental Protection, the U.S. Environmental Protection Agency, and stakeholder groups. The mill's ap-proach in the 1990s evolved further to essentially follow prin-ciples of industrial ecology. New approaches focused on "clos-ing the loop" by finding beneficial uses for previously landfilled wastes, replacements for most hazardous chemicals, and re-ductions in solid and hazardous waste generation. The mill also pursued the establishment of symbiotic relationships with a facility that began using a mill by-product on-site and an on-site natural gas burning facility that provided part of the mill's steam demand. IP also established a public advisory committee in 1992 to advise management on operational and "big-picture" issues, which later included the application of sustain-ability criteria to the mill. IP has since formed community advisory committees at each of their integrated pulp and paper mills.     

Differing Populations of Endemic Bacteriophages in Cattle Shedding High and Low Numbers of Escherichia coli O157:H7 Bacteria in Feces

The objectives of this study were to identify endemic bacteriophages (phages) in the feedlot environment and determine relationships of these phages to Escherichia coli O157:H7 from cattle shedding high and low numbers of naturally occurring E. coli O157:H7. Angus crossbred steers were purchased from a southern Alberta (Canada) feedlot where cattle excreting ≥10⁴ CFU · g⁻¹ of E. coli O157:H7 in feces at a single time point were identified as supershedders (SS; n = 6), and cattle excreting <10⁴ CFU · g⁻¹ of feces were identified as low shedders (LS; n = 5). Fecal pats or fecal grabs were collected daily from individual cattle for 5 weeks. E. coli O157:H7 in feces was detected by immunomagnetic separation and enumerated by direct plating, and phages were isolated using short- and overnight-enrichment methods. The total prevalence of E. coli O157:H7 isolated from feces was 14.4% and did not differ between LS and SS (P = 0.972). The total prevalence of phages was higher in the LS group (20.9%) than in the SS group (8.3%; P = 0.01). Based on genome size estimated by pulsed-field gel electrophoresis and morphology determined by transmission electron microscopy, T4- and O1-like phages of Myoviridae and T1-like phage of Siphoviridae were isolated. Compared to T1- and O1-like phages, T4-like phages exhibited a broad host range and strong lytic capability when targeting E. coli O157:H7. Moreover, the T4-like phages were more frequently isolated from feces of LS than SS, suggesting that endemic phages may impact the shedding dynamics of E. coli O157:H7 in cattle.     

Environmental Life Cycle Assessment of a Swedish Dissolving Pulp Mill Integrated Biorefinery

The pulp industry plays an important role in the structure of the European economy and society. The production of pulp has been traditionally considered an important source of pollution due to the use of large amounts of chemicals, fuels, and water and its intensive energy consumption. Currently, this situation is changing due to the potential use of biomass to produce value‐added products, which minimizes environmental impacts and increases sustainability. This article uses life cycle assessment (LCA) to identify and quantify the environmental impacts associated with a Swedish softwood‐based biorefinery where total chlorine‐free (TCF) dissolving cellulose is produced together with ethanol and lignosulfonates. The system was defined according to a cradle‐to‐gate perspective—that is to say, from forest activities to the output of the biorefinery mill. According to the results, forest activities associated with the production of soft roundwood play a minor role in all the environmental impact categories under study. In contrast, the production of chemicals consumed in the cooking and bleaching stages, the sludge treatment generated in the wastewater treatment plant, and the on‐site energy production system were identified as the elements that negatively contribute the most to all impact categories. The production of steam from biorefinery wastes, biogas, and methanol in external boilers reduces the environmental impact in all categories. Specific actions associated with the reuse of wastes and improved gas treatment systems would improve the environmental profile of this production activity.     

Growth of Lactic Acid Bacteria in Waste Waters of Vegetable-Processing Plants

Waters used in washing, blanching, cooling, and conveying vegetables during processing for freezing were filtered, sterilized, and inoculated with Streptococcus faecalis, S. lactis, or Lactobacillus plantarum. The contents of total nitrogen and total solids were determined, and ninhydrin tests and Benedict's tests for reducing sugars were performed. Substances positive to the ninyhydrin tests and also capable of supporting the growth of the bacteria to high levels of population were found in waters used to blanch cut green beans, but not in the cooling or conveying waters. They were found only in waters following slicing of blanched whole beans. They were also present in waters used in processing purple hull peas at all stages, but only in the waters used to blanch and cool lima beans. The substances were present in waters used to wash and blanch squash, but only in the waters used to blanch greens; they were not found in the cooling waters during the handling of either vegetable. No waters used in the processing of okra yielded a positive ninhydrin test, nor did they support the growth of the lactic acid bacteria.     

Prosthecodimorpha staleyi gen. nov., sp. nov., Novel Prosthecate Bacteria within the Family Ancalomicrobiaceae and Reclassification of the Polyphyletic Genus Prosthecomicrobium

Microbiology - An isolate of prosthecate bacteria was isolated from the takyr soil of a rice field on the Akdala massif (Kazakhstan) and designated as strain 22T. Strain 22T was represented by...