Direct Fluorescent Labeling of Microorganisms as a Possible Life-Detection Technique

Microorganisms and selected proteinaceous substances were directly tagged with fluorescein isothiocyanate. This approach suggested a possible application for detection of extraterrestrial life. A stable and apparently specific linkage was formed with protein, and nonprotein substances were readily destained. Soil and atmospheric debris did not exhibit any significant affinity for the dye.     

Degradation of Linseed Oil Vapors by Soil Bacteria in Trickling Biofilters

Oxidation products of linseed oil were produced by impinging a stream of air onto the surface of pure linseed oil and injecting the vapor-laden air into soil percolation columns to enrich the population of bacteria capable of degrading linseed oil vapors. As the populations of bacteria increased, the linseed oil vapors were consumed by these organisms, and the air that emerged from the columns was free of linseed oil contaminants. Five different kinds of bacteria capable of growing on the linseed oil oxidation products as sole source of carbon and energy were found and isolated in pure culture. Chromatographic analyses showed that individual organisms removed specific components of the vapor at specific rates, but none was able to remove them all within a 30-day period of time. When the five were grown together and presented the linseed oil vapor, all vapor constituents were utilized, and the rate of utilization was greater than that seen when the isolates were tested in pure culture. This indicated that the five organisms operated as a bacterial consortium in the degradation of linseed oil vapors. Trickling biofilters prepared from pregrown populations of the five organisms challenged with linseed oil vapors were able to remove all volatile constituents found in linseed oil vapor. Bioremediation of the air was complete and it was accomplished in a single pass of the air through the filter.

This work shows that bacteria found in the soil are capable of degrading linseed oil vapors and that they can be grown in the laboratory and used successfully in bench scale trickling biofilters.     

Metabolism of Styrene Oxide and 2-Phenylethanol in the Styrene-Degrading Xanthobacter Strain 124X

Styrene oxide and 2-phenylethanol metabolism in the styrene-degrading Xanthobacter sp. strain 124X was shown to proceed via phenylacetaldehyde and phenylacetic acid. In cell extracts 2-phenylethanol was oxidized by a phenazine methosulfate-dependent enzyme, probably a pyrroloquinoline quinone enzyme. Xanthobacter sp. strain 124X also contains a novel enzymatic activity designated as styrene oxide isomerase. Styrene oxide isomerase catalyzes the isomerization of styrene oxide to phenylacetaldehyde. The enzyme was partially purified and shown to have a very high substrate specificity. Of the epoxides tested, styrene oxide was the only substrate transformed. The initial step in styrene metabolism in Xanthobacter sp. strain 124X is oxygen dependent and probably involves oxidation of the aromatic nucleus.     

The Determination of Comparable Labeling Densities in Quantitative Immunoelectron Microscopic Double Labeling Studies

In quantitative ultrastructural studies using colloidal gold immunocytochemical techniques, labeling intensities vary according to the size of the probe used. Using postembedded indirect two-sided double labeling and single labeling protocols, the labeling characteristics of four antigens were studied using two probe sizes commonly used in double labeling studies. It was determined that the labeling intensity variation resulting from the use of different probe sizes was unpredictable after correcting for the increased probe size alone. It was possible, however, to obtain comparable labeling densities by first determining the labeling intensities for each probe size with its antigen in single label studies on serial sections and using the same procedure as the double labeling studies. A probe size correction factor for each antigen was calculated from these data. This factor was used to obtain comparable measurements of the relative abundance of each label.     

Temporal and Spatial Stability of Ammonia-Oxidizing Archaea and Bacteria in Aquarium Biofilters

Nitrifying biofilters are used in aquaria and aquaculture systems to prevent accumulation of ammonia by promoting rapid conversion to nitrate via nitrite. Ammonia-oxidizing archaea (AOA), as opposed to ammonia-oxidizing bacteria (AOB), were recently identified as the dominant ammonia oxidizers in most freshwater aquaria. This study investigated biofilms from fixed-bed aquarium biofilters to assess the temporal and spatial dynamics of AOA and AOB abundance and diversity. Over a period of four months, ammonia-oxidizing microorganisms from six freshwater and one marine aquarium were investigated at 4–5 time points. Nitrogen balances for three freshwater aquaria showed that active nitrification by aquarium biofilters accounted for ≥81–86% of total nitrogen conversion in the aquaria. Quantitative PCR (qPCR) for bacterial and thaumarchaeal ammonia monooxygenase (amoA) genes demonstrated that AOA were numerically dominant over AOB in all six freshwater aquaria tested, and contributed all detectable amoA genes in three aquarium biofilters. In the marine aquarium, however, AOB outnumbered AOA by three to five orders of magnitude based on amoA gene abundances. A comparison of AOA abundance in three carrier materials (fine sponge, rough sponge and sintered glass or ceramic rings) of two three-media freshwater biofilters revealed preferential growth of AOA on fine sponge. Denaturing gel gradient electrophoresis (DGGE) of thaumarchaeal 16S rRNA genes indicated that community composition within a given biofilter was stable across media types. In addition, DGGE of all aquarium biofilters revealed low AOA diversity, with few bands, which were stable over time. Nonmetric multidimensional scaling (NMDS) based on denaturing gradient gel electrophoresis (DGGE) fingerprints of thaumarchaeal 16S rRNA genes placed freshwater and marine aquaria communities in separate clusters. These results indicate that AOA are the dominant ammonia-oxidizing microorganisms in freshwater aquarium biofilters, and that AOA community composition within a given aquarium is stable over time and across biofilter support material types.     

Methodologies for Pre-Validation of Biofilters and Wetlands for Stormwater Treatment

Background

Water Sensitive Urban Design (WSUD) systems are frequently used as part of a stormwater harvesting treatment trains (e.g. biofilters (bio-retentions and rain-gardens) and wetlands). However, validation frameworks for such systems do not exist, limiting their adoption for end-uses such as drinking water. The first stage in the validation framework is pre-validation, which prepares information for further validation monitoring.

Objectives

A pre-validation roadmap, consisting of five steps, is suggested in this paper. Detailed methods for investigating target micropollutants in stormwater, and determining challenge conditions for biofilters and wetlands, are provided.

Methods

A literature review was undertaken to identify and quantify micropollutants in stormwater. MUSIC V5.1 was utilized to simulate the behaviour of the systems based on 30-year rainfall data in three distinct climate zones; outputs were evaluated to identify the threshold of operational variables, including length of dry periods (LDPs) and volume of water treated per event.

Results

The paper highlights that a number of micropollutants were found in stormwater at levels above various worldwide drinking water guidelines (eight pesticides, benzene, benzo(a)pyrene, pentachlorophenol, di-(2-ethylhexyl)-phthalate and a total of polychlorinated biphenyls). The 95^th percentile LDPs was exponentially related to system design area while the 5^th percentile length of dry periods remained within short durations (i.e. 2–8 hours). 95^th percentile volume of water treated per event was exponentially related to system design area as a percentage of an impervious catchment area.

Conclusions

The out-comings of this study show that pre-validation could be completed through a roadmap consisting of a series of steps; this will help in the validation of stormwater treatment systems.     

Thiobarbiturate-reacting Materials in Microorganisms

The amount of thiobarbiturate-reacting material in 51 strains of bacteria and three yeasts was determined. Reactive material was found to be present in all of the gram-negative bacteria examined. It was assumed that the reactive material in this case was primarily 2-keto-3-deoxyoctonate (KDO), an eight-carbon sugar acid which is usually associated with the cell wall lipolysaccharide of members of the Salmonella-Escherichia group. Very little reactive material could be detected in the gram-positive species and yeasts that were examined. When expressed as per cent dry weight, the gram-negative bacteria exhibited about eight times more reactive material than the gram-positive species. It is suggested that the small amount of reactive material detected in gram-positive cells and yeasts is due to compounds other than KDO.     

Metabolism of Hydrocarbons in Microorganisms

The fatty acid composition of the lipid of yellowfin tuna (Thunnus albacares) caught in two different localities, Philippine Sea (a tropical zone) and the Pacific coast area of Japan (a temperate zone) is described. The total lipids of various organs (dorsal ordinary muscle, ventral ordinary muscle, dark muscle, liver, heart, pyloric cecum, and orbital region) and of the stomach contents were extracted, and the fatty acid comosition was analyzed by gas-liquid chromatography (GLC).

Docosahexaenoic acid (DHA; 22:6n-3) was the major unsaturated fatty acid in the lipid of all organs in the specimens examined from both localities, the mean DHA content accounting for more than 25% (mean ± S.D. of 26.9 ±5.7%) of the total fatty acids. This value is markedly different from the fatty acid profile of other fish species, because, in general, the fatty acid composition of other species is variable and the DHA content is less than 20% of total fatty acids.

Although the mean DHA content of the total fatty acids in the lipid of yellowfin tuna caught in the tropical and temperate zones was markedly higher than that in other fish species, there was a small difference between that in the northern samples (temperate waters, 30.5 ±6.1%) and the southern samples (tropical waters, 25.9 ± 5.2%). It is suggested that this difference may be due to environmental effects, e.g., the fatty acid composition of the lipids of prey organisms, because there is also a small difference between the mean DHA content of northern prey fish (22.7 ±6.1%) and that of southern prey fish (19.2 ±4.0%).     

Transport of Metabolites in Microorganisms

This paper describes, not ionic movement, but rather the transferof organic nutrients from environment to cell in microorganisms.A section on methods for studying such transfer is followedby a discussion of generalized models derived from kinetic data.Control mechanisms, involving regulation of synthesis of membraneproteins and feedback modification of their activity, are described.Some recent work on specific models for transport of amino acidsand carbohydrates is also discussed.     

Cryopreservation of Microorganisms

Abstract

A great deal of recent interest has been shown in the ability of some microbes to synthesize exopolysaccharides. Most attention has been directed toward the prokaryote producers, yet many filamentous fungi also produce exopolysaccharides that have chemical and physical properties of considerable commercial potential. Surprisingly little is known about how and why fungi overproduce these metabolites and how yields are affected by both the physical and chemical environments. This review attempts to critically appraise the current literature on fungal exopolysaccharides, considers their chemical diversity, and examines factors that seem to affect their production. Although much of the published work has been carried out with the α-glucan pullulan, there is considerable literature on the β-glucans and, hence, both of these are discussed.     

黄土高原水体浮游微生物及沉积微生物研究进展

黄土高原跨越我国半干旱、干旱及半湿润地区,是世界上面积最大的黄土分布区。近些年来,由于气候变化和人类活动的加剧,黄土高原的河流湖库面临着水少、水浑、水污等威胁,因此亟需开展此区域水体的生态健康评价以提出生态保护和管理措施。除了鱼类、底栖动物和浮游植物生态学外,以微生物为核心的微生物生态学研究在环境现状指示、生态健康评价方面也占有重要作用。本文首先探讨黄土高原的范围及其水体的生态学研究进展,选择了黄土高原的"广义"划分区域(北纬33°41′～41°16′,东经100°54′～114°33′),其次整合国内外不同水体中浮游及沉积微生物的群落结构、功能及生物完整性指数(index of biological integrity, IBI)生态指示作用的相关研究,指出微生物群落结构、功能特征及IBI指数的动态变化是自然与人为因素综合作用的结果,同样也指示了生态、环境现状。目前,针对黄土高原微生物生态指示作用的研究主要集中于土壤环境中微生物群落结构指标,利用功能参数以及IBI指数进行生态指示的研究较少。黄土高原横跨7个省,范围广,面积大,未来针对黄土高原中部地带、西北地区核心——陕西省的河流湖库微生物进行研究能够以小见大,更全面地反映区域水生态系统健康状况。     

Quantitation of Microorganisms in Sputum

A method of quantitating microbial cultures of homogenized sputum has been devised. Possible application of this method to the problem of determining the etiologic agent of lower-respiratory-tract infections has been studied to determine its usefulness as a guide in the management of these infections. Specimens were liquefied by using an equal volume of 2% N-acetyl-L-cysteine. The liquefied sputum suspension was serially diluted to 10^-1, 10^-3, 10^-5, and 10^-7. These dilutions were plated on appropriate media by using an 0.01-ml calibrated loop; they were incubated and examined by standard diagnostic methods. Quantitation of fresh sputum from patients with pneumonia prior to antimicrobial therapy revealed that probable pathogens were present in populations of 10⁷ organisms/ml or greater. Normal oropharyngeal flora did not occur in these numbers before therapy. Comparison of microbial counts on fresh and aged sputum showed that it is necessary to use only fresh specimens, since multiplication or death alters both quantitative and qualitative findings. Proper collection and quantitative culturing of homogenized sputum provided information more directly applicable to patient management than did qualitative routine methods. Not only was the recognition of the probable pathogenic organism in pneumonia patients improved, but serial quantitative cultures were particularly useful in recognizing the emergence of superinfections and in evaluating the efficacy of antimicrobial therapy.     

Metabolism of Hydrocarbons in Microorganisms

The crude extract obtained from Pseudomonas aeruginosa grown on xylene as sole carbon source converted p-xylene-methyl-¹⁴C or toluene-methyl-¹⁴C to p-toluic or benzoic acid, respectively. However, addition of p-methylbenzyl or benzyl alcohol to the reaction mixture resulted in accumulation of p-methylbenzyl or benzyl alcohol. This indicates that in the crude extract, p-xylene or toluene is metabolized via its corresponding alcohol to p-toluic or benzoic acid, respectively. The enzyme system responsible for these reactions required NAD or NADH and FAD, and could be stabilized by the presence of glutathione. When the crude extract was fractionated by the use of DEAE-cellulose chromatography, it was demonstrated that two distinct protein fractions and two cofactors (NADH and FAD) were required for the step of the hydroxylation of p-xylene or toluene to its corresponding alcohol. NAD, NADP or NADPH had very few or little activity. FMN partially replaced FAD.     

Fatty Acid Metabolism in Microorganisms

The production of pimelic acid from azelaic acid by microorganisms was studied. About 100 strains of bacteria which were able to utilize azelaic acid as a sole carbon source were isolated from soil and other natural materials. Among these bacteria, several strains produced a large quantity of an organic acid (pimelic acid) from azelaic acid in their culture fluids during the cultivation. The acid was isolated from the culture fluid of strain A133 in crystalline form. The crystal was identified as pimelic acid by physicochemical and biological methods.

From the results of investigations on the morphological and physiological characters, the bacterial strain A133 was assumed to be Micrococcus sp.     

Metabolism of Pyridine Coenzymes in Microorganisms

NADP was enzymatically synthesized from NAD and p-nitrophenyl phosphate or nucleoside monophosphate with the enzyme preparation of Proteus mirabilis (IFO 3849). In this phosphotransferring reaction, ATP did not serve as phosphoryl donor.

In addition to NADP, an unidentified substance (Compound I) showing fluorescence with methyl ethyl ketone and having no coenzyme activity to glutamic dehydrogenase was synthesized. The yield of NADP was usually below 30 per cent of Compound I.

NADP was isolated from the reaction mixture and its coenzyme activity to some dehydrogenases was demonstrated.

A new derivative of NAD (Compound I) synthesized from NAD and p-nitrophenyl phosphate by the enzyme preparation of Proteus mirabilis (IFO 3849), was isolated from the reaction mixture.

After degradation of this compound with snake venom nucleotide pyrophosphatase, Compound III was obtained. 5′-NMN was phosphorylated to Compound IV by the same enzyme preparation of P. mirabilis. By the determination of chemical constituents and the degradation with phosphomonoesterases, Compounds III and IV were identified as nicotinamide riboside 2′(3′),5′-diphosphate, and Compound I was identified as NADP analog which was formed by phosphorylation at the 2′ or 3′ position of the nicotinamide ribose moiety, not at the 2′ position of adenosine moiety of NAD.     

Investigation of Microorganisms in Bentonite Deposits

Microbiological characteristics of bentonite deposits were investigated as a natural analogue of microbial behavior in the buffer material for geological disposal of radioactive waste. Distributions of microorganisms in bentonite were examined at four sites in two different bentonite deposits in Japan. The sites included pond bottom, wetland, and wet mine gallery environments where bentonite layers have been left undisturbed for 2 to 30 years. Excavation was performed without using drilling water and the center parts of the cores were used for microbial examination. Plate counts with R2A medium of aerobic and anaerobic bacteria at the drilling mouth ranged from 10⁵ to 10⁷/g DW (dry weight) and from 10³ to 10⁶/g DW, respectively. The CFDA-AM (Carboxyfluorescein diacetate acetoxymethyl ester) cell counts ranged from 10⁶ to 10⁹/g DW. Bacterial numbers in the bentonite layers declined with distance from the drilling mouth; both aerobes and anaerobes were less than 10²/g DW and CFDA-AM cell counts less than 10⁶/g DW for core samples taken from approximately 1 m depth, except at the pond bottom. These results suggest that microbial activity in natural bentonite is lower than in typical soils and aquatic sediments and does not spread easily.     

Metabolism of Pyridine Coenzymes in Microorganisms

NADP and NADP analog were phosphorylated to NAD diphosphate and NADP analog phosphate, respectively, by an enzyme preparation of Proteus mirabilis (IFO 3849). The degradation products from NAD-diphosphate and NADP analog phosphate by the snake venom nucleotide pyrophosphatase were identical with nicotinamide riboside diphosphate and adenosine 2′(3′), 5′-diphosphate.     

Fatty Acid Metabolism in Microorganisms

During the investigation on the metabolism of azelaic acid by Micrococcus sp., it was found that the bacterium produced a large amount of keto acid (α-ketoglutaric acid) under the restricted condition for nitrogen source. The acid was identified as α-ketoglutaric acid by physico-chemical and biological methods. The mechanism of the production of α-ketoglutaric acid from azelaic acid was investigated. From the result, it was suggested that α-ketoglutaric acid production proceeded thrpugh the further oxidation of acetic acid produced from azelaic acid and that the production might be functioned by TCA cycle enzymes of the bacterium. Similarly, α-ketoglutaric acid was found to be produced remarkably from other various fatty acids.