Seasonal changes in numbers and the location of a particular bacterial strain of Alteromonas sp. in seagrass sediments.

Many variables must be considered in seeking to describe differences in population sizes for native aquatic bacterial populations. In this study of seagrass- and nearby plant-free sediments, seasonal effects on total bacterial counts were found to be highly significant, outweighing the significance of factors such as geographic variability, but on populations of a chosen Alteromonas sp., they were not significant at the 5% level. Summer counts for both populations were higher than those for winter; this result is likely to reflect the higher productivity of the host Zostera capricomi in summer months, resulting in the exudation of increased amounts of organic nutrients. The Alteromonas sp. occurred in greatest abundance (1.8% of the total population) at the seagrass sediment site from which it was originally isolated and formed up to 1.5% of the population in adjacent plant-free sediments. In fluorescent microscopy studies with labeled antibodies, the Alteromonas sp. was found to be ubiquitous in seagrass and plant-free sediments but was found closely associated in much higher numbers with seagrass root-rhizome tissue, suggesting a possible nutritional relationship between plant and bacterium. In associated trials of sediment preservation techniques, bacterial counts of replicate sediments preserved with glutaraldehyde (3% v/v) were higher than those obtained using Lugol's iodine or freezing.

Kinetics of Colonization of Adult Queensland Fruit Flies (Bactrocera tryoni) by Dinitrogen-Fixing Alimentary Tract Bacteria.

The average total population of bacteria remained constant in the alimentary tracts of adult laboratory-raised Queensland fruit flies (Bactrocera tryoni) although the insects had ingested large numbers of live bacteria as part of their diet. The mean number of bacteria (about 13 million) present in the gut of the insects from 12 to 55 days after emergence was not significantly modified when, at 5 days after emergence, the flies were fed antibiotic-resistant bacteria belonging to two species commonly isolated from the gut of field-collected B. tryoni. Flies were fed one marked dinitrogen-fixing strain each of either Klebsiella oxytoca or Enterobacter cloacae, and the gastrointestinal tracts of fed flies were shown to be colonized within 7 days by antibiotic-resistant isolates of K. oxytoca but not E. cloacae. The composition of the microbial population also appeared to be stable in that the distribution and frequency of bacterial taxa among individual flies exhibited similar patterns whether or not the flies had been bacteria fed. Isolates of either E. cloacae or K. oxytoca, constituting 70% of the total numbers, were usually dominant, with oxidative species including pseudomonads forming the balance of the population. Antibiotic-resistant bacteria could be spread from one cage of flies to the adjacent surfaces of a second cage within a few days and had reached a control group several meters distant by 3 weeks. Restriction of marked bacteria to the population of one in five flies sampled from the control group over the next 30 days suggested that the bacterial population in the gut of the insect was susceptible to alteration in the first week after emergence but that thereafter it entered a steady state and was less likely to be perturbed by the introduction of newly encountered strains. All populations sampled, including controls, included at least one isolate of the dinitrogen-fixing family Enterobacteriaceae; many were distinct from the marked strains fed to the flies. Nitrogenase activity detected by the acetylene reduction assay was associated with flies fed dinitrogen-fixing bacteria as well as with control groups given either no supplement or free access to a yeast hydrolysate preparation. Nitrogen fixed from the atmosphere may supplement the nutrition of the alimentary tract microbial population of B. tryoni. Transmission electron microscopy showed that the principal site of bacterial colonization in the abdominal alimentary tract was the lumen of the midgut inside the peritrophic membrane. No intracellular symbionts were seen in the gut tissues nor were bacteria found attached to the cuticular folds of the hindgut. The ultrastructure of the gut resembled that of other fly genera except that the intercellular spaces between rectal epithelial cells were more extensive, suggesting a role for unspecialized epithelium in water and solute uptake in B. tryoni.

Cytotoxic and haemagglutinating activities of motile Aeromonas species.

Cytotoxic and haemagglutinating properties were determined in 114 Aeromonas strains isolated from various sites in slaughtered lambs and from processed lamb meat. Cytotoxic activity on Vero cells was observed in 48 (42%) of the strains. It was more common in A. sobria and A. hydrophila isolates than with A. caviae isolates. Haemagglutination (HA) activity was found frequently in motile aeromonads irrespective of species; it was present in 50% of A. sobria strains, 51% of A. hydrophila strains and 48% of A. caviae strains. HA was inhibited by fucose, galactose and mannose at low concentration, and in most cases, two or three of these sugars were inhibitory. A significant association was found between certain HA-inhibition patterns and the production of cytotoxin by Aeromonas spp.

Degradation of triglycerides by a pseudomonad isolated from milk: molecular analysis of a lipase-encoding gene and its expression in Escherichia coli.

Psychrotrophic lipolytic bacteria represent a significant problem in the storage of refrigerated dairy products. A lipase-encoding gene has been cloned and characterized from a highly lipolytic strain of Pseudomonas. The nucleotide sequence of the gene predicts a polypeptide of M(r) 49,905, which was identified when the gene was expressed in Escherichia coli.

Molecular analysis of an esterase-encoding gene from a lipolytic psychrotrophic pseudomonad.

An esterase gene (estA) from a lipolytic psychotroph (Pseudomonas sp. LS107d2), was cloned in Escherichia coli and its nucleotide sequence was determined, revealing an ORF encoding a polypeptide of 389 amino acid residues, with a molecular mass of 42276 Da. Labelling of plasmid-encoded proteins with [35S]methionine, using the maxicell procedure, gave a single polypeptide of molecular mass 42 kDa, consistent with that calculated from the ORF. Colonies of E. coli cells containing estA produced a clear halo when grown on solid media containing tributyrin; no clearance was produced when cells were grown on media containing triolein. Extracts of cells containing estA also hydrolysed water-soluble nitrophenol esters, but were unable to cleave water-insoluble substrates. The preference for water-soluble substrates indicates that the gene product is an esterase.

Characteristics of 0/129-sensitive motile Aeromonas strains isolated from freshwater on starch-ampicillin agar.

Motile Aeromonas hydrophila strains were recovered from several freshwater sources by spread-plating water samples on starch-ampicillin agar, originally described as a medium for recovering Aeromonas hydrophila quantitatively from foods. Starch-ampicillin agar was compared with membrane Aeromonas medium and Rimler-Shotts medium for selectivity for, and recovery of, Aeromonas strains from freshwater. Thirty-four Aeromonas strains thus isolated were identified to species level by their phenotypic characteristics, and the Mol% G+C of representative strains was determined. Although resistant to 10 µg of the vibriostatic agent 0/129, all these strains showed sensitivity to 150 µg 0/129, which brings into question the use of this agent for distinguishing aeromonads from vibrios. The ability of these strains to produce extracellular virulence factors was generally similar to that reported for environmental strains isolated by other methods from various geographical locations within and beyond Australia. Ten of the 20 A. sobria strains, but none of the A. hydrophila or A. caviae strains, produced enterotoxin as shown by the suckling mouse test. Haemolysin was produced by 9/10 of the enterotoxigenic A. sobria strains and 2/9 A. hydrophila strains. Hemagglutinating activity was detected in 5/20 A. sobria and 7/9 A. hydrophila strains, and was inhibited by fucose and mannose, but not by galactose. The characteristics of these strains were comparable with those of Aeromonas strains isolated from other freshwater environments apart from their sensitivity to 0/129.

Rhizopus oligosporus grown on natural rubber waste serum for production of single cell protein: a preliminary study.

Maximum production of mycelium and utilization of total organic carbon byR. oligosporus grown on natural rubber waste serum was achieved at 28°C with an inoculum size of 7.5% (v/v) and grown for 144 h with an initial pH of 4.0. The maximum production of total crude protein, however, was when culture medium was inoculated with 2% (v/v) of spore suspension under the same conditions. Natural rubber waste serum may be a potential substrate for the production of single cell protein.

Fermentation patterns of forage sorghum ensiled under different environmental conditions.

The effects of temperature, aerobic and anaerobic conditions in the silo and plant characteristics [water-soluble carbohydrate (WSC) contents, growing season] on the fermentation characteristics of a tropical forage species, Sorghum bicolor cv. sugar-drip, were investigated. Silages fermented in oxygen-impermeable bags were well preserved and had low pH (3.7), high lactic acid [72 g kg(-1) dry matter (DM) ≡ 80% of total acids], and low butyric acid (0.12 g kg(-1) DM) and ammonia nitrogen (NH3-N) (57 g kg(-1) total nitrogen contents. Conversely, the use of oxygen-permeable bags as silos allowed aerobic decomposition of the ensiled forages. Increasing the incubation temperature lowered the population of lactic acid bacteria, reduced lactic acid production and caused the pH to rise. The heterofermentative Leuconostoc spp. predominated on fresh forages but homofermentative Lactobacillus plantarum began to dominate after 5 and 8 days of fermentation. Heterofermentative lactobacilli, notably Lactobacillus brevis, were dominant among the isolates obtained from 100-day silages. Varying the WSC contents, by crushing and/or chopping the forage, and growing season did not significantly affect the fermentation quality of the resulting silages. It was concluded that the maintenance of anaerobic conditions is essential if good quality silage is to be produced from tropical forage species.

Enterotoxigenic aeromonads on retail lamb meat and offal.

Enrichment in alkaline peptone water was compared with the direct plating method for the isolation of Aeromonas spp. from lamb meat and offal samples. The enrichment method significantly increased the isolation rate of aeromonads. Motile Aeromonas species (A. hydrophila, A. sobria and A. caviae) were present in all kinds of samples investigated. Seventy-three Aeromonas strains isolated in this survey were characterized to species level and examined for their ability to produce virulence factors. Strains identified as A. sobria were the strongest producers of haemolysin and enterotoxin, whereas A. caviae strains were consistently non-haemolytic and non-enterotoxigenic. Thus it is likely that lamb meat and offal are potentially significant sources of virulent Aeromonas species and may play an important role in the aetiology of Aeromonas-associated gastro-enteritis.

Microbial metabolism of pesticides and structurally related compounds.

This chapter provides a review concerning the microbial metabolism of pesticides and substances that are either major metabolites from pesticides or have structural similarity to certain pesticides, and covers the period 1981 to 1987. While reference has only been made to work published during this period, it should be realized that in some instances the results cited may confirm or expand upon earlier findings rather than being entirely novel. Therefore, the reader is referred to earlier reviews. The metabolism of pesticides in natural environments, water and wastewater, mixed microbial cultures, and pure cultures has been discussed. Attention has been drawn to the meager amount of information concerning the biodegradation of pesticides in anaerobic and marine environments. Issues such as the importance of cometabolism of pesticides in natural environments and a clear understanding of enhanced degradation of pesticides in soil still remain unresolved. Separate sections have been devoted to methodology in biodegradation studies, bound residues and removal of pesticides from soil and water. While pure culture studies have an important place in investigations into microbial metabolism of pesticides, increasing emphasis has been placed on the use of microbial consortia, either natural or artificial and microcosms to provide an understanding of pesticide biodegradation in natural environments. Another dimension in bound residue formation, one of physical entrapment in humic materials has been described. Various questions regarding the bioavailability of bound residues and whether they pose an environmental problem have not been answered fully. The microbiological removal of pesticides from soil and water by selected or genetically-engineered strains is discussed. It has been emphasized that the future success of such methods for the decontamination of soil and water depends very heavily on an improved knowledge of microbial ecology.

Growth of psychrotrophic bacteria in raw and UHT-treated goats' milk.

The growth of six strains of Pseudomonas fluorescens, two of Ps. fragi, and one of Serratia liquefaciens was followed in raw and UHT-treated goats' milk, held at 4 degrees C. Generation times for Ps. fluorescens in UHT milk ranged from 5.19 to 5.81 h, increasing markedly in raw milk (8.34-21.49 h). Growth of Ps. fragi did not differ significantly between raw (4.56, 4.65 h) and UHT (5.04, 7.24 h) milk. Generation times for S. liquefaciens were 6.63 and 14.07 h, for UHT and raw milk respectively.

Comparison of a simple butterfat agar medium with other media used for isolation and enumeration of lipolytic bacteria from dairy products.

A nutrient agar medium containing 0.1% of a low melting point fraction of butterfat was shown to be suitable for detection, enumeration and isolation of lipolytic bacteria from milk. Bacterial growth was not inhibited by the butterfat and lipolytic reactions were clearly visible and easily interpreted. Lipolytic counts on the butterfat agar compared favourably with lipolytic counts obtained with other commonly used media.

A numerical taxonomic study of psychrotrophic bacteria associated with lipolytic spoilage of raw milk.

Raw milk samples were stored for 1-4 d and examined for bacterial growth and lipase activity. Thirty-six samples in which an increase in the heat-stable lipase activity was observed during storage were selected for further study. From these raw milk samples 205 lipolytic psychrotrophic strains were selected using butterfat agar and subsequently characterized with 86 taxonomic tests. Complete linkage cluster analysis of the taxonomic data produced two major and six minor clusters at the 83% similarity level. Pseudomonas fluorescens and Ps. fragi accounted for 63.9 and 31.2%, respectively, of the isolates.

Growth of lipolytic psychrotrophic pseudomonads in raw and ultra-heat-treated milk.

The lipolytic floras of 36 raw milk samples showing lipolytic defects were dominated by pseudomonads. Representative lipolytic isolates were selected and tested for growth, lipase activity and lipolysis in ultra-heat-treated milk at temperatures ranging from 5 degrees to 30 degrees C. Pseudomonas fluorescens was the most frequently encountered species but Ps. fragi was found to cause more severe lipolytic defects in both single and mixed strain milk cultures. A representative strain of Ps. fragi multiplied faster in cold-stored milk than did three representative strains of Ps. fluorescens. The lipases produced by Ps. fragi strains were more heat-stable than those produced by Ps. fluorescens strains.

Conversion of fensulfothion by Klebsiella pneumoniae to fensulfothion sulfide and its accumulation.

In a detailed study it was shown that washed cell suspensions of K. pneumoniae reduced the organophosphorus pesticide fensulfothion to fensulfothion sulfide. Temperature and pH optima for this conversion plus sensitivity to sulfhydryl-reacting agents strongly suggested enzyme involvement. The reaction was also quite sensitive to molecular oxygen, only proceeding under conditions of low oxygen tension. Once formed, the fensulfothion sulfide was rapidly bound by living and heat-killed cells. A combination of lysozyme treatment and differential centrifugation showed 90% of the sulfide to be concentrated in the cell membrane fraction of exposed cells.

Plant yield and nitrogen content of a digitgrass in response to azospirillum inoculation.

Two Australian soils, a vertisol (pH 6.8, 0.299% N) and a sandy yellow podzol (pH 6.2, 0.042% N), were used with digitgrass, Digitaria sp. X46-2 (PI 421785), in a growth room experiment. Comparisons were made between plants inoculated with live and autoclaved bacterial suspensions of Australian and Brazilian isolates of Azospirillum brasilense. Seedlings were inoculated on days 10 and 35. Acetylene-reducing activity was measured five times during the experiment. Dry matter yields of the digitgrass on the podzol (low N) inoculated with live bacteria were 23% higher than those of the controls. On the vertisol (high N), yield increases from inoculation with live bacteria were 8.5%. The higher-yielding plants had significantly lower percent nitrogen, but when total nitrogen of the tops was calculated, the inoculated plants had a higher total N than did the controls (P=0.04). Acetylene-reducing activity was variable in the experiment, ranging from 0.5 to 11.9 mumol of C(2)H(4) core day. Live bacterial treatment induced a proliferation of roots, possible earlier maturity, higher percent dry matter, and a higher total N in the tops.

Influence of partial pressures of acetylene and nitrogen upon nitrogenase activity of species of Beijerinckia.

Acetylene reduction and nitrogen fixation by strains of Beijerinckia indica and B. lacticogenes increased with increased partial pressures of acetylene and nitrogen up to 80 kPa. The optical emission spectrophotometric method was used for the determination of 14N:15N ratios. The molar ratios of acetylene to nitrogen varied greatly from the theoretical value.

Reduction of fensulfothion to fensulfothion sulfide by Klebsiella pneumoniae.

A cell suspension of Klebsiella pneumoniae converted the organophosphorus pesticide fensulfothion to a product that was shown by chemical oxidation, gas-liquid chromatography, infrared spectrophotometry, and mass spectrometry to be fensulfothion sulfide. Further alteration of this metabolite was not noted.