Quantitative assessment of total and Gram-positive aerobic bacteria in fresh and ambient-temperature-stored sub-tropical marine fish

The current study was undertaken to enumerate Gram-positive bacteria in fresh sub-tropical marine fish and determine the effect of ambient storage (25°C) on the Gram-positive bacterial count. Total and Gram-positive bacteria were enumerated in the muscles, gills and gut of fresh and stored Pseudocaranx dentex, Pagrus auratus and Mugil cephalus on tryptone soya agar (TSA) and TSA with 0.25% phenylethyl alcohol (PEA), respectively. Initial studies indicated that PEA significantly reduced total aerobic bacterial count (TABC) whereas control Gram-positive bacteria were not affected by 0.25% PEA. TABC significantly increased in all fish body parts, whereas Gram-positive aerobic bacterial count (GABC) significantly increased only in the muscles and gills during ambient storage for 15 h. The TABC of the fish species increased from 4.00, 6.13 and 4.58 log cfu g-1, respectively in the muscles, gills, and gut to 6.31, 7.31 and 7.23 log cfu g-1 by the end of storage. GABC increased from 2.00, 3.52 and 2.20 log cfu g-1 to 4.70, 5.85 and 3.36 log cfu g-1. Within each species, TABC were significantly higher in the gills compared to that of muscles and gut; however, no significant differences were found in GABC between muscles and gills. This study demonstrated the potential importance of Gram-positive bacteria in sub-tropical marine fish and their spoilage.

Spoilage pattern of five species of Australian prawns: Deterioration is influenced by environment of capture and mode of storage

Five species of commercial prawns Penaeus plebejus, P. merguiensis, P. semisulcatus/P. esculentus and M. bennettae, were obtained from South-East and North Queensland, chilled soon after capture and then stored either whole or deheaded on ice and ice slurry, until spoilage. Total bacterial counts, total volatile nitrogen, K-values and total demerit scores were assessed at regular intervals. Their shelf lives ranged from 10-17 days on ice and >20 days on ice slurry. Initial bacterial flora on prawns from shallower waters (4-15m) were dominated by Gram-positives and had lag periods around 7 days, whereas prawns from deeper waters (100m) were dominant in Pseudomonas spp. with no lag periods in bacterial growth. The dominant spoiler in ice was mainly Pseudomonas fragi whereas the main spoiler in ice slurry was Shewanella putrefaciens. Bacterial interactions seem to play a major role in the patterns of spoilage in relation to capture environment and pattern of storage

New records of the River Shark Glyphis (Carcharhinidae) reported from Cape York Peninsula, northern Australia

The distribution of the river shark Glyphis in northern Australia is extended with new records of occurrence in the Gulf of Carpentaria and a reassessment of historical survey data from Cape York Peninsula. Nine new specimens of Glyphis sp. A were collected in 2005 from the Weipa region on the Queensland coast of the Gulf of Carpentaria. A re-examination of archival records from 1978-86 marine and estuarine fish surveys in the Gulf of Carpentaria and along the northern Queensland East Coast allowed a further nineteen Glyphis specimens to be identified. Combined this gives twenty-eight new records of Glyphis specimens from the coasts of Cape York Peninsula, Queensland. Common habitat characteristics for all captures were turbid, shallow, fast running tidal water in the upper reaches of coastal rivers. The substrate was generally muddy and the rivers lined with mangrove. In all surveys the representation of Glyphis was low, being less than 1% of the total shark captures historically and 0.002 sharks 50 m net hour-1 in Weipa 2005. The size range captured was 1000-1800 mm total length historically and 705-1200 mm total length from Weipa 2005, with none recorded as sexually mature. Diagnostic characteristics of the Weipa specimens, identified as Glyphis sp. A, were: lower jaw teeth protruding and "spear-like"; second dorsal fin greater than half the height of the first dorsal fin; the snout relatively short and fleshy in the lateral view; pectoral fin ventral surface black in colouration; the precaudal vertebral count between 118 and 123; and the total vertebral count between 204 and 209.

Ruminococcus bromii, identification and isolation as a dominant community member in the rumen of cattle fed a barley diet

Aims: To identify dominant bacteria in grain (barley)-fed cattle for isolation and future use to increase the efficiency of starch utilization in these cattle. Methods and Results: Total DNA was extracted from samples of the rumen contents from eight steers fed a barley diet for 9 and 14 days. Bacterial profiles were obtained using denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V2/V3 region of the 16S rRNA genes from total bacterial DNA. Apparently dominant bands were excised and cloned, and the clone insert sequence was determined. One of the most common and dominant bacteria present was identified as Ruminococcus bromii. This species was subsequently isolated using traditional culture-based techniques and its dominance in the grain-fed cattle was confirmed using a real-time Taq nuclease assay (TNA) designed for this purpose. In some animals, the population of R. bromii reached densities above 1010R. bromii cell equivalents per ml or approximately 10% of the total bacterial population. Conclusions: Ruminococcus bromii is a dominant bacterial population in the rumen of cattle fed a barley-based diet. Significance and Impact of the Study: Ruminococcus bromii YE282 may be useful as a probiotic inoculant to increase the efficiency of starch utilization in barley-fed cattle. The combination of DGGE and real-time TNA has been an effective process for identifying and targeting for isolation, dominant bacteria in a complex ecosystem.

Volatile components associated with bacterial spoilage of tropical prawns

Analysis of headspace volatiles by gas chromatography/mass spectrometry from king (Penaeus plebejus), banana (P. merguiensis), tiger (P. esculentus/semisulcatus) and greasy (Metapenaeus bennettae) prawns stored in ice or ice slurry, which is effectively an environment of low oxygen tension, indicated the presence of amines at the early stages of storage (less than 8 days) irrespective of the nature of the storage media. Esters were more prevalent in prawns stored on ice (normal oxygen conditions) at the latter stages of storage (more than 8 days) and were only produced by Pseudomonas fragi, whereas sulphides and amines occurred whether the predominant spoilage organism was Ps.fragi or Shewanella putrefaciens. The free amino acid profiles of banana and king prawns were high in arginine (12–14%) and low in cysteine (0.1–0.17%) and methionine (0.1–0.2%). Filter sterilised raw banana prawn broth inoculated with a total of 15 cultures of Ps. fragi and S. putrefaciens and incubated for two weeks at 5°C, showed the presence of 17 major compounds in the headspace volatiles analysed using gas chromatography/mass spectrometry (GC/MS). These were mainly amines, sulphides, ketones and esters. Principal Component Analysis of the results for the comparative levels of the volatiles produced by pure cultures, inoculated into sterile prawn broth, indicated three subgroupings of the organisms; I, Ps. fragi from a particular geographic location; II, S. putrefaciens from another geographic location; and III, a mixture of Ps. fragi and S. putrefaciens from different geographic locations. The sensory impression created by the cultures was strongly related to the chemical profile as determined by GC/MS. Organisms, even within the same subgrouping classified as identical by the usual tests, produced a different range of volatiles in the same uniform substrate.

Predicting survival of western rock lobsters Panulirus cygnus using discriminant analysis of hemolymph parameters taken immediately following simulated handling treatments

Instances of morbidity amongst rock lobsters (Panulirus cygnus) arriving at factories in Western Australia (WA) have been attributed to stress during post-harvest handling. This study used discriminant analysis to determine whether physiological correlates of stress following a period of simulated post-harvest handling had any validity as predictors of future rejection or morbidity of western rock lobsters. Groups of 230 western rock lobsters were stored for 6 h in five environments (submerged/flowing sea water, submerged/re-circulating sea water, humid air, flowing sea water spray, and re-circulated sea water spray). The experiment was conducted in late spring (ambient sea water 22°C), and repeated again in early autumn (ambient sea water 26°C). After 6 h treatment, each lobster was graded for acceptability for live export, numbered, and its hemolymph was sampled. The samples were analysed for a number of physiological and health status parameters. The lobsters were then stored for a week in tanks in the live lobster factory to record mortality. The mortality of lobsters in the factory was associated with earlier deviations in hemolymph parameters as they emerged from the storage treatments. Discriminant analysis (DA) of the hemolymph assays enabled the fate of 80-90% of the lobsters to be correctly categorised within each experiment. However, functions derived from one experiment were less accurate at predicting mortality when applied to the other experiments. One of the reasons for this was the higher mortality and the more severe patho-physiological changes observed in lobsters stored in humid air or sprays at the higher temperature. The analysis identified lactate accumulation during emersion and associated physiological and hemocyte-related effects as a major correlate of mortality. Reducing these deviations, for example by submerged transport, is expected to ensure high levels of survival. None of the indicators tested predicted mortality with total accuracy. The simplest and most accurate means of comparing emersed treatments was to count the mortality afterwards.

Microbial ecology of the equine hindgut during oligofructose-induced laminitis

Alimentary carbohydrate overload is a significant cause of laminitis in horses and is correlated with drastic shifts in the composition of hindgut microbiota. Equine hindgut streptococcal species (EHSS), predominantly Streptococcus lutetiensis, have been shown to be the most common microorganisms culturable from the equine caecum prior to the onset of laminitis. However, the inherent biases of culture-based methods are estimated to preclude up to 70% of the normal caecal microbiota. The objective of this study was to evaluate bacterial population shifts occurring in the equine caecum throughout the course of oligofructose-induced laminitis using several culture-independent techniques and to correlate these with caecal lactate, volatile fatty acid and degrees of polymerization 3-7 fructo-oligosaccharide concentrations. Our data conclusively show that of the total microbiota present in the equine hindgut, the EHSS S. lutetiensis is the predominant microorganism that proliferates prior to the onset of laminitis, utilizing oligofructose to produce large quantities of lactate. Population shifts in lactobacilli and Escherichia coli subpopulations occur secondarily to the EHSS population shifts, thus confirming that lactobacilli and coliforms have no role in laminitis. A large, curved, Gram-negative rod previously observed during the early phases of laminitis induction was most closely related to the Anaerovibrio genus and most likely represents a new, yet to be cultured, genus and species. Correlation of fluorescence in situ hybridization and quantitative real-time PCR results provide evidence supporting the hypothesis that laminitis is associated with the death en masse and rapid cell lysis of EHSS. If EHSS are lysed, liberated cellular components may initiate laminitis.

Quantitative assessment of total phenol contents of European oak (Quercus petraea and Quercus robur) by diffuse reflectance NIR spectroscopy on solid wood surfaces

Near infrared spectroscopy (NIRS) combined with multivariate analysis techniques was applied to assess phenol content of European oak. NIRS data were firstly collected directly from solid heartwood surfaces: in doing so, the spectra were recorded separately from the longitudinal radial and the transverse section surfaces by diffuse reflectance. The spectral data were then pretreated by several pre-processing procedures, such as multiplicative scatter correction, first derivative, second derivative and standard normal variate. The tannin contents of sawmill collected from the longitudinal radial and transverse section surfaces were determined by quantitative extraction with water/methanol (1:4, by vol). Then, total phenol contents in tannin extracts were measured by the Folin-Ciocalteu method. The NIR data were correlated against the Folin-Ciocalteu results. Calibration models built with partial least squares regression displayed strong correlation - as expressed by high determination correlation coefficient (r2) and high ratio of performance to deviation (RPD) - between measured and predicted total phenols content, and weak calibration and prediction errors (RMSEC, RMSEP). The best calibration was provided with second derivative spectra (r2 value of 0.93 for the longitudinal radial plane and of 0.91 for the transverse section plane). This study illustrates that the NIRS technique when used in conjunction with multivariate analysis could provide reliable, quick and non-destructive assessment of European oak heartwood extractives.

NIR model development and robustness in prediction of melon fruit total soluble solids

Near infrared spectroscopy (NIRS) can be used for the on-line, non-invasive assessment of fruit for eating quality attributes such as total soluble solids (TSS). The robustness of multivariate calibration models, based on NIRS in a partial transmittance optical geometry, for the assessment of TSS of intact rockmelons (Cucumis melo) was assessed. The mesocarp TSS was highest around the fruit equator and increased towards the seed cavity. Inner mesocarp TSS levels decreased towards both the proximal and distal ends of the fruit, but more so towards the proximal end. The equatorial region of the fruit was chosen as representative of the fruit for near infrared assessment of TSS. The spectral window for model development was optimised at 695-1045 nm, and the data pre-treatment procedure was optimised to second-derivative absorbance without scatter correction. The 'global' modified partial least squares (MPLS) regression modelling procedure of WINISI (ver. 1.04) was found to be superior with respect to root mean squared error of prediction (RMSEP) and bias for model predictions of TSS across seasons, compared with the 'local' MPLS regression procedure. Updating of the model with samples selected randomly from the independent validation population demonstrated improvement in both RMSEP and bias with addition of approximately 15 samples.

Metabolite mobilization in the stem galls of Parthenium hysterophorus induced by Epiblema strenuana inferred from the signatures of isotopic carbon and nitrogen and concentrations of total non-structural carbohydrates

Parthenium hysterophorus L. (Asteraceae) is a weed of national significance in Australia. Among the several arthropod agents introduced into Australia to control populations of P. hysterophorus biologically, Epiblema strenuana Walker (Lepidoptera: Tortricidae) is the most widespread and abundant agent. By intercepting the normal transport mechanisms of P. hysterophorus, the larvae of E. strenuana drain nutrients, other metabolic products, and energy, and place the host plant under intense metabolic stress. In this study, determinations of total non-structural carbohydrates (TNC) levels and carbon and nitrogen isotope ratios of fixed products in different parts of the plant tissue, including the gall, have been made to establish the function of gall as a sink for the nutrients. Values of δ13C and δ15N in galls were significantly different than those in proximal and distal stems, whereas the TNC levels were insignificant, when measured in the total population of P. hysterophorus, regardless of plant age. However, carbon, nitrogen, and TNC signatures presented significant results, when assayed in different developmental stages of P. hysterophorus. Carbon isotope ratios in galls were consistently more negative than those from the compared plant organs. Nitrogen isotope ratios in galls, on the contrary, were either similar to or less negative than the compared plant organs, especially within a single host-plant stage population (i.e., either rosette, preflowering, or flowering stage). TNC levels varied within compared plant populations. The stem distal to the gall functioned more efficiently as a nodal channel than the stem proximal to the gall, especially in the translocation of nitrogenous nutrients. Our findings indicate that the gall induced by E. strenuana functions as a sink for the assayed nutrients, although some variations have been observed in the patterns of nutrient mobilization. By creating a sink for the nutrients in the gall, E. strenuana is able to place the overall plant metabolism under stress, and this ability indicates E. strenuana has the necessary potential for use as a biological-control agent.

Modelling the distribution and relative abundance of feral camels in the Northern Territory using count data

The objectives of this study were to predict the potential distribution, relative abundance and probability of habitat use by feral camels in southern Northern Territory. Aerial survey data were used to model habitat association. The characteristics of ‘used’ (where camels were observed) v. ‘unused’ (pseudo-absence) sites were compared. Habitat association and abundance were modelled using generalised additive model (GAM) methods. The models predicted habitat suitability and the relative abundance of camels in southern Northern Territory. The habitat suitability maps derived in the present study indicate that camels have suitable habitat in most areas of southern Northern Territory. The index of abundance model identified areas of relatively high camel abundance. Identifying preferred habitats and areas of high abundance can help focus control efforts.

Impact of tillage and residues from rotation crops on the nematode community in soil and surface mulch during the following sugarcane crop.

The impact of three cropping histories (sugarcane, maize and soybean) and two tillage practices (conventional tillage and direct drill) on plant-parasitic and free-living nematodes in the following sugarcane crop was examined in a field trial at Bundaberg. Soybean reduced populations of lesion nematode (Pratylenchus zeae) and root-knot nematode (Meloidogyne javanica) in comparison to previous crops of sugarcane or maize but increased populations of spiral nematode (Helicotylenchus dihystera) and maintained populations of dagger nematode (Xiphinema elongatum). However the effect of soybean on P zeae and M. javanica was no longer apparent 15 weeks after planting sugarcane, while later in the season, populations of these nematodes following soybean were as high as or higher than maize or sugarcane. Populations of P zeae were initially reduced by cultivation but due to strong resurgence tended to be higher in conventionally tilled than direct drill plots at the end of the plant crop. Even greater tillage effects were observed with M. javanica and X. elongatum, as nematode populations were significantly higher in conventionally tilled than direct drill plots late in the season. Populations of free-living nematodes in the upper 10 cm of soil were initially highest following soybean, but after 15, 35 and 59 weeks were lower than after sugarcane and contained fewer omnivorous and predatory nematodes. Conventional tillage increased populations of free-living nematodes in soil in comparison to direct drill and was also detrimental to omnivorous and predatory nematodes. These results suggest that crop rotation and tillage not only affect plant-parasitic nematodes directly, but also have indirect effects by impacting on natural enemies that regulate nematode populations. More than 2 million nematodes/m(2) were often present in crop residues on the surface of direct drill plots. Bacterial-feeding nematodes were predominant in residues early in the decomposition process but fungal-feeding nematodes predominated after 15 weeks. This indicates that fungi become an increasingly important component of the detritus food web as decomposition proceeds, and that that the rate of nutrient cycling decreases with time. Correlations between total numbers of free-living nematodes and mineral N concentrations in crop residues and surface soil suggested that the free-living nematode community may provide an indication of the rate of mineralisation of N from organic matter.

Effects of secondary crops on bacterial growth and nitrogen removal in shrimp farm effluent treatment systems

Secondary crops provide a means of assimilating some effluent nitrogen from eutrophic shrimp farm settlement ponds. However, a more important role may be their stimulation of beneficial bacterial nitrogen removal processes. In this study, bacterial biomass, growth and nitrogen removal capacity were quantified in shrimp farm effluent treatment systems containing vertical artificial substrates and either the banana shrimp Penaeus merguiensis (de Man) or the grey mullet, Mugil cephalus L. Banana shrimp were found to actively graze biofilm on the artificial substrates and significantly reduced bacterial biomass relative to a control (24.5 ± 5.6mgCm−2 and 39.2 ± 8.7mgCm−2, respectively). Bacterial volumetric growth rates, however, were significantly increased in the presence of the shrimp relative to the control 45.2±11.3mgCm−2 per day and 22.0±4.3mgCm−2 per day, respectively). Specific growth rate, or growth rate per cell, of bacteria was therefore appreciably stimulated by the banana shrimp. Nitrate assimilation was found to be significantly higher on grazed substrate biofilm relative to the control (223±54 mgNm−2 per day and 126±36 mg Nm−2 per day, respectively), suggesting that increased bacterial growth rate does relate to enhanced nitrogen uptake. Regulated banana shrimp feeding activity therefore can increase the rate of newbacterial biomass production and also the capacity for bacterial effluent nitrogen assimilation. Mullet had a negligible influence on the biofilm associated with the artificial substrate but reduced sediment bacterial biomass (224 ± 92 mgCm−2) relative to undisturbed sediment (650 ± 254 mgCm−2). Net, or volumetric bacterial growth in the sediment was similar in treatments with and without mullet, suggesting that the growth rate per cell of bacteria in grazed sediments was enhanced. Similar rates of dissolved nitrogen mineralisation werefound in sediments with and without mullet but nitrificationwas reduced. Presence of mullet increased water column suspended solids concentrations, water column bacterial growth and dissolved nutrient uptake. This study has shown that secondary crops, particularly banana shrimp, can play a stimulatory role in the bacterial processing of effluent nitrogen in eutrophic shrimp effluent treatment systems.

Growing banana prawns in prawn farm settlement ponds to utilise and help remove waste nutrients.

To assess their utility for profitable wastewater bioremediation, banana prawns, Penaeus (Fenneropenaeus) merguiensis (de Man), were stocked at low densities (1 – 5 m-2) and grown without supplemental feeding in five commercial-prawn-farm settlement ponds (0.3 to 6.0 ha). The prawns free-ranged in the variously designed ponds for 160 to 212 days after stocking as PL15. Survival estimates ranged from 12% to 60% with production of 50 – 528 kg ha-1. Over 1150 kg of marketable product was produced in the study. Exceptional growth was monitored at one farm where prawns reached an average size of 17g in 80 days. Nutrients in water flowing into (8 - 40 ML d-1) and out of the settlement pond at that farm were assessed twice weekly along with routine water quality measurements. Only small differences in water qualities were detected between waters running into and out of this settlement pond. Total nitrogen levels gradually increased from 1 - 1.5 mg L-1 early in the season to over 3 mg L-1 towards the end of the season. Total phosphorus levels similarly rose from 0.1 - 0.2 mg L-1 to 0.3 - 0.4 mg L-1 in the middle of the season, but fell to 0.2 – 0.3 mg L-1 towards the end when approximately 12,000 prawns were harvested with a total weight of 175 kg. No significant differences (P > 0.05) were detected in the overall acceptability of prawns harvested from each of the 5 settlement ponds in small-scale consumer sensory analyses. The prawns from settlement ponds were rated similarly to banana prawns grown with commercial diets at two other establishments. Microbiological analyses of prawns from all farms showed bacterial levels to be well within food-grade standards and lower than prawns produced in a normal growout pond. These results demonstrate that high quality food grade banana prawns can be produced in these wastewater treatment systems.

Develop added value uses of high anti-bacterial honey

Honey is valued for its medicinal properties. There interest from industry to develop a rapid method to detect the bioactivity of honey and also to develop value added products from high antibacterial honey.