Ta McMeekin

Evaluation of extraction methods for recovery of fatty acids from lipid-producing microheterotrophs.

The effect of different extraction techniques on the recovery of fatty acids from freeze-dried biomass of two lipid-producing microheterotrophs was examined. Two procedures were used: the extraction of lipids from biomass followed by transesterification of the fatty acids (extraction-transesterification); and the direct transesterification of biomass to produce fatty acid methyl esters (i.e. without the initial extraction step). Variable factors in the extraction-transesterification experiment were the sequence in which solvents were added to the samples, the relative amount of methanol in the solvent mix, and sonication of biomass while in the solvent mix. Variable factors in the direct transesterification experiment were sample size, and reaction duration. Statistical analysis of data (level of significance P<0.05) showed that: (1) extraction of total fatty acids prior to transesterification was significantly more efficient when solvents were added in the order of increasing polarity; (2) neither sonication nor increasing the proportion of methanol in the extraction solvent significantly affected extraction of fatty acids prior to transesterification; (3) efficiency of direct transesterification of fatty acids increased significantly with reaction time; (4) efficiency of direct transesterification of fatty acids was not significantly affected by sample size; (5) the most efficient method for extraction of fatty acids prior to transesterification yielded significantly less fatty acids than the most effective direct transesterification method. While the study examined only two strains, our results suggest that fatty acid analysis methodology for microheterotrophs under consideration for biotechnological exploitation requires optimisation and validation.

Shewanella gelidimarina sp. nov. and Shewanella frigidimarina sp. nov., novel Antarctic species with the ability to produce eicosapentaenoic acid (20:5 omega 3) and grow anaerobically by dissimilatory Fe(III) reduction

A polyphasic taxonomic study was performed to characterize dissimilatory iron-reducing strains mostly isolated from Antarctic sea ice. The strains were isolated from samples of congelated (land-fast) sea ice, grease ice, and ice algal biomass collected from the coastal areas of the Vestfold Hills in eastern Antarctica (68 degrees S 78 degrees E). The strains were facultatively anaerobic, motile, and rod shaped, were capable of anaerobic growth either by fermentation of carbohydrates or by anaerobic respiration, and utilized a variety of electron acceptors, including nitrate, ferric compounds, and trimethylamine N-oxide. A phylogenetic analysis performed with 16S rRNA sequences showed that the isolates formed two groups representing novel lineages in the genus Shewanella. The first novel group included seawater-requiring, psychrophilic, chitinolytic strains which had DNA G + C contents of 48 mol%. The members of the second strain group were psychrotrophic and did not require seawater but could tolerate up to 9% NaCl. The strains of this group were also unable to degrade polysaccharides but could utilize a number of monosaccharides and disaccharides and had G + C contents of 40 to 43 mol%. The whole-cell-derived fatty acid profiles of the sea ice isolates were found to be similar to the profiles obtained for other Shewanella species. The omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) (20:5 omega 3) was detected in all of the sea ice isolates at levels ranging from 2 to 16% of the total fatty acids. EPA was also found at high levels in Shewanella hanedai (19 to 22%) and Shewanella benthica (16 to 18%) but was absent in Shewanella alga and Shewanella putrefaciens. On the basis of polyphasic taxonomic data, the Antarctic iron-reducing strains are placed in two new species, Shewanella frigidimarina sp. nov. (type strain, ACAM 591) and Shewanella gelidimarina sp. nov. (type strain, ACAM 456).

Growth Limits of Listeria monocytogenes as a Function of Temperature, pH, NaCl, and Lactic Acid

ABSTRACT Models describing the limits of growth of pathogens under multiple constraints will aid management of the safety of foods which are sporadically contaminated with pathogens and for which subsequent growth of the pathogen would significantly increase the risk of food-borne illness. We modeled the effects of temperature, water activity, pH, and lactic acid levels on the growth of two strains ofListeria monocytogenes in tryptone soya yeast extract broth. The results could be divided unambiguously into “growth is possible” or “growth is not possible” classes. We observed minor differences in growth characteristics of the two L. monocytogenes strains. The data follow a binomial probability distribution and may be modeled using logistic regression. The model used is derived from a growth rate model in a manner similar to that described in a previously published work (K. A. Presser, T. Ross, and D. A. Ratkowsky, Appl. Environ. Microbiol. 64:1773–1779, 1998). We used “nonlinear logistic regression” to estimate the model parameters and developed a relatively simple model that describes our experimental data well. The fitted equations also described well the growth limits of all strains of L. monocytogenesreported in the literature, except at temperatures beyond the limits of the experimental data used to develop the model (3 to 35°C). The models developed will improve the rigor of microbial food safety risk assessment and provide quantitative data in a concise form for the development of safer food products and processes.

Estimation of bacterial growth rates from turbidimetric and viable count data

The relationship between maximum specific growth rates (mu max) determined from viable counts and turbidimetric measurements for a range of bacterial species is examined in order to assess the potential of turbidimetric methods in predictive microbiology. Two methods for the estimation of mu max from turbidimetric data are presented. One is based on absorbance and the other on transmittance measurements. Both are compared to estimates obtained by viable count methods. Calibration factors, a function to correct the non-linearity of absorbance measurements, and variance stabilising transformations for corrected absorbance measurements and for viable count data, are determined. It is concluded that turbidimetric measurements may be used reliably for estimation of mu max.

The Biotechnological Potential of Thraustochytrids

Abstract: Thraustochytrids are common marine microheterotrophs, taxonomically aligned with heterokont algae. Recent studies have shown that some thraustochytrid strains can be cultured to produce high biomass, containing substantial amounts of lipid rich in polyunsaturated fatty acid (PUFA). It is also evident that cell yield and PUFA production by some thraustochytrid strains can be varied by manipulation of physical and chemical parameters of the culture. At present, fish oils and cultured phototrophic microalgae are the main commercial sources of PUFA. The possible decline of commercial fish stocks and the relatively complex technology required to commercially produce microalgae have prompted research into possible alternative sources of PUFA. The culture of thraustochytrids and other PUFA-producing microheterotrophs is seen as one such alternative. Indeed, several thraustochytrid-based products are already on the market, and research into further applications is continuing. Many fish and microalgal oils currently available have relatively complex PUFA profiles, increasing the cost of preparation of high-purity PUFA oils. In contrast, some of the thraustochytrids examined to date have simpler PUFA profiles. If these or other strains can be grown in sufficient quantities and at an appropriate cost, the use of thraustochytrid-derived oils may decrease the high expense currently involved with producing high-purity microbial oils. As more is learned about the health and nutritional benefits of PUFA, demand for PUFA-rich products is expected to increase. Results to date suggest that thraustochytrids could form an important part in the supply of such products.

Psychroflexus torquis gen. nov., sp. nov. a psychrophilic species from Antarctic sea ice, and reclassification of Flavobacterium gondwanense (Dobson et al. 1993) as Psychroflexus gondwanense gen. nov., comb. nov.

A group of sea-ice-derived psychrophilic bacterial strains possessing the unusual ability to synthesize the polyunsaturated fatty acids eicosapentaenoic acid (20:5 omega 3) and arachidonic acid (20:4 omega 6) belong to the Family Flavobacteriaceae (Flexibacter-Bacteroides-Flavobacterium phylum), according to 16S rRNA sequence analysis. Surprisingly, the isolates were also found to cluster closely to the moderately halophilic and psychrotrophic species [Flavobacterium] gondwanense (sequence similarity 97.8-98.1%). The whole-cell fatty acid profiles of this group and [Flavobacterium] gondwanense were very similar and distinct from other related flavobacteria. The sea ice strains and [Flavobacterium] gondwanense differed substantially in terms of ecophysiology, possibly representing divergent adaptations to sympagic and planktonic marine habitats, respectively. Evidence based on phylogeny and fatty acid profiles supports the conclusion that the taxa are close relatives distinct from other bacterial groups. It is thus proposed that the sea ice strains represent a novel taxon designated Psychroflexus torquis gen. nov., sp. nov. (type strain ACAM 623T) while [Flavobacterium] gondwanense becomes Psychroflexus gondwanense gen. nov., comb. nov.

Halobacterium lacusprofundi sp. nov., a Halophilic Bacterium Isolated from Deep Lake, Antarctica

Summary Halobacterium lacusprofundi sp. nov. is described based on the characteristics of two strains (ACAM 32, ACAM 34) of red halophilic bacteria isolated from Deep Lake, a hypersaline, Antarctic lake. Bacteria which could grow on media prepared with undiluted Deep Lake water have not been isolated previously from Deep Lake. Cells were pleomorphic rods which lysed when suspended in distilled water. Unlike other halobacteria, the strains grew at 4°C, albeit very slowly. Fastest generation times (11 h) occurred between 31–37°C. The strains were not proteolytic, did not produce acids from sugars, but utilized a wide range of carbon sources including sugars, alcohols, and organic acids for growth. The strains grew in media with Mg ++ concentrations ranging from 0.005 to 1.0 mol/l and with NaCl concentrations ranging from 1.5 mol/l to saturation. Cells contained isoprenoid neutral lipids typical of halophilic archaebacteria. The major constituents were diphytanyl glyceryl ether, squalene and dihydrophytol. Approximately 30% of the total ether lipids consisted of at least four glyceryl ethers containing one or more double bonds in the phytenyl group. Also present were squalane, dihydrosqualene, tetrahydrosqualene, dihydrophytol and phytol. The G+C content of the major component DNA was 65 to 66 mol%. Satellite band DNA was present with G+C contents ranging from 54 to 57 mol%. Analysis of the 16S rRNA catalogue showed strain ACAM 34 was most closely related to Halobacterium saccharovorum ATCC 29252 (S AB value 0.74) but only distantly related to Haloferax volcanii ATCC 29605 (S AB value 0.43), Haloarcula vallismortis ATCC 29715 (S AB value 0.36), Halococcus morrhuae ATCC 17082 (S AB value 0.39) or Natronococcus occultus NCMB 2192 (S AB value 0.47). The type strain is UQM 3107 (ACAM 34).

Psychroserpens burtonensis gen. nov., sp. nov., and Gelidibacter algens gen. nov., sp. nov., psychrophilic bacteria isolated from antarctic lacustrine and sea ice habitats.

Psychrophilic, yellow-pigmented, seawater-requiring bacteria isolated from the pycnocline of meromictic Burton Lake and from sea ice cores obtained in the Vestfold Hills (68 degrees S, 78 degrees E) in eastern Antarctica were characterized. Phenotypic analysis showed that the strains isolated formed two distinct taxa. The first taxon included nonmotile, nutritionally fastidious strains that were isolated from the pycnocline of Burton Lake. The cells of these strains were morphologically variant, ranging from vibrioid to ring shaped to coiled and filamentous; in addition, the strains were unable to metabolise carbohydrates or polysaccharides and had DNA G + C contents of 27 to 29 mol%. The strains of the second taxon, which were isolated from sea ice cores and from ice aigal biomass, were saccharolytic, exhibited rapid gliding motility, were rodlike to filamentous, and had DNA G + C contents of 36 to 38 mol%. A 16S ribosomal DNA (rDNA) sequence analysis revealed that the two Antarctic taxa formed related but distinct lineages within the [Flexibacter] maritimus rRNA branch of the family Flavobacteriacrae. The levels of 16S rDNA sequence similarity between the taxa were 90.5 to 91.3%, while the levels of similarity to other members of the [F.] maritimus rRNA branch were 85 to 90%. The whole-cell lipid profiles of the Antarctic strains were mainly comprised of branched and unbranched monounsaturated C15 to C17 fatty acids. The presence of significant levels of the lipids a 15:1 omega 10c and a17:1 omega 7c appeared to be useful biomarkers for the new Antarctic taxa and for differentiating these organisms from other members of the family Flavobacteriaceae. On the basis of polyphasic taxonomic data we propose that the new taxa are novel bacterial species designated Psychroserpens burtonensis gen. nov., sp. nov. (type strain, ACAM 188) and Gelidibacter algens gen. nov., sp. nov. (type strain, ACAM 536).

Colwellia demingiae sp. nov., Colwellia hornerae sp. nov., Colwellia rossensis sp. nov. and Colwellia psychrotropica sp. nov.: psychrophilic Antarctic species with the ability to synthesize docosahexaenoic acid (22 : 6 omega 3)

As part of a general survey of the biodiversity and inherent ecophysiology of bacteria associated with coastal Antarctic sea-ice diatom assemblages, eight strains were identified by 16S rRNA sequence analysis as belonging to the genus Colwellia. The isolates were non-pigmented, curved rod-like cells which exhibited psychrophilic and facultative anaerobic growth and possessed an absolute requirement for sea water. One isolate was able to form gas vesicles. All strains synthesized the 3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid (22:63, DHA) (0·7–8·0% of total fatty acids). Previously, DHA has only been detected in strains isolated from deep-sea benthic and faunal habitats and is associated with enhanced survival in permanently cold habitats. The G+C content of the DNA from the Antarctic Colwellia strains ranged from 35 to 42 mol% and DNA-DNA hybridization analyses indicated that the isolates formed five genospecies, including the species Colwellia psychrerythraea (ACAM 550T). 16S rRNA sequence analysis indicated that the strains formed a cluster in the gM-subclass of the Proteobacteria with Colwellia psychrerythraea. Sequence similarities ranged from 95·2 to 100% between the various Antarctic Colwellia isolates. Phenotypic characterization confirmed distinct differences between the different genospecies. These studies indicate that the DHA-producing Antarctic isolates consist of five different Colwellia species: Colwellia psychrerythraea and four novel species with the proposed names Colwellia demingiae sp. nov. (ACAM 459T), Colwellia psychrotropica sp. nov. (ACAM 179T), Colwellia rossensis sp. nov. (ACAM 608T) and Colwellia hornerae sp. nov. (ACAM 607T).

DEVELOPMENTS WITH ANTARCTIC MICROORGANISMS : CULTURE COLLECTIONS, BIOACTIVITY SCREENING, TAXONOMY, PUFA PRODUCTION AND COLD-ADAPTED ENZYMES

There have been recent research developments with Antarctic prokaryotes in the areas of isolations of novel bacterial, culture collections, bioactivity screening, taxonomy, production of polyunsaturated fatty acids (PUFAs), cold-adapted enzymes and bioremediation. Research to date confirms the novelty of bacteria isolated from this extreme environment. Opportunities now exist to exploit these and other findings to develop possible new biotechnological products from Antarctic microorganisms.