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Applied Microbiology and Biotechnology (v.63, #2)

Anaerobic ammonium oxidation by marine and freshwater planctomycete-like bacteria by M. S. M. Jetten; O. Sliekers; M. Kuypers; T. Dalsgaard; L. van Niftrik; I. Cirpus; K. van de Pas-Schoonen; G. Lavik; B. Thamdrup; D. Le Paslier; H. J. M. Op den Camp; S. Hulth; L. P. Nielsen; W. Abma; K. Third; P. Engström; J. G. Kuenen; B. B. Jørgensen; D. E. Canfield; J. S. Sinninghe Damsté; N. P. Revsbech; J. Fuerst; J. Weissenbach; M. Wagner; I. Schmidt; M. Schmid; M. Strous (pp. 107-114).

Recently, two fresh water species, "Candidatus Brocadia anammoxidans" and "Candidatus Kuenenia stuttgartiensis", and one marine species, "Candidatus Scalindua sorokinii", of planctomycete anammox bacteria have been identified. "Candidatus Scalindua sorokinii" was discovered in the Black Sea, and contributed substantially to the loss of fixed nitrogen. All three species contain a unique organelle—the anammoxosome—in their cytoplasm. The anammoxosome contains the hydrazine/hydroxylamine oxidoreductase enzyme, and is thus the site of anammox catabolism. The anammoxosome is surrounded by a very dense membrane composed almost exclusively of linearly concatenated cyclobutane-containing lipids. These so-called 'ladderanes' are connected to the glycerol moiety via both ester and ether bonds. In natural and man-made ecosystems, anammox bacteria can cooperate with aerobic ammonium-oxidising bacteria, which protect them from harmful oxygen, and provide the necessary nitrite. The cooperation of these two groups of ammonium-oxidising bacteria is the microbial basis for a sustainable one reactor system, CANON (completely autotrophic nitrogen-removal over nitrite) to remove ammonia from high strength wastewater.

Bacterial heme biosynthesis and its biotechnological application by N. Frankenberg; J. Moser; D. Jahn (pp. 115-127).

Proteins carrying a prosthetic heme group are vital parts of bacterial energy conserving and stress response systems. They also mediate complex enzymatic reactions and regulatory processes. Here, we review the multistep biosynthetic pathway of heme formation including the enzymes involved and reaction mechanisms. Potential biotechnological implications are discussed.

Phytoremediation of toxic aromatic pollutants from soil by O. V. Singh; R. K. Jain (pp. 128-135).

The enormous growth of industrialization, and the use of numerous aromatic compounds in dyestuffs, explosives, pesticides and pharmaceuticals has resulted in serious environmental pollution and has attracted considerable attention continuously over the last two decades. Many aromatic hydrocarbons, nitroaromatic compounds, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, diauxins and their derivatives are highly toxic, mutagenic and/or carcinogenic to natural microflora as well as to higher systems including humans. The increasing costs and limited efficiency of traditional physicochemical treatments of soil have spurred the development of new remediation technologies. Phytoremediation is emerging as an efficient treatment technology that uses plants to bioremediate pollutants from soil environments. Various modern tools and analytical devices have provided insight into the selection and optimization of remediation processes by various plant species. Sites heavily polluted with organic contaminants require hyperaccumulators, which could be developed by genetic engineering approaches. However, efficient hyperaccumulation by naturally occurring plants is also feasible and can be made practical by improving their nutritional and environmental requirements. Thus, phytoremediation of organics appears a very promising technology for the removal of contaminants from polluted soil. In this review, certain aspects of plant metabolism associated with phytoremediation of organic contaminants and their relevant phytoremediation efforts are discussed.

Overproduction of lipase by Yarrowia lipolytica mutants by P. Fickers; J. M. Nicaud; J. Destain; P. Thonart (pp. 136-142).

Non-genetically modified mutants with increased capacities of extracellular lipase production were obtained from Yarrowia lipolytica strain CBS6303 by chemical mutagenesis. Of the 400 mutants isolated, LgX64.81 had the highest potential for the development of an industrial lipase production process. This mutant exhibits lipase production uncoupled from catabolite repression by glucose, and a 10-fold increased productivity upon addition of oleic acid. Using a LIP2-LacZ reporter gene, we demonstrate that the mutant phenotype originates from a trans-acting mutation. The glucose uptake capacity of LgX64.81 is reduced 2.5-fold compared to the wild-type-strain, and it exhibits high lipase production on glucose medium. A trans-acting mutation in a gene involved in glucose transport could thus explain this mutant phenotype.

Microbial fed-batch production of 1,3-propanediol by Klebsiella pneumoniae under micro-aerobic conditions by X. Chen; D.-J. Zhang; W.-T. Qi; S.-J. Gao; Z.-L. Xiu; P. Xu (pp. 143-146).

The microbial production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae under micro-aerobic conditions was investigated in this study. The experimental results of batch fermentation showed that the final concentration and yield of 1,3-PD on glycerol under micro-aerobic conditions approached values achieved under anaerobic conditions. However, less ethanol was produced under microaerobic than anaerobic conditions at the end of fermentation. The batch micro-aerobic fermentation time was markedly shorter than that of anaerobic fermentation. This led to an increment of productivity of 1,3-PD. For instance, the concentration, molar yield, and productivity of 1,3-PD of batch micro-aerobic fermentation by K. pneumoniae DSM 2026 were 17.65 g/l, 56.13%, and 2.94 g l⁻¹ h⁻¹, respectively, with a fermentation time of 6 h and an initial glycerol concentration of 40 g/l. Compared with DSM 2026, the microbial growth of K. pneumoniae AS 1.1736 was slow and the concentration of 1,3-PD was low under the same conditions. Furthermore, the microbial growth in fed-batch fermentation by K. pneumoniae DSM 2026 was faster under micro-aerobic than anaerobic conditions. The concentration, molar yield, and productivity of 1,3-PD in fed-batch fermentation under micro-aerobic conditions were 59.50 g/l, 51.75%, and 1.57 g l⁻¹ h⁻¹, respectively. The volumetric productivity of 1,3-PD under microaerobic conditions was almost twice that of anaerobic fed-batch fermentation, at 1.57 and 0.80 g l⁻¹ h⁻¹, respectively.

Using banana to generate lactic acid through batch process fermentation by Y. Chan-Blanco; A. R. Bonilla-Leiva; A. C. Velázquez (pp. 147-152).

We evaluated the usefulness of waste banana for generating lactic acid through batch fermentation, using Lactobacillus casei under three treatments. Two treatments consisted of substrates of diluted banana purée, one of which was enriched with salts and amino acids. The control treatment comprised a substrate suitable for L. casei growth. When fermentation was evaluated over time, significant differences (P<0.05) were found in the three treatments for each of five variables analyzed (generation and productivity of lactic acid, and consumption of glucose, fructose, and sucrose). Maximum productivity was (in g l⁻¹ h⁻¹) 0.13 for the regular banana treatment, 1.49 for the enriched banana, and 1.48 for the control, with no significant differences found between the latter two treatments. Glucose consumption curves showed that L. casei made greater use of the substrate in the enriched banana and control treatments than in the regular banana treatment. For fructose intake, the enriched banana treatment showed significantly better (P<0.05) results than the regular one. Sucrose consumption was insignificant (P<0.05), probably because fermentation time was too short. Even when enriched, diluted banana purée is an ineffective substrate for L. casei, probably because it lacks nutrients.

Primary structure and transcription analysis of a laccase-encoding gene from the basidiomycete Trametes trogii by M. Ch. Colao; A. M. Garzillo; V. Buonocore; A. Schiesser; M. Ruzzi (pp. 153-158).

A cDNA coding for laccase was isolated from the white-rot fungus Trametes trogii 201. This cDNA corresponded to the lcc1 gene, which coded for a precursor protein of 517 amino acids with a 21 amino acid signal peptide. Comparison of the deduced sequence with known laccases showed that this enzyme was most closely related to Lac1 from basidiomycete PM1 and Trametes C30 (98% similarity). The expression of lcc1 was analysed under different growth conditions; transcription of this gene was enhanced by the addition of organic nitrogen to the medium. The level of lcc1 transcription was higher when T. trogii was grown on synthetic medium supplemented with yeast extract rather than mycological peptone or tryptone. The transcription data were in agreement with total laccase activity measured in the supernatant and suggested that laccase production and lcc1 transcription are coordinately regulated in this organism. The lcc1 cDNA was expressed in the methylotrophic yeast Pichia pastoris and the detection of laccase activity indicated that this cDNA encodes a laccase.

Azotobacter vinelandii mutants that overproduce poly-β-hydroxybutyrate or alginate by D. Segura; J. Guzmán; G. Espín (pp. 159-163).

Azotobacter vinelandii produces two polymers of industrial importance, i.e. alginate and poly-β-hydroxybutyrate (PHB). Alginate synthesis constitutes a waste of substrate when seeking to optimize PHB production and, conversely, synthesis of PHB is undesirable when optimizing alginate production. In this study we evaluated the effect of a mutation in algA, the gene encoding the enzyme that catalyzes the first step of the alginate biosynthetic pathway in the production of PHB. We also evaluated production of alginate in strain AT6 carrying a phbB mutation that impairs PHB synthesis. The algA mutation prevented alginate production and increased PHB accumulation up to 5-fold, determined in milligrams per milligram of protein. Similarly, the phbB mutation increased alginate production up to 4-fold.

Metabolic changes during cell growth inhibition by p27 overexpression by A. V. Carvalhal; I. Marcelino; M. J. T. Carrondo (pp. 164-173).

The overexpression of p27, a cyclin-dependent kinase (CDK) inhibitor, has been shown to effectively inhibit cell growth at the G1-phase of different cell lines, potentiating a valid genetic strategy for cell proliferation control. In order to characterize the energy requirements after p27 overexpression in CHO cells expressing SEAP (secreted form of the human alkaline phosphatase enzyme), key metabolic parameters were evaluated. Cell growth inhibition led to a significant increase in cell size concomitant with a 2-fold increase in cell protein content. The simultaneous increase of the intracellular proteolytic activity with protein content suggests higher protein synthesis. A general 2-fold increase in oxygen, glutamine and glucose consumption rates, coupled with an increase in lactate and ammonia production was observed. p27 overexpression led to a significant increase in the intracellular pool of AMP (8.5-fold), ADP (6-fold) and, more uncommonly, ATP (4.5-fold). Nevertheless, cells were able to maintain the equilibrium among the three adenine nucleotides since both the ATP/ADP ratio and the energy charge values remained similar to those observed with non-growth inhibited cells. This work shows that the observed 4-fold increase in SEAP specific productivity after cell growth inhibition by p27, occurred concomitantly with a higher expenditure of cell energy. This characterization of cell metabolism becomes important in demonstrating the applicability of growth inhibition systems.

Molecular cloning and characterization of a laccase gene from the basidiomycete Fome lignosus and expression in Pichia pastoris by W. Liu; Y. Chao; S. Liu; H. Bao; S. Qian (pp. 174-181).

A cDNA encoding for a laccase was isolated from the white-rot fungus Fome lignosus by RT-PCR. It contained an open reading frame of 1,557 bp. The deduced mature protein consisted of 497 amino acids and was preceded by a signal peptide of 21 amino acids. The genomic DNA of the laccase, containing 11 introns, was cloned by PCR. The cDNA was cloned into the vectors pGAPZαA and pGAPZA, and expressed in the Pichia pastoris GS115. Laccase-secreting transformants were selected by their ability to oxidize the substrate 2′2-azinobis-(3-ethylbenzthiaoline-6-sufonic acid) (ABTS). The laccase activity obtained with the native signal peptide was found to be fivefold higher than that obtained with the α-factor secretion signal peptide. The presence of 0.4 mM copper was necessary for optimal activity of the enzyme. The highest activity value reached 9.03 U ml⁻¹, and the optimal secreting time was 2~3 days at 20°C. The crude laccase was stable in a pH range from 6.0 to 10.0 and at temperatures lower than 30°C in pH4.5 for 24 h. The molecular mass of the enzyme was estimated to be 66.5 kDa by SDS-PAGE. The optimum pH and temperature were 2.4 and 55°C. The K _m and V _max values for ABTS were 177 μM and 23.54 μmol min⁻¹ respectively. The extent of glycosylation of the purified enzyme was 58.6%.

Bioadsorption of cadmium ion by cell surface-engineered yeasts displaying metallothionein and hexa-His by K. Kuroda; M. Ueda (pp. 182-186).

The Cd²⁺-chelating abilities of yeast metallothionein (YMT) and hexa-His displayed on the yeast-cell surface were compared. Display of YMT and hexa-His by α-agglutinin-based cell-surface engineering was confirmed by immunofluorescent labeling. Surface-engineered yeast cells with YMT and hexa-His fused in tandem showed superior cell-surface adsorption and recovery of Cd²⁺ under EDTA treatment on the cell surface than hexa-His-displaying cells. YMT was demonstrated to be more effective than hexa-His for the adsorption of Cd²⁺. Yeast cells displaying YMT and/or hexa-His exhibited a higher potential for the adsorption of Cd²⁺ than Escherichia coli cells displaying these molecules. In order to investigate the effect of the displayed YMT and hexa-His on sensitivity to toxic Cd²⁺, growth in Cd²⁺-containing liquid medium was monitored. Unlike hexa-His-displaying cells, cells displaying YMT and hexa-His fused in tandem induced resistance to Cd²⁺ through active and enhanced adsorption of toxic Cd²⁺. These results indicate that YMT-displaying yeast cells are a unique bioadsorbent with a functional chelating ability superior to that of E. coli.

Metabolism of hexahydro-1,3,5-trinitro-1,3,5-triazine through initial reduction to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine followed by denitration in Clostridium bifermentans HAW-1 by J.-S. Zhao; L. Paquet; A. Halasz; J. Hawari (pp. 187-193).

A fast hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)-degrading [28.1 μmol h⁻¹ g (dry weight) cells⁻¹; biomass, 0.16 g (dry weight) cells⁻¹] and strictly anaerobic bacterial strain, HAW-1, was isolated and identified as Clostridium bifermentans using a 16S-rRNA-based method. Based on initial rates, strain HAW-1 transformed RDX to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) with yields of 56, 7.3 and 0.2%, respectively. Complete removal of RDX and its nitroso metabolites produced (%, of total C or N) methanol (MeOH, 23%), formaldehyde (HCHO, 7.4%), carbon dioxide (CO₂, 3.0%) and nitrous oxide (N₂O, 29.5%) as end products. Under the same conditions, strain HAW-1 transformed MNX separately at a rate of 16.9 μmol h⁻¹ g (dry weight) cells⁻¹ and produced DNX (25%) and TNX (0.4%) as transient products. Final MNX transformation products were (%, of total C or N) MeOH (21%), HCHO (2.9%), and N₂O (17%). Likewise strain HAW-1 degraded TNX at a rate of 7.5 μmol h⁻¹ g (dry weight) cells⁻¹to MeOH and HCHO. Furthermore, removal of both RDX and MNX produced nitrite (NO₂ ⁻) as a transient product, but the nitrite release rate from MNX was quicker than from RDX. Thus, the predominant pathway for RDX degradation is based on initial reduction to MNX followed by denitration and decomposition. The continued sequential reduction to DNX and TNX is only a minor route.

Naphthalene-utilizing and mercury-resistant bacteria isolated from an acidic environment by S. Y. Dore; Q. E. Clancy; S. M. Rylee; C. F. Kulpa Jr (pp. 194-199).

Soil samples were taken from areas of low pH (2.5–3.5) surrounding an outdoor coal storage pile. These samples were added to medium with naphthalene as the sole carbon source to enrich for organisms capable of degrading polycyclic aromatic hydrocarbons (PAH) at low pH. Five such bacterial strains were isolated. Sequencing of the 16S rDNA showed them to be members of the genera Clavibacter, Arthrobacter and Acidocella. These organisms were all capable of growth with naphthalene as a sole carbon source at low pH. The genes nahAc, nahAd, phnAc, nahH, xylE or GST, which are known to be associated with PAH degradation were not detected. Isolate 10, the Acidocella strain, tolerated high levels of mercury. PCR amplification and sequencing of genes from the mer operon from isolate 10 DNA suggested that mercury is transported into the bacterial cell and subsequently detoxified since the enzymes encoded by genes in this operon are involved in these processes.

A novel polygalacturonic acid bioflocculant REA-11 produced by Corynebacterium glutamicum: a proposed biosynthetic pathway and experimental confirmation by Y. Li; N. He; H. Guan; G. Du; J. Chen (pp. 200-206).

Corynebacterium glutamicum CCTCC M201005 produces a novel polygalacturonic acid bioflocculant, REA-11, consisting of galacturonic acid as the main structural unit. A biosynthetic pathway of REA-11 in C. glutamicum CCTCC M201005 was proposed. Evidence for the biosynthetic pathway was provided by: (1) analyzing the response upon addition of UDP-glucose to the culture medium; (2) detecting the presence of several key intermediates in the pathway; and (3) correlating the activities of several key enzymes involved in the pathway with the yields of polygalacturonic acid. The production of polygalacturonic acid was improved by 24%, while the activities of UDP-galactose epimerase and UDP-galactose dehydrogenase were improved by 200% and 50%, respectively, upon addition of 100 μM UDP-glucose. In addition, the key intermediates in the proposed biosynthetic pathway, such as UDP-glucose, UDP-galactose, and UDP-glucuronic acid, were detected in cell-free extracts. Furthermore, the activities of UDP-glucose pyrophosphorylase (R ²=0.97), UDP-galactose epimerase (R ²=0.75) and UDP-galactose dehydrogenase (R ²=0.89) were well correlated with the yields of polygalacturonic acid when different sugars were used as sole carbon sources. Therefore, the biosynthetic pathway of REA-11 in C. glutamicum CCTCC M201005 starts from phosphate-1-glucose, which was then converted to UDP-glucose by UDP-pyrophosphorylase. Predominantly, the UDP-glucose was converted to UDP-galactose by UDP-galactose epimerase; the latter was further converted to UDP-galacturonic acid by UDP-galactose dehydrogenase, which was presumably polymerized to polygalacturonic acid bioflocculant REA-11 by an unknown glucosyltransferase and a polymerase.

Keywords: Corynebacterium glutamicum ; Polysaccharide-protein bioflocculant; Galacturonic acid; Proposed biosynthetic pathway; Experimental confirmation

Effect of malic acid on the growth kinetics of Lactobacillus plantarum by F. V. Passos; H. P. Fleming; H. M. Hassan; R. F. McFeeters (pp. 207-211).

The fermentation kinetics of Lactobacillus plantarum were studied in a specially designed broth formulated from commercially available, dehydrated components (yeast extract, trypticase, ammonium sulfate) in batch and continuous culture. During batch growth in the absence of malic acid, the specific growth rate was 0.20 h⁻¹. Malic acid in the medium, at 2 mM or 10 mM, increased the specific growth rate of L. plantarum to 0.34 h⁻¹. An increase in the maximum cell yield due to malic acid also was observed. Malic acid in the medium (12 mM) reduced the non-growth-associated (maintenance energy) coefficient and increased the biomass yield in continuous culture, based on calculations from the Luedeking and Piret model. The biomass yield coefficient was estimated as 27.4 mg or 34.3 mg cells mmol⁻¹ hexose in the absence or presence of malic acid, respectively. The maintenance coefficient was estimated as 3.5 mmol or 1.5 mmol hexose mg⁻¹ cell h⁻¹ in the absence or presence of malic acid. These results clearly demonstrate the energy-sparing effect of malic acid on the growth- and non-growth-associated energy requirements for L. plantarum. The quantitative energy-sparing effect of malic acid on L. plantarum has heretofore not been reported, to our knowledge.

Physiology of a colony of Pleurotus pulmonarius grown on medium overlaid with a Cellophane membrane by M. Téllez-Téllez; G. Díaz-Godínez; C. Sánchez (pp. 212-216).

The physiology of a colony of Pleurotus pulmonarius grown on potato dextrose agar overlaid with a Cellophane membrane (PDA-WC) was studied. On PDA-WC, the colony presented higher biomass density and productivity of fruit body formation (4.84±0.23 mg mycelial biomass/cm² and 23.7±3.12 μg ml⁻¹ h⁻¹, respectively) than that which developed on potato dextrose agar without Cellophane (PDA-OC; 0.26±0.01 mg mycelial biomass/cm² and 10.8±1.57 μg ml⁻¹ h⁻¹, respectively). In cultures developed on PDA-WC, intracellular laccases and β-1,3-glucanases activities were lower [12±0.9 arbitrary units (AU)/g mycelial biomass and 1.33±0.1 international units (IU)/g mycelial biomass, respectively] than those observed on PDA-OC (20.65±1.0 AU/g mycelial biomass and 3.67±0.2 IU/g mycelial biomass, respectively). In cultures developed on PDA-WC, intracellular protein and glycogen concentrations were lower (1.9±0.9 and 117±3.5 mg/g mycelial biomass, respectively) than those observed on PDA-OC (14.3±1.1 and 347±2.9 mg/g mycelial biomass, respectively). The radial growth rate and the content of glucans in the cell wall were not significantly different between cultures developed on PDA-WC and PDA-OC. These results show that the use of the Cellophane as a tool to study in vitro fungal physiology might affect the interpretation of experimental results, since the physiology under otherwise similar conditions was different on medium with and without Cellophane.

Improved method for determination of ammonia and nitrite oxidation activities in mixed bacterial cultures by M. S. Moussa; H. J. Lubberding; C. M. Hooijmans; M. C. M. van Loosdrecht; H. J. Gijzen (pp. 217-221).

A simple and reliable method to measure the activity of ammonia and nitrite oxidisers in mixed bacterial cultures was developed. The developed method differentiates between the ammonia and nitrite oxidisers by consecutive injection of NO₂ ⁻ and NH₄ ⁺. The main advantage of this method is that it avoids the use of metabolic inhibitors for ammonia or nitrite oxidisers, as used by other methods. Moreover, it allows measuring of the short-term effect of an inhibitor on both the ammonia and nitrite oxidisers in one test under controlled environmental conditions (pH, temperature). The developed method was applied to determine the inhibitory effects of salt (NaCl up to 15 g Cl/l) on an enriched culture of nitrifying bacteria. The results of the method demonstrate its potential to accurately determine the individual activities of nitrite and ammonia oxidisers.

Polychlorinated biphenyl (PCB) degradation and persistence of a gfp-marked Ralstonia eutropha H850 in PCB-contaminated soil by A.-M. Irwin Abbey; L. A. Beaudette; H. Lee; J. T. Trevors (pp. 222-230).

Ralstonia eutropha H850 was labelled chromosomally with a gfp marker gene encoding for the green fluorescent protein, and designated R. eutropha H850g13. Visual observation of green fluorescent cells under an epifluorescence microscope, and PCR amplification products, confirmed that the bacterium was labelled with gfp. Southern blot hybridization products further confirmed the gfp was chromosomally labelled. Using resting cell assays, it was determined that insertion of the gfp gene decreased the microorganisms' ability to degrade biphenyl compared to the parent strain. However, this marker facilitated the identification and monitoring of R. eutropha H850g13 survival in soil microcosm experiments. Survival and polychlorinated biphenyl degradation by R. eutropha H850g13 was analysed in soil microcosms spiked with 2,2′,5,5′-tetrachlorobiphenyl (TeCB). R. eutropha H850g13 was detected by viable plate counts and most-probable-number/PCR after 102 days in TeCB-contaminated soil microcosms, and was likely outcompeted by indigenous soil microorganisms in microcosms amended with oil and Daramend (an organic amendment, http://www.adventusremediation.com ). R. eutropha H850g13 did not degrade TeCB in any of the soil microcosms. This research confirmed that gfp was useful as a marker to distinguish R. eutropha H850g13 from indigenous soil microorganisms over a 102 day period and that, under the experimental conditions used, R. eutropha H850g13 did not degrade TeCB.

A biomarker for the identification of swine fecal pollution in water, using the STII toxin gene from enterotoxigenic Escherichia coli by L. A. Khatib; Y. L. Tsai; B. H. Olson (pp. 231-238).

This research developed a PCR method to identify swine fecal pollution in water, using a portion of the STII toxin gene from enterotoxigenic Escherichia coli as the target sequence. This method showed the gene to have a wide-spread geographical distribution and temporal stability; and the primers demonstrated high specificity, sensitivity, and reliability. A total of 110 DNA extracts from different animal fecal and human sewage samples were screened using the primers and no positives resulted. Centrifugation and filtration methods for concentrating E. coli seeded into stream, ocean, secondary effluent, and dairy lagoon waters resulted in detection limits at the femtogram and attogram levels. E. coli with the biomarker seeded into stream, ocean, and secondary effluent waters remained stable for approximately 2 weeks for all water types. Of the farm lagoon and waste samples tested, 94% were positive for the STII trait, regardless of the number of E. coli screened and 100% were positive when ≥35 E. coli isolates were screened. As the PCR product of the target sequence yielded a single band, the method is applicable to dot blot detection methodology, yielding great accuracy in determining the presence of swine fecal sources.

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Applied Microbiology and Biotechnology (v.63, #2)