Altered Protozoan and Bacterial Communities and Survival of Escherichia coli O157:H7 in Monensin-Treated Wastewater from a Dairy Lagoon

Surviving predation is a fitness trait of Escherichia coli O157:H7 (EcO157) that provides ample time for the pathogen to be transported from reservoirs (e.g. dairies and feedlots) to farm produce grown in proximity. Ionophore dietary supplements that inhibit rumen protozoa may provide such a selective advantage for EcO157 to proliferate in lagoons as the pathogen is released along with the undigested supplement as manure washings. This study evaluated the fate of an outbreak strain of EcO157, protozoan and bacterial communities in wastewater treated with monensin. Although total protozoa and native bacteria were unaffected by monensin, the time for 90% decrease in EcO157 increased from 0.8 to 5.1 days. 18S and 16S rRNA gene sequencing of wastewater samples revealed that monensin eliminated almost all colpodean and oligohymenophorean ciliates, probably facilitating the extended survival of EcO157. Total protozoan numbers remained high in treated wastewater as monensin enriched 94% of protozoan sequences undetected with untreated wastewater. Monensin stimulated 30-fold increases in Cyrtohymena citrina, a spirotrichean ciliate, and also biflagellate bicosoecids and cercozoans. Sequences of gram-negative Proteobacteria increased from 1% to 46% with monensin, but gram-positive Firmicutes decreased from 93% to 46%. It is noteworthy that EcO157 numbers increased significantly (P<0.01) in Sonneborn medium containing monensin, probably due to monensin-inhibited growth of Vorticella microstoma (P<0.05), a ciliate isolated from wastewater. We conclude that dietary monensin inhibits ciliate protozoa that feed on EcO157. Feed supplements or other methods that enrich these protozoa in cattle manure could be a novel strategy to control the environmental dissemination of EcO157 from dairies to produce production environments.     

Extractable organic components and nutrients in wastewater from dairy lagoons influence the growth and survival of Escherichia coli O157:H7

The influence of nutrients in wastewater from dairy lagoons on the survival of Escherichia coli O157:H7 was monitored. Initially, the survival of E. coli O157:H7 in wastewater from which the competing native organisms had been removed by filter sterilization or autoclaving was compared with that in wastewater from which competing organisms had not been removed. Numbers of E. coli O157:H7 or E. coli ONT (O-nontypeable):H32 cells declined rapidly in filter-sterilized water and exhibited a slower decline in nonsterile water, while the organisms proliferated in autoclaved water. Subsequently, the growth of E. coli O157:H7 strains was monitored in 300 mul of Luria-Bertani (LB) broth supplemented with incremental proportions of filter-sterilized wastewater. E. coli O157:H7 and E. coli ONT:H32 strains failed to grow in filter-sterilized wastewater, and their growth was reduced incrementally with wastewater supplementation of LB broth. Consequently, the influence of organic extracts of wastewater on the growth of E. coli O157:H7 and E. coli ONT:H32 in reduced-strength LB was monitored, followed by scale-up tests in wastewater. Acidic and basic extracts inhibited growth of both strains, while the neutral aqueous extract improved growth. However, a scale-up with a threefold increase in the acidic components supplementing the wastewater did not result in any additional decline in numbers of E. coli O157:H7 cells. When protected inside a 300-kDa dialysis tube and exposed to diffusible components, E. coli O157:H7 survived longer, with a decimal reduction time of 18.1 days, compared to 3.5 days when inoculated directly into wastewater. Although wastewater can potentially provide nutrients to naturally occurring human pathogens, the chemical components, protozoa, and coliphages in wastewater can inhibit the growth of freshly introduced pathogens from manure.     

Strain Differences in Fitness of Escherichia coli O157:H7 to Resist Protozoan Predation and Survival in Soil

Escherichia coli O157:H7 (EcO157) associated with the 2006 spinach outbreak appears to have persisted as the organism was isolated, three months after the outbreak, from environmental samples in the produce production areas of the central coast of California. Survival in harsh environments may be linked to the inherent fitness characteristics of EcO157. This study evaluated the comparative fitness of outbreak-related clinical and environmental strains to resist protozoan predation and survive in soil from a spinach field in the general vicinity of isolation of strains genetically indistinguishable from the 2006 outbreak strains. Environmental strains from soil and feral pig feces survived longer (11 to 35 days for 90% decreases, D-value) with Vorticella microstoma and Colpoda aspera, isolated previously from dairy wastewater; these D-values correlated (P<0.05) negatively with protozoan growth. Similarly, strains from cow feces, feral pig feces, and bagged spinach survived significantly longer in soil compared to clinical isolates indistinguishable by 11-loci multi-locus variable-number tandem-repeat analysis. The curli-positive (C⁺) phenotype, a fitness trait linked with attachment in ruminant and human gut, decreased after exposure to protozoa, and in soils only C⁻ cells remained after 7 days. The C⁺ phenotype correlated negatively with D-values of EcO157 exposed to soil (r _s = −0.683; P = 0.036), Vorticella (r _s = −0.465; P = 0.05) or Colpoda (r _s = −0.750; P = 0.0001). In contrast, protozoan growth correlated positively with C⁺ phenotype (Vorticella, r _s = 0.730, P = 0.0004; Colpoda, r _s = 0.625, P = 0.006) suggesting a preference for consumption of C⁺ cells, although they grew on C⁻ strains also. We speculate that the C⁻ phenotype is a selective trait for survival and possibly transport of the pathogen in soil and water environments.     

Extractable Organic Components and Nutrients in Wastewater from Dairy Lagoons Influence the Growth and Survival of Escherichia coli O157:H7

The influence of nutrients in wastewater from dairy lagoons on the survival of Escherichia coli O157:H7 was monitored. Initially, the survival of E. coli O157:H7 in wastewater from which the competing native organisms had been removed by filter sterilization or autoclaving was compared with that in wastewater from which competing organisms had not been removed. Numbers of E. coli O157:H7 or E. coli ONT (O-nontypeable):H32 cells declined rapidly in filter-sterilized water and exhibited a slower decline in nonsterile water, while the organisms proliferated in autoclaved water. Subsequently, the growth of E. coli O157:H7 strains was monitored in 300 μl of Luria-Bertani (LB) broth supplemented with incremental proportions of filter-sterilized wastewater. E. coli O157:H7 and E. coli ONT:H32 strains failed to grow in filter-sterilized wastewater, and their growth was reduced incrementally with wastewater supplementation of LB broth. Consequently, the influence of organic extracts of wastewater on the growth of E. coli O157:H7 and E. coli ONT:H32 in reduced-strength LB was monitored, followed by scale-up tests in wastewater. Acidic and basic extracts inhibited growth of both strains, while the neutral aqueous extract improved growth. However, a scale-up with a threefold increase in the acidic components supplementing the wastewater did not result in any additional decline in numbers of E. coli O157:H7 cells. When protected inside a 300-kDa dialysis tube and exposed to diffusible components, E. coli O157:H7 survived longer, with a decimal reduction time of 18.1 days, compared to 3.5 days when inoculated directly into wastewater. Although wastewater can potentially provide nutrients to naturally occurring human pathogens, the chemical components, protozoa, and coliphages in wastewater can inhibit the growth of freshly introduced pathogens from manure.     

Survival of Escherichia coli O157:H7 in wastewater from dairy lagoons

AIM: To determine the survival of Escherichia coli O157:H7 in dairy wastewater from on-site holding lagoons equipped with or without circulating aerators. METHODS AND RESULTS: Survival was monitored in dairy lagoon microcosms equipped with or without scale-size circulators. Both laboratory strains of E. coli O157:H7 and an isolate of E. coli H7 from wastewater had poor survival rates and none proliferated in water from waste lagoons with or without circulators. Furthermore, the decline of E. coli O157:H7 was not enhanced in those microcosms equipped with circulators. Strain variation in survival was observed in both circulated and settling waters. The decline rate of E. coli O157:H7 Odwalla strain increased proportionately with the inoculum load. Escherichia coli failed to establish itself in wastewater even after four sequential inoculations simulating continuous faecal input into the lagoon. The native aerobic bacteria survived longer with a decimal reduction time of 21.3 days vs either introduced or native E. coli, which declined rapidly with decimal reduction time of 0.5-9.4 days. CONCLUSIONS: Escherichia coli O157:H7 failed to establish and proliferate in dairy wastewater microcosms equipped with or without circulating aerators. SIGNIFICANCE AND IMPACT OF THE STUDY: This study furthers our knowledge of pathogen survival in wastewater, and suggests that proper management of wastewater before its use in irrigation is essential to reduce pathogen transfer to crops.     

Functional Metagenomics of Escherichia coli O157:H7 Interactions with Spinach Indigenous Microorganisms during Biofilm Formation

The increase in foodborne outbreaks worldwide attributed to fresh fruit and vegetables suggests that produce may serve as an ecological niche for enteric pathogens. Here we examined the interaction of E. coli O157:H7 (EcO157) with spinach leaf indigenous microorganisms during co-colonization and establishment of a mixed biofilm on a stainless steel surface. Stainless steel surface was selected to mimic the surface of produce-processing equipment, where retention of foodborne pathogens such as EcO157 could serve as a potential source for transmission. We observed a positive effect of spinach-associated microbes on the initial attachment of EcO157, but an antagonistic effect on the EcO157 population at the later stage of biofilm formation. Metagenomic analyses of the biofilm community with the GeoChip revealed an extremely diverse community (gene richness, 23409; Shannon-Weiner index H, 9.55). Presence of EcO157 in the mixed biofilm resulted in a significant decrease in the community α-diversity (t test, P<0.05), indicating a putative competition between the pathogen and indigenous spinach microbes. The decrease in the β-diversity of the EcO157-inoculated biofilm at 48 h (ANOVA, P<0.05) suggested a convergent shift in functional composition in response to EcO157 invasion. The success of EcO157 in the mixed biofilm is likely associated with its metabolic potential in utilizing spinach nutrients: the generation time of EcO157 in spinach lysates at 28°C is ～ 38 min, which is comparable to that in rich broth. The significant decrease in the abundance of many genes involved in carbon, nitrogen, and phosphorus cycling in the EcO157-inoculated biofilms (t test, P<0.05) further support our conclusion that competition for essential macronutrients is likely the primary interaction between the EcO157 and indigenous spinach-biofilm species.     

Effect of dichromate on population and growth of various protozoa isolated from industrial effluents

Three protozoa belonging to genera Euglena, Vorticella and Stylonychia collected from industrial wastes were cultured in a medium containing inorganic salts, basically meant for the growth of algae. Protozoa showed rapid growth in the medium. Hexavalent chromium (K2Cr2O7) at a concentration of 5 micrograms/L in the medium adversely affected the growth of protozoa. At the end of eight days of Cr administration, the population of Euglena, Vorticella and Stylonychia increased 8-, 4.5- and 10-fold, respectively, as against 30-, 6.75- and 50-fold increase in the control cultures. No apparent death phase and no change in activity or morphology of protozoa was observed at this Cr concentration. The protozoa were also exposed to different metal ions, viz. Pb (2.42 mmol/L), Cr (0.48 mmol/L), Cd (0.36 mmol/L), administered in the culture medium for a period of 2 years. The metal tolerance for S. mytilus and V. microstoma was Pb > Cr > Cd. E. proxima could not tolerate any of the long-term metal treatments. Because of the ability of these protozoa to tolerate high concentrations of heavy metals, their potential role in remediation of heavy metals from industrial wastewater is considered.     

Studies on the survival of enterohemorrhagic and environmental Escherichia coli strains in wastewater and in activated sludges from dairy sewage treatment plants

Survival of Escherichia coli O157:H7 strain isolated from milk in Poland and an environmental E. coli strain in wastewater from Garwolin and ?owicz dairies and in activated sludges from dairy sewage treatment plants as well as in dairy wastewater with activated sludges was examined. Environmental materials were contaminated with about 10(8) of target bacteria/ml of sample. The experiments were performed under temperature conditions typical of autumn-winter (6 degrees) and spring-summer (24 degrees C) seasons. It was found that the non-pathogenic E. coli strain survived longer in all media than the enterohemorrhagic serotype. E. coli O157:H7 bacteria were not detected (in direct plating method) in activated sludges after 21-28 days; in dairy wastewater as well as in wastewater with activated sludges after 21-25 days. These periods for environmental E. coli strain were 35-42 days (activated sludges), 25-28 days (wastewater with activated sludges). At higher temperature environmental E. coli were not detected in wastewater from ?owicz dairy sewage treatment plant after 25 days, but the bacteria were still present in wastewater from Garwolin dairy sewage tratment plant after 34 days. The obtained results show that the lack of environmental E. coli bacteria (as a indicator bacteria of fecal contamination) in dairy wastewater and in dairy wastewater with activated sludges could indicate the absence of pathogenic E. coli bacteria. Prolonged existence of the enterohemorrhagic serotype in activated sludges shows the need to treat activated sludges prior to the utilization of these materials as fertilizer.     

Incidence and tracking of Escherichia coli O157:H7 in a major produce production region in California

Fresh vegetables have become associated with outbreaks caused by Escherichia coli O157:H7 (EcO157). Between 1995-2006, 22 produce outbreaks were documented in the United States, with nearly half traced to lettuce or spinach grown in California. Outbreaks between 2002 and 2006 induced investigations of possible sources of pre-harvest contamination on implicated farms in the Salinas and San Juan valleys of California, and a survey of the Salinas watershed. EcO157 was isolated at least once from 15 of 22 different watershed sites over a 19 month period. The incidence of EcO157 increased significantly when heavy rain caused an increased flow rate in the rivers. Approximately 1000 EcO157 isolates obtained from cultures of>100 individual samples were typed using Multi-Locus Variable-number-tandem-repeat Analysis (MLVA) to assist in identifying potential fate and transport of EcO157 in this region. A subset of these environmental isolates were typed by Pulse Field Gel Electrophoresis (PFGE) in order to make comparisons with human clinical isolates associated with outbreak and sporadic illness. Recurrence of identical and closely related EcO157 strains from specific locations in the Salinas and San Juan valleys suggests that transport of the pathogen is usually restricted. In a preliminary study, EcO157 was detected in water at multiple locations in a low-flow creek only within 135 meters of a point source. However, possible transport up to 32 km was detected during periods of higher water flow associated with flooding. During the 2006 baby spinach outbreak investigation, transport was also detected where water was unlikely to be involved. These results indicate that contamination of the environment is a dynamic process involving multiple sources and methods of transport. Intensive studies of the sources, incidence, fate and transport of EcO157 near produce production are required to determine the mechanisms of pre-harvest contamination and potential risks for human illness.     

Interactions between food-borne pathogens and protozoa isolated from lettuce and spinach

The survival of Salmonella enterica was recently shown to increase when the bacteria were sequestered in expelled food vacuoles (vesicles) of Tetrahymena. Because fresh produce is increasingly linked to outbreaks of enteric illness, the present investigation aimed to determine the prevalence of protozoa on spinach and lettuce and to examine their interactions with S. enterica, Escherichia coli O157:H7, and Listeria monocytogenes. Glaucoma sp., Colpoda steinii, and Acanthamoeba palestinensis were cultured from store-bought spinach and lettuce and used in our study. A strain of Tetrahymena pyriformis previously isolated from spinach and a soil-borne Tetrahymena sp. were also used. Washed protozoa were allowed to graze on green fluorescent protein- or red fluorescent protein-labeled enteric pathogens. Significant differences in interactions among the various protist-enteric pathogen combinations were observed. Vesicles were produced by Glaucoma with all of the bacterial strains, although L. monocytogenes resulted in the smallest number per ciliate. Vesicle production was observed also during grazing of Tetrahymena on E. coli O157:H7 and S. enterica but not during grazing on L. monocytogenes, in vitro and on leaves. All vesicles contained intact fluorescing bacteria. In contrast, C. steinii and the amoeba did not produce vesicles from any of the enteric pathogens, nor were pathogens trapped within their cysts. Studies of the fate of E. coli O157:H7 in expelled vesicles revealed that by 4 h after addition of spinach extract, the bacteria multiplied and escaped the vesicles. The presence of protozoa on leafy vegetables and their sequestration of enteric bacteria in vesicles indicate that they may play an important role in the ecology of human pathogens on produce.     

Removal of Escherichia coli in wastewater by activated sludge.

Removal of bacteria from wastewater treated with activated sludge was studied by the use of a streptomycin-resistant Escherichia coli strain. The removal appeared to be a biphasic process. A rapid sorption of bacteria to the sludge flocs took place in the first hour after seeding mixed liquor with E. coli. Thereafter, slower elimination of E. coli was observed. The latter process was due to predation on E. coli by ciliated protozoa. This was shown by: (i) appearance of fluorescent food vacuoles of ciliates when fluorescent E. coli cells were added to mixed liquor; (ii) inhibition of predation either in the presence of cycloheximide or under anaerobic conditions; and (iii) absence of predation in bulking and washed sludge.     

Removal of Escherichia coli in wastewater by activated sludge.

Removal of bacteria from wastewater treated with activated sludge was studied by the use of a streptomycin-resistant Escherichia coli strain. The removal appeared to be a biphasic process. A rapid sorption of bacteria to the sludge flocs took place in the first hour after seeding mixed liquor with E. coli. Thereafter, slower elimination of E. coli was observed. The latter process was due to predation on E. coli by ciliated protozoa. This was shown by: (i) appearance of fluorescent food vacuoles of ciliates when fluorescent E. coli cells were added to mixed liquor; (ii) inhibition of predation either in the presence of cycloheximide or under anaerobic conditions; and (iii) absence of predation in bulking and washed sludge.     

Fluctuations in Populations of 3 Vorticella Species from an Activated-Sludge Sewage Plant

SYNOPSIS. To evaluate Vorticella populations in an activated sludge sewage-disposal plant as a ) indicators and b ) contributors to purification, population behavior in the plant was compared with behavior in clonaI cultures maintained with mixed sludge microflora. In a 12-month survey of protozoa in the plant, of 37 spp recorded only 9—all ciliates—were indigenous. Ciliate populations fluctuated cyclically. Alternations in dominance of Vorticella convallaria (L.) with V. octava (Stokes) + V. aequitata (Kahl) apparently followed changes in bacterial activity as indicated by changes in reduction of biochemical oxygen demand and suspended solids of the wastes. This correlation of Vorticella and bacterial activities was also investigated with clonal populations of the vorticellas and their accompanying mixed microflora—the bacteria being isolated together with the vorticellas from sludge samples. The mixed microflora which consistently supported healthy vorticellas was predominantly proteolytic. Varying periods of dominance in Vorticella species thus indicated activity of their supporting microflora. Log-phase bacteria activity stimulated V. octava ; stationary and declining phases stimulated V. convallaria. Thus, V. octava appeared predatory and V. convallaria appeared to require bacterial conditioning of the medium. Bacterial activity was stimulated by V. octava predation. Vorticella activity may thus promote purification.     

Comparative genomics of enterohemorrhagic Escherichia coli O145:H28 demonstrates a common evolutionary lineage with Escherichia coli O157:H7

Background

Although serotype O157:H7 is the predominant enterohemorrhagic Escherichia coli (EHEC), outbreaks of non-O157 EHEC that cause severe foodborne illness, including hemolytic uremic syndrome have increased worldwide. In fact, non-O157 serotypes are now estimated to cause over half of all the Shiga toxin-producing Escherichia coli (STEC) cases, and outbreaks of non-O157 EHEC infections are frequently associated with serotypes O26, O45, O103, O111, O121, and O145. Currently, there are no complete genomes for O145 in public databases.

Results

We determined the complete genome sequences of two O145 strains (EcO145), one linked to a US lettuce-associated outbreak (RM13514) and one to a Belgium ice-cream-associated outbreak (RM13516). Both strains contain one chromosome and two large plasmids, with genome sizes of 5,737,294 bp for RM13514 and 5,559,008 bp for RM13516. Comparative analysis of the two EcO145 genomes revealed a large core (5,173 genes) and a considerable amount of strain-specific genes. Additionally, the two EcO145 genomes display distinct chromosomal architecture, virulence gene profile, phylogenetic origin of Stx2a prophage, and methylation profile (methylome). Comparative analysis of EcO145 genomes to other completely sequenced STEC and other E. coli and Shigella genomes revealed that, unlike any other known non-O157 EHEC strain, EcO145 ascended from a common lineage with EcO157/EcO55. This evolutionary relationship was further supported by the pangenome analysis of the 10 EHEC str ains. Of the 4,192 EHEC core genes, EcO145 shares more genes with EcO157 than with the any other non-O157 EHEC strains.

Conclusions

Our data provide evidence that EcO145 and EcO157 evolved from a common lineage, but ultimately each serotype evolves via a lineage-independent nature to EHEC by acquisition of the core set of EHEC virulence factors, including the genes encoding Shiga toxin and the large virulence plasmid. The large variation between the two EcO145 genomes suggests a distinctive evolutionary path between the two outbreak strains. The distinct methylome between the two EcO145 strains is likely due to the presence of a BsuBI/PstI methyltransferase gene cassette in the Stx2a prophage of the strain RM13514, suggesting a role of horizontal gene transfer-mediated epigenetic alteration in the evolution of individual EHEC strains.     

Discharge of disinfected wastewater in recipient aquatic systems: fate of allochthonous bacterial and autochthonous protozoa populations

The discharge of disinfected effluents affects the bacterivorous ability of protozoa and the effect depends on the disinfectant applied. Chlorine provokes a decrease in the number of protozoa and a delay in the bacterivorous ability. The discharge of ozonated and peracetic acid-treated wastewater provokes only an initial slight decrease in bacterivorous ability. No correlation was found between toxicity values detected using the Microtox^™ assay and the effect of disinfected effluents on freshwater protozoa populations. After the disinfection processes, recipient systems (fresh and marine water) have different effects on the survival of Escherichia coli populations discharged to them. The effect of the freshwater recipient system is less negative than the effect provoked by sea-water, and the differences detected depend on the disinfection treatment applied. The wastewater bacterial population as a whole is able to grow after discharge of disinfected wastewater to receiving waters. However, in the absence of predation or competition, the recipient systems exert selection, with rod-shaped bacteria predominating.     

Real-time PCR for the detection of Escherichia coli O157:H7 in dairy and cattle wastewater

AIMS: Developing and evaluating a rapid real-time polymerase chain reaction (PCR) method for the identification of Escherichia coli O157:H7 in cattle and dairy wastewater samples produced from mozzarella cheese factories, without pre-enrichment step before DNA extraction. METHODS AND RESULTS: Wastewater samples were collected from a dairy farm producing mozzarella cheese and located in Puglia (south of Italy). Plate count and other microbial assays were performed 1 h after sampling. Wastewater samples were artificially inoculated with 10(4), 10(7) and 10(8) cells ml(-1) of E. coli O157:H7, strain EDL933. PCR protocols for stx1, stx2 and eae genes were first tested on pure DNA extracted from type strains, in order to optimize the amplification conditions and reagent concentration before real-time PCR experiments. Three specific fragments of ca 106, 150 and 200 bp corresponding to genes eae, stx1 and stx2, respectively, were obtained. Real-time PCR experiments were performed with DNA extracted from dairy and manure wastewater samples inoculated with 10(4), 10(7) and 10(8) colony-forming units (CFU) ml(-1) of E. coli O157:H7 strain EDL 933. The sensitivity limit of the assay was 10(-1) pg microl(-1) for eae, stx2 and 16SrRNA, and 1 pg microl(-1) for stx1 gene respectively. CONCLUSIONS: A real-time PCR protocol has been developed and used in order to identify potential pathogens in dairy wastewater, in which previous methods (including standard PCR) failed to work. SIGNIFICANCE AND IMPACT OF THE STUDY: Cattle and dairy wastewater samples produced from mozzarella cheese factories may harbour verocytotoxin-producing E. coli. The availability of rapid and sensitive molecular methods may be useful to monitor the persistence of verocytotoxin-producing E. coli in general and to assess the effectiveness of wastewater treatments.     

Role of Pore Size Location in Determining Bacterial Activity during Predation by Protozoa in Soil

The predation of a luminescence-marked strain of Pseudomonas fluorescens by the soil ciliate Colpoda steinii was studied in soil microcosms. Bacterial cells were introduced in either small (neck diameter, <6 (mu)m) or intermediate-sized (neck diameter, 6 to 30 (mu)m) pores in the soil by inoculation at appropriate matric potentials, and ciliates were introduced into large pores (neck diameter, 30 to 60 (mu)m). Viable cell concentrations of bacteria introduced into intermediate-sized pores decreased at a greater rate than those in small pores, with reductions in bacterial populations being accompanied by an increase in viable cell numbers of the ciliate. The data indicate that the location of bacteria in small pores provides significant protection from predation. In the absence of C. steinii, the level of metabolic activity of the bacterial population, measured by luminometry, decreased at a greater rate than cell number, and the level of luminescence cell(sup-1) consequently decreased. The decrease in levels of luminescence indicates a loss of activity due to starvation. During predation by C. steinii, the level of the activity of cells introduced into small pores fell in a similar manner. The level of cell activity was, however, significantly greater for cells introduced into intermediate-sized pores, despite their greater susceptibility to predation. The data suggest that increased activity arises from a release of nutrients by the predator and the greater accessibility of bacteria to nutrients in larger pores. Nutrient amendment of microcosms resulted in increases in bacterial populations to sustained, higher levels, while levels of luminescence increased transiently. The predation of cells introduced into intermediate-sized pores was greater, and there was also evidence that the level of activity of surviving bacteria was greater for bacteria in intermediate-sized but not small pores.     

Rapid effect of an elicitor on uptake and intracellular distribution of phosphate in cultured parsley cells

Cell suspension cultures of parsley (Petroselinum hortense) grown in synthetic medium take up most of the inorganic phosphate supplied with the medium within the initial 5 days after transfer. Nuclear magnetic resonance spectra of intact parsley cells from this growth stage revealed that approximately half of the phosphate was located within the vacuoles, whereas after 7 days of growth phosphate content of the vacuoles was relatively low. At both times, addition of an elicitor preparation from Alternaria carthami, which is not toxic to the cells, led to a temporary increase of vacuolar phosphate at the expense of cytoplasmic phosphate, even when excess phosphate was added to the medium. The rapid decrease of cytoplasmic phosphate might play a role in the redirection of phenylpropanoid metabolism reported for elicitor-treated parsley cells.     

Induction of purple sulfur bacterial growth in dairy wastewater lagoons by circulation

Aims:  To determine whether circulation of dairy wastewater induces the growth of phototrophic purple sulfur bacteria (PSB).
Methods and Results:  Two dairy wastewater lagoons that were similar in size, geographic location, number and type of cattle loading the lagoons were chosen. The only obvious visual difference between them was that one was stagnant and the water was brown in colour (Farm 1), and the other was circulated and the water was red in colour because of the presence of PSB that contained carotenoid pigments (Farm 2). Both wastewaters were sampled monthly for 3 months and assayed for PSB and extractable carotenoid pigments (ECP). After this point, circulators were placed in the wastewater lagoon on Farm 1, and samples were taken monthly for 9 months and assayed for PSB and ECP. Before the installation of circulators, no PSB-like 16S rRNA sequences or ECP were observed in the wastewater from Farm 1; however, both were observed in the wastewater from Farm 2. After the installation of circulators, statistically greater levels of PSB and extractable carotenoid pigments were observed in the wastewater from Farm 1.
Conclusions:  Circulation enhances the growth of PSB in dairy wastewater.
Significance and Impact of this Study:  Because PSB utilize H₂S and volatile organic acids (VOA) as an electron source for photosynthesis, and VOA and alcohols as a carbon source for growth, the increase in these bacteria should reduce H₂S, volatile organic compounds and alcohol emissions from the lagoons, enhancing the air quality in dairy farming areas.