Environmental algal phage isolates and their impact on production potential for food and biofuel applications.

AIMS: The United States Department of Energy is aiming to bring microalgal biofuels into commercial use by 2030 at the price of $3 per gasoline gallon equivalent. Large-scale production of biofuel faces many challenges including naturally occurring algal phages; and characterizing this threat is the aim of this study. METHODS AND RESULTS: Bench-scale experiments were performed to study the impact of viral infectivity on the production of microalgal in bioreactors. All environmental samples were tested positive for algal phages which showed various levels of infectivity against Synechocystis PCC 6803 and the environmental isolates of microalgae. The viral attachment to algal cells was observed under transmission electron microscopy (TEM) and to determine the shape and size of the viral particles. All the viruses detected were c. 50-60 nm icosahedral particles. Viral infection resulted in 48% reduction in the biomass of the infected algal culture in 22 days. CONCLUSIONS: This study has lead to the conclusion that the microalgal phages prevalent in natural environment may cause infections in broad range of microalgae used for biofuel production. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has detected and quantified the phages that can infect algal populations in natural freshwater habitats and laboratory cultures of microalgal strains. The impact of viral threat to health of commercial algal production operations has been identified in this study.

Impact of milk components on recovery of viral RNA from MS2 bacteriophage.

Noroviruses are responsible for approximately 44 % of outbreaks involving dairy products for which causative agents are reported. Recovery of viruses from milk and dairy products is a difficult task. The role of different components of milk in the recovery of viral RNA was evaluated in this study. Four model milk formulations (A-D) were prepared by mixing different combinations of lactose, whey protein, casein, and fat in water. Each model formulation was spiked with five concentrations of bacteriophage MS2. The phenol-guanidine thiocyanate-chloroform protocol was used for extracting viral RNA from the model milk formulations and then extracted RNA was measured by a nanodrop spectrophotometer in ng/µl. The results showed that casein and whey protein had the highest negative impact on RNA yield, especially when the number of MS2 was less than 1.3 pfu/ml. The highest RNA recovery was obtained from the model milk formulation containing all four components; lactose, whey protein, casein, and fat. The amount of extracted RNA was closely correlated with the dry matter content of each formulation and the spiked concentration of coliphage using response surface modeling (R²:0.93). It was determined that milk fat is the most effective component in facilitating RNA extraction and the highest RNA yield can be achieved via elimination of whey protein and casein from milk by centrifugation at 40,000×g for 60 min. To achieve the highest viral RNA recovery efficiency by the proposed method, milk fat must be recombined with the supernatant of the centrifuged sample and then homogenized before performing the extraction protocol.

Riverbank filtration: comparison of pilot scale transport with theory.

Pilot-scale column experiments were conducted in this study using natural soil and river water from Ohio river to assess the removal of microbes of size ranging over 2 orders of magnitude, i.e., viruses (0.025-0.065 microm), bacteria (1-2 microm), and Cryptosporidium parvum oocysts (4-7 microm) under conditions representing normal operation and flood scour events. Among these different organisms, the bacterial indicators were transported over the longest distances and highest concentrations; whereas much greater retention was observed for smaller (i.e., viral indicators) and larger (i.e., Cryptosporidium parvum oocysts) microbes. These results are in qualitative agreement with colloid filtration theory (CFT) which predicts the least removal for micrometer size colloids, suggesting that the respective sizes of the organisms was a dominant control on their transport despite expected differences in their surface characteristics. Increased fluid velocity coupled with decreased ionic strength (representative of major flood events) decreased colloid retention, also in qualitative agreement with CFT. The retention of organisms occurred disproportionately near the source relative to the log-linear expectations of CFT, and this was true both in the presence and absence of a colmation zone, suggesting that microbial removal by the RBF system is not necessarily vulnerable to flood scour of the colmation zone.

Assessment of the risk of infection by Cryptosporidium and Giardia in non-potable reclaimed water.

Quantitative risk assessment for Cryptosporidium oocysts and Giardia cysts was performed to determine the public health significance of non-potable use of tertiary treated reclaimed water. Seven reclaimed water treatment plants in the southwestern United States participated in this study. The average public exposure to oocysts and cysts was estimated, based on concentrations, recovery efficiency, viability and three exposure scenarios. The exponential dose-response model was chosen to determine the probability of infection from ingestion of various numbers of oocysts and cysts. The risks of infection for Giardia were approximately one or two orders of magnitude higher than those for Cryptosporidium. The combined risks of infection from oocysts and cysts at sites using a combination of chlorination and UV disinfection would meet the annual acceptable risk of 1.00E-04, whereas those at the other utilities using only chlorination indicated higher probability of infection than the 1.00E-04 resulting from accidental consumption of a small amount of non-potable reclaimed water.

Laboratory testing protocol to identify critical factors in bacterial compliance monitoring.

This research focused on providing guidelines for water utilities on the collection and handling of routine bacteriological samples and in developing scientifically-based approaches in selecting the most representative sampling locations. A laboratory-scale pilot distribution system was designed comprising two parallel loops, one using unlined cast-iron pipe and one using PVC pipe. Each loop contained six sampling ports, including (1) a distribution main dead end faucet, (2) one long (5.5 m; 18 feet) and (3) one short (0.3 m; 1 foot) household copper service line with threaded hose-bibb taps, (4) one hose-bibb with welded faucet, (5) one dedicated sampling port (modeled after a manufacturer's specifications) and (6) one laboratory-style (PVC) stop-cock sampling port. Residual chlorine concentrations were maintained at 0, 0.5, 1.5 and 2.0 mg/L stages during the course of the experiment. Bacterial samples were collected from the different sampling ports and assayed by membrane filtration and/or spread plate. Nutrient and R2A agars were used for heterotrophic plate counts (HPC), m-Endo agar for total coliform (TC) counts and Chromocult agar for injured bacterial analyses. Several methods of sample collection were tested using various combinations of flushing and tap disinfection, including "first flush" (no flushing, without tap disinfection), flushing only, tap disinfection only (using alcohol or hypochlorite solution) and flushing coupled with tap disinfection. The results indicated that the bacterial counts in samples drawn from dead ends were not significantly different from counts in samples from the other sample port configurations. First flush samples consistently produced the highest bacterial count results. Bacterial counts in samples from the long household copper service line were typically three orders of magnitude higher than in samples from the other sample ports. Thus, there is evidence that long copper household service connections may be unsuitable sample tap configurations for collecting samples intended to represent microbial quality in the distribution system.