Wastewater microbiology in Nannochloropsis salina algal biofuel production systems

The recent use of wastewater as a medium to grow biofuel microalgae on a large scale presents challenges and opportunities. The fate of the complex microbial consortia that wastewater might harbor is a significant question that need to be addressed. Any algal-bacterial interactions should be evaluated for presumed health risks associated with fecal wastewater microbes, including putative pathogens, enteric viruses, and for issues of nutrient use efficiency as algae and bacteria compete for the same resources. The goal of this dissertation is to investigate the interactions of wastewater microbes and biofuel algae.;Chapter 1 reviews the known information on the interactions between wastewater and microbes, both free-living and algae associated. The alga Nannochloropsis sauna which is a good candidate for biofuel production has been grown in chlorinated and advanced secondary treated wastewater in a large scale outdoor Solix BioSystems Lumian AGS4000 photo-bioreactor panels (PBR). Bacterial diversity in these systems was documented by 454 pyrosequencing of 16s rDNA amplicons and subsequent metagenomic analysis (Chapter 2). This research evaluated bacterial diversity in the presence of algae in wastewater and in standard f12 algal growth media. The work reveals that algae-bacteria interactions affect bacterial diversity and it controls exclusion or dominance of particular bacterial populations, including potential pathogens.;Enteric viruses and the associated risk assessment for algal biofuel systems reusing wastewaters was monitored. MS2 bacteriophage often used as a surrogate indicator for the transport, disinfection and fate of human enteric viruses, was spiked into algal-wastewater cultures. This research addresses the role algae might play in partitioning the virus between surface and solution fates, and addresses MS2 virus survival in wastewater, standard f/2 media or phosphate buffered saline (Chapter 3). MS2 viability rate was assessed by the culture based plaque assay methods and MS2 RNA was detected using culture independent assays via quantitative PCR (qRT PCR). Results indicate that there is no specific attachment of virus to algal cells. Recovery rates of viable virus in the supernatant are affected by algal age. The presence of algae and wastewater organics marginally enhance viral viability.;During the experimental work with viruses methodological concerns were raised. The persistence of RNA genomic fragments of non-viable MS2 bacteriophage in autoclave sterilized wastewater led to the concern of false positive signals in qRT PCR based protocols. The occurrence of false positives in disinfected wastewater samples may lead to the misinterpretations of the health hazards of the samples. The goal of the research was to firstly identify this problem by determining the efficiency of autoclaving, and secondly to evaluate protocols that may minimize false positives. For this 16 distinct primer sets were used to amplify MS2 virus that had undergone single to multiple autoclave events (Chapter 4). Detection and quantification was done by qRT PCR, melting curve analysis, gel electrophoresis, cloning and subsequent sequencing. This work revealed the importance of the amplicon length, melting temperature on the occurrence of false positives and also showed that a single autoclaving is not effective in removing small genomic fragments. An enzymatic pre- tea ment of wastewater samples to avoid false positives were also investigated as a possible resolution to this problem (Chapter 5). The enzymes RNase A and Proteinase K were used in different concentrations and combinations to find the most effective treatment to degrade the inactivated viruses before nucleic acid extraction and qRT PCR. It was revealed that Rnase A at 5ng microL -1 was most effective in removing the false positives though it was unable to digest the RNA completely as shown by the gel electrophoresis. This can lead to the conclusion that there are high chances for the occurrence of false positives in qRT PCR protocols and while enzyme pre-treatment will drastically reduce these signals they are unable to eliminate them completely. Overall, the results of this research indicate that the risks associated with wastewater bacterial and viral loads are diminished in biofuel algal production system.