Microbial diversity associated with single cell protein produced from a brewery wastewater pilot plant

Abstract

Background: Brewery wastewater typically contains large untapped amounts of useful dissolved carbon measured as Biological Oxygen Demand (BOD) that can be utilized for protein production for fish feed in the form of Single Cell Protein (SCP). Protein is produced from the growth of bacteria as it consumes carbon and is harvested and dried into a fishmeal replacement. Here we present results of a 1-year biodiversity monitoring study of a brewery wastewater treatment pilot plant tuned to produce a dried bacterial Single Cell Protein (SCP) fishfeed replacement product. The plant consistently produced 55-60% (w/w) crude protein SCP at about 15 kg/day. The key to this consistency was the addition of micronutrients to the wastewater during aerobic growth, but the exact microbial response to this addition was not well understood. Materials and Methods: An initial survey of the brewery wastewater operations was conducted over the year 2008 using Sanger sequencing of the 16S SSU rRNA gene with the 8F/1492R primer. Samples were taken from throughout the brewery treatment works and pilot plant to establish a time course. Next, A 454 FLX pyrosequencing run was also completed using normalized DNA from the same samples as above with the bacterial 27F/338R primers and sample barcoding. Pyrotags were processed and clustered into Operational Taxonomic Units (OTUs) by the MOTHUR bioinformatics package. Results: Ribosomal Database Project classifications of Sanger data showed that while the order level diversity was relatively simple, the consortia varied considerably both in time and in location. Pyrotag data (55,000 sequences) was characterized by a high degree of singleton OTUs. No single sequence comprised more than 2% of all sequences and no two samples (in either time or space) contained more than 10% OTU similarity. Phylum-level pyrotag diversity of the pilot production tank revealed dominance by Bacteroidetes followed by Firmicutes and beta/gamma-Proteobacteria. Fast UniFrac results show that SCP product and pilot plant environments sometimes clustered together, and that some temporal clustering also occurs. More significantly, Fast UniFrac results show that each segment of the treatment works was highly selective. In order to understand where variations in Fast UniFrac data exist, a taxonomic rank abundance plot was made which details distributions of sequences within various phylum. Results show that major contributors to community structure lie in Firmicutes and Beta-proteobacteria, but the majority of dominant organisms come from Bacteroidetes, particularly from genus Prevotella, a group of carbohydrate metabolizing anaerobes commonly associated with tooth decay. Conclusion: These results indicated that the bulk of diversity in the pilot plant were low count species and that high turnover led to considerable shifts in diversity within several major phyla, particularly from phylum Bacteroidetes, though overall protein concentration of the system remained consistent for the production of SCP. These results indicate that minute changes in reactor conditions commonly seen in day-to-day operations at any treatment plant can cause wide fluctuations in reactor diversity without impacting process stability.