Impact of Biostimulation and Bioaugmentation on Microbial Communities in Woodchip Bioreactors

Abstract

Woodchip bioreactors are used to remove nutrients from the subsurface drainage.However, the nitrogen removal performance and efficiency of woodchips are limited by low temperature and limited availability of liable carbon. Bioaugmentation (i.e., adding cold-adapted denitrifying microbes) and biostimulation (i.e., adding substrates to enhance the activities of indigenous microbial populations) are potential approaches to enhance nitrate removal of woodchip bioreactors at cold conditions, but their effectiveness is still unclear. My thesis research was done to clarify the effects of bioaugmentation and biostimulation on the microbial communities in woodchip bioreactors. I also examined how microbial community change influenced the overall nitrate removal. Previously isolated cold-adapted denitrifying bacteria Cellulomonas sp. strain WB94 and Microvirgula sp. strain BE2.4 were used in this study. These strains were obtained from woodchip bioreactors and were verified to reduce nitrate at cold temperatures. Based on the whole genome analysis, Microvirgula sp. strain BE2.4 has a complete set of denitrification genes, while Cellulomonas sp. strain WB94 has genes for cellulose degradation. These two strains were grown in the lab condition and inoculated back into the woodchip bioreactors located at Willmar, MN (i.e., bioaugmentation). In addition, acetate was added into the woodchip bioreactors as a liable carbon source to promote denitrification (i.e., biostimulation). Woodchip samples were taken before and after the bioaugmentation and biostimulation treatments to examine the changes in the abundance of inoculated strains and the impact of the two treatments on the microbial communities in woodchip bioreactors. Strain-specific TaqMan probe quantitative PCR assays were designed to quantify the abundance of inoculated strains. A high-throughput Nitrogen v Cycle Evaluation (NiCE) chip was also used to quantify various N cycle-related genes in many samples. Furthermore, microbial communities in the woodchip bioreactors were analyzed by sequencing the V4 region of the 16S rRNA gene on the Illumina MiSeq platform. TaqMan probe qPCR results showed that the abundance of inoculant Cellulomonas sp. strain WB94 increased in all the treatment and control bioreactors throughout the sampling period (p < 0.05), suggesting that genera Cellulomonas plays a key role in the cellulose degradation process inside aged woodchip bioreactors. The abundance of inoculant Microvirgula sp. strain BE2.4 tended to increase in the bioaugmentation bioreactors, although this increase was not statistically significant, most likely due to considerable variation between samples. 16S rRNA gene amplicon sequencing results showed that the treatments account for 4.07% of the difference in the microbial community between samples. NiCE chip results showed that the abundance of amoA, hao/hdh, and nosZ can be used to predict the nitrate removal rate of woodchip bioreactors. In addition, there was a positive relationship between the abundance of inoculant Microvirgula sp. strain BE2.4 and the abundance of denitrification gene norB and nosZ in the woodchip bioreactors. These results showed that bioaugmentation and biostimulation treatments could change the microbial communities in the woodchip bioreactors and increase the abundance of denitrifiers to optimize the denitrification efficiency of the bioreactors.