24Remediation of industrial wastewater is important for preventing environmental 25 contamination and enabling water reuse. Biological treatment for one industrial 26 contaminant, thiocyanate (SCN -), relies upon microbial hydrolysis, but this process is 27 sensitive to high loadings. To examine the activity and stability of a microbial community 28 over increasing SCN -loadings, we established and operated a continuous-flow bioreactor 29 fed increasing loadings of SCN -. A second reactor was fed ammonium sulfate to mimic 30 breakdown products of SCN -. Biomass was sampled from both reactors for 31 metagenomics and metaproteomics, yielding a set of genomes for 144 bacteria and one 32 rotifer that constituted the abundant community in both reactors. We analyzed the 33 metabolic potential and temporal dynamics of these organisms across the increasing 34 loadings. Microbial communities in biotechnology have historically been treated as black boxes, 46 but as molecular methods have improved, our knowledge of these systems has deepened. 47Increasingly, 'meta-omics' methods are being used to investigate critical processes and 48 potential weak points in biotechnology, such as nitrogen and phosphorus removal or 49 bulking in wastewater treatment. [1][2][3] In particular, specialized treatment of industrial 50 wastewater has benefited from a genome-resolved meta-omics approach 4-7 using high-51 throughput sequencing of community genomic DNA (metagenomics) or RNA 52 (metatranscriptomics), or spectral characterization of proteins (metaproteomics). These 53 data can be used to identify the key species involved in processes of interest. Improved 54 understanding of the activities and abundances of these organisms under varying 55 conditions could inform design and operation of these systems. (Figure 1) and small amounts of 5 N KOH were added directly to reactors if 100 observed pH was ≤ 6.5. Bicarbonate (4 g/L) was added to the feed to buffer the system 101 from day 112 to day 136. 102The reactors were run in batch-fed mode until SCN -degradation was stably 103 observed in the SCN -reactor, at which time both reactors were switched to continuous 104 feeding at a residence time of 42 hours (day 5). The hydraulic retention time (HRT) of 105 both reactors was lowered from 42 hours to 12 hours (days 5-68) and then maintained at 106 12 hours while the feed concentration of SCN -or equivalent NH 4 (SO 4 ) 1/2 was increased 107 stepwise. The reactor was allowed to stabilize between each step to reach steady state 108 ( Figure S1). 109 110Sampling: Samples of biomass from each reactor were taken for metagenomic 111 sequencing just before increases in feed concentration (Figure 1 and Table S1). 112Approximately 0.5 g (wet-weight) of biofilm was scraped from the wall of each reactor 113 with sterile spatula and stored at -60 C. Paired samples of planktonic biomass were 114

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Thiocyanate (SCN−) is a toxic compound that forms when cyanide (CN−), used to recover gold, reacts with sulfur species. SCN−‐degrading microbial communities have been studied, using bioreactors fed synthetic wastewater. The inclusion of suspended solids in the form of mineral tailings, during the development of the acclimatized microbial consortium, led to the selection of an active planktonic microbial community. Preliminary analysis of the community composition revealed reduced microbial diversity relative to the laboratory‐based reactors operated without suspended solids. Despite minor upsets during the acclimation period, the SCN− degradation performance was largely unchanged under stable operating conditions. Here, we characterized the microbial community in the SCN− degrading bioreactor that included solid particulate tailings and determined how it differed from the biofilm‐based communities in solids‐free reactor systems inoculated from the same source. Genome‐based analysis revealed that the presence of solids decreased microbial diversity, selected for different strains, suppressed growth of thiobacilli inferred to be primarily responsible for SCN− degradation, and promoted growth of Trupera, an organism not detected in the reactors without solids. In the solids reactor community, heterotrophy and aerobic respiration represent the dominant metabolisms. Many organisms have genes for denitrification and sulfur oxidation, but only one Thiobacillus sp. in the solids reactor has SCN− degradation genes. The presence of the solids prevented floc and biofilm formation, leading to the observed reduced microbial diversity. Collectively the presence of the solids and lack of biofilm community may result in a process with reduced resilience to process perturbations, including fluctuations in the influent composition and pH. The results from this investigation have provided novel insights into the community composition of this industrially relevant community, giving potential for improved process control and operation through ongoing process monitoring.

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