###### Abstract  
Wastewater biosolids are a promising feedstock for production of value-added renewable chemicals. Methane-producing archaea (methanogens) are already used to produce renewable biogas via the anaerobic treatment of wastewater. The ability of methanogens to efficiently convert dissolved organic carbon into methane makes them an appealing potential platform for biofenning using metabolic engineering. We have engineered a strain of the methanogen _Methanosarcina acetivorans_ to produce the volatile hemiterpene isoprene in addition to methane. The engineered strain was adapted to grow in municipal wastewater through cultivation in a synthetic wastewater medium. When introduced to municipal wastewater the engineered methanogens were able to compete with the indigenous microorganisms and produce 0.97 mM of isoprene (65.9 \(\pm\) 21.3 g per m\({}^{3}\) of effluent). The production of isoprene in wastewater appears to be dependent on the quantity of available methanogenic substrate produced during upstream digestion by heterotrophic fermenters. This shows that with minimal adaptation it is possible to drop-in engineered methanogens to existing wastewater environments and attain value-added products in addition to the processing of wastewater. This shows the potential for utilizing methanogens as a platform for low-cost production of renewable materials without expensive feedstocks or the need to build or adapt existing facilities. biorefining; isoprene; methanogen; archaea; _Methanosarcina acetivorans_; synthetic biology +
Footnote †: journal: Medical  
acids. These one- and two-carbon compounds are utilized by methanogens to complete the decomposition process [6,11]. In addition to removing polluting organic carbon from the water, anaerobic digestion has the added benefit of producing methane which is often captured as renewable biogas [11, 12, 13, 14]. Due to the low energetic potential of methanogenic feedstocks, methanogens utilize a highly efficient central metabolism which greatly favors the production of methane over biomass and heat. Anaerobic treatment of wastewater results in 95% conversion of the initial substrate into available biogas with 5% being utilized for microbial biomass [15,16]. We hypothesized that the highly efficient metabolism of methanogens may have potential to produce high yields of other value-added products in addition to methane. Isoprene (2-methyl-1,3-butadiene) is the primary component of natural rubber and an important chemical precursor utilized in the production of synthetic rubber as well as adhesives, flavorings, cosmetics, and pharmaceuticals. Traditionally, isoprene is harvested from natural rubber from tree sap or produced industrially through the thermal cracking of petroleum. By producing renewable isoprene via engineered microbes, it could be possible to reduce the need to rely on the harvesting of plant biomass or the mining of fossil fuels. Recently our laboratory demonstrated that the methanogen _Methanosarcina acettivorans_ can be engineered to efficiently produce bioisoprene as a methane coproduct under laboratory conditions [9]. The gene for isoprene synthase, _ispS_, was stably inserted into the chromosome of _M. acettivorans_ (_att::ispS_) and the production of isoprene as well as methane was confirmed via gas chromatography. The production of isoprene showed no detrimental effect on growth rate or metabolic efficiency of the engineered strains compared with a vector-only control. We surmised that without an obvious decrease in fitness it may be possible to drop-in these engineered methanogens into an existing anaerobic wastewater treatment consortium to produce bioisoprene. However, any inoculated methanogens would have to compete for substrate with wild methanogens in the mixed microbial community of the anaerobic digester. _M. acettivorans_ has the largest genome of any characterized methanogen as well as the widest range of substrate utilization, allowing for growth from methanol, methyl-amines, carbon monoxide, and acetate [17,18].