## 4 Discussion  
Our results confirm the hypothesis that engineered _Methanosarcina acetivorans_ can survive in municipal wastewater and produce isoprene at detectable levels. Methane production was greater in wastewater biosolids which had undergone anaerobic digestion compared with those samples grown in preanaerobic digestion biosolids. While there is an incidental enrichment process during fermentation over time, the majority of microbes found in municipal wastewater are known bacterial gut symbionts such as _Proteobacteria_, _Firmicutes_, and _Bacteroides_[28], as well as a diverse collection of methanogens including _Methanomicrobiales_, _Methanosarcinaules_, _Methanobacteriales_, and _Methanobrevbincter_ spp. [29; 30; 31]. It has been well documented that these organisms exchange nutrients via syntrophy both in the gut and during wastewater treatment [32; 33; 34; 35] and methanogen growth is dependent on metabolic byproducts of upstream microbial metabolism. The increase in methane production observed in samples grown in wastewater postanaerobic digestate suggests the engineered _M. acetivorans ispS+_ strains may be capable of participating in syntrophic relationships with other microbes in the digestate. Figure 4: Methane and isoprene production from waste biosolids. Endpoint methane assays after _M. acetivorans_ strains were added to unsterilized wastewater biosolids digestate or synthetic wastewater (SWW) amended with methanol. (**a**) Methane production by isoprene-producing _M. acetivorans_ (_att:ispS_) and vector only control (_att:_VOC) strains added to predigest effluent, postdigest effluent, or SWW with (w/MeOH) and without (w/o MeOH) added methanol. (**b**) Isoprene production by _M. acetivorans_ _att:_VOC and _att:ispS_ strains added to SWW or postdigest effluent amended with methanol and postdigest effluent without added substrates. Blue, _att:_VOC strain in which empty vector pNB730 is integrated onto the chromosome. Red, _att:ispS_ strain in which plasmid pJA2 expressing isoprene synthase is integrated onto the chromosome. All data were obtained from triplicate biological and technical replicates (_n_ = 9). After four days of incubation at 35 \({}^{\circ}\)C methane was detected though no isoprene production was identified in samples grown in preanaerobic digested effluent, indicating that there may not be enough freely available methanogenic substrates for the engineered strains to compete with the existing microbial population. However, the postdigester effluent inoculated with our isoprene-producing _M. accitovans_ incubated under the same culture conditions yielded detectable bioisoprene (0.144 \(\pm\) 0.273 mM). When this postdigest effluent was supplemented with 125 mM of MeOH the yield was increased to 0.968 \(\pm\) 0.144 mM. This indicates that isoprene production from wastewater is primarily determined by the available substrate rather than environmental stressors. These results demonstrate that engineered methanogens are viable as a drop-in additive to wastewater treatment, and that the rate limiting factor for isoprene production is the rate at which the syntrophic microbial community can produce metabolites necessary for methanogen growth. By stimulating the microbial community, higher titers of bioisoprene production may be achieved. Methanogens are a compelling source of renewable bioproducts due to their high substrate to product ratio efficiency. Here, we have demonstrated that with minor 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. Due to existing capabilities for methane capture, many wastewater treatment facilities are already equipped with the infrastructure necessary for the capture of gaseous products such as isoprene. Separation of isoprene from biogas would require additional investment in biogas refining.