applied sciences Communication Isoprene Production from Municipal Wastewater Biosolids by Engineered Archaeon Methanosarcina acetivorans Sean Carr, Jared Aldridge and Nicole R. Buan * Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0664, USA; [email protected] (S.C.); [email protected] (J.A.) * Correspondence: [email protected] Featured Application: Engineered Methanosarcina acetivorans can be introduced to municipal wastewater to produce renewable bioisoprene. 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 biorefining 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 ± 21.3 g per m3 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 Citation: Carr, S.; Aldridge, J.; Buan, existing wastewater environments and attain value-added products in addition to the processing of N.R. Isoprene Production from wastewater. This shows the potential for utilizing methanogens as a platform for low-cost production Municipal Wastewater Biosolids by of renewable materials without expensive feedstocks or the need to build or adapt existing facilities. Engineered Archaeon Methanosarcina acetivorans. Appl. Sci. 2021, 11, 3342. Keywords: biorefining; isoprene; methanogen; archaea; Methanosarcina acetivorans; synthetic biology https://doi.org/10.3390/app11083342 Academic Editor: Faisal I. Hai 1. Introduction Received: 13 March 2021 Methane-producing archaea (methanogens) are obligate anaerobes which inhabit a Accepted: 5 April 2021 Published: 8 April 2021 keystone niche in the global carbon cycle, utilizing the endpoint degradation products of complex organic material and liberating otherwise inaccessible carbon [1–4]. Their Publisher’s Note: MDPI stays neutral unique metabolism and their potential to utilize a wide array of plentiful substrates make with regard to jurisdictional claims in methanogens a subject of particular interest in industrial applications such as wastewater published maps and institutional affil- treatment [5–7], and the production of value-added products (Figure1)[ 8,9]. Methanogens iations. are used worldwide to reduce dissolved organic carbon in effluent as part of the wastewater treatment process. Wastewater treatment is a multistage process which is highly variant depending on the substrate being treated, though the end goal is largely the same: The detoxification of water by degrading complex biomass and pollutants before reintroducing the effluent into the water cycle. For the purpose of this study, we focused on the anaerobic Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. digestion of municipal wastewater which primarily aims to remove dissolved carbon and This article is an open access article suspended solids from a city’s water supply. distributed under the terms and Municipal wastewater treatment generally occurs in three distinct stages based upon conditions of the Creative Commons the aerobicity of the wastewater and the activity of microorganisms involved in the mul- Attribution (CC BY) license (https:// tistage process. In the first stage, aerobic microorganisms breakdown complex biomass creativecommons.org/licenses/by/ into simpler organic material [10]. The deconvoluted material is further anaerobically di- 4.0/). gested by a second consortia of microbes into one- or two-carbon compounds and organic Appl. Sci. 2021, 11, 3342. https://doi.org/10.3390/app11083342 https://www.mdpi.com/journal/applsci Appl. Sci. 2021, 11, 3342 2 of 9 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 [11–14]. 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 Appl. Sci. 2021, 11, x FOR PEER REVIEWfor microbial biomass [15,16]. We hypothesized that the highly efficient metabolism of 2 of 9 methanogens may have potential to produce high yields of other value-added products in addition to methane. FigureFigure 1. 1.Schematic Schematic representation representation of anaerobic of anaerobic digestion digest of wasteion biomass of waste at thebiomass Theresa at Street the Theresa Street WaterWater Resource Resource Recovery Recovery facility facility in Lincoln, in Lincoln, NE. After NE. aerobic After incubation, aerobic waste incubation, biomass waste is anaer- biomass is obicallyanaerobically digested digested in a two-step in a process.two-step First, process. the complex First, the biomolecules complex arebiomolecules degraded through are degraded through heterotrophicheterotrophic fermentation fermentation to less to complex less complex substrates subs for methanogenictrates for methanogenic growth in the second growth stage. in the second In the methanogenic bioreactor dissolved organic carbon is converted to biogas that can be recouped stage. In the methanogenic bioreactor dissolved organic carbon is converted to biogas that can be as a biofuel. Introduction of isoprene-synthesizing methanogens (e.g., strain NB 394 in which plasmid recouped as a biofuel. Introduction of isoprene-synthesizing methanogens (e.g., strain NB 394 in pJA2 expressing isoprene synthase is integrated onto the chromosome) to the second stage digester haswhich potential plasmid to produce pJA2 renewable expressing isoprene isoprene in the synthase captured biogas. is integrated onto the chromosome) to the second stage digester has potential to produce renewable isoprene in the captured biogas. Isoprene (2-methyl-1,3-butadiene) is the primary component of natural rubber and an importantMunicipal chemical wastewater precursor utilized treatment in the productiongenerally ofoccurs synthetic in rubber three as distinct well as stages based adhesives, flavorings, cosmetics, and pharmaceuticals. Traditionally, isoprene is harvested fromupon natural the aerobicity rubber from of tree the sap wastewater or produced and industrially the activity through of themicroorganisms thermal cracking involved in the ofmultistage petroleum. process. By producing In the renewable first isoprenestage, aerobic via engineered microorganisms microbes, itcould breakdown be complex possiblebiomass to reduceinto simpler the need toorganic rely on thematerial harvesting [10] of. plantThe biomass deconvoluted or the mining material of is further fossilanaerobically fuels. Recently digested our laboratory by a demonstratedsecond consortia that the of methanogen microbesMethanosarcina into one- or two-carbon acetivoranscompoundscan beand engineered organic to acids. efficiently These produce one- bioisoprene and two-carbon as a methane compounds coproduct are utilized by under laboratory conditions [9]. The gene for isoprene synthase, ispS, was stably inserted intomethanogens the chromosome to complete of M. acetivorans the decomposition(att::ispS) and the productionprocess [6,11]. of isoprene In addition as well to removing aspolluting methane wasorganic confirmed carbon via gasfrom chromatography. the water, anaerobic The production digestion of isoprene has showedthe added benefit of noproducing detrimental methane effect on which growth is rate often or metaboliccaptured efficiency as renewable of the engineeredbiogas [11– strains14]. Due to the low comparedenergetic with potential a vector-only of methanogenic control. We surmised feedstoc thatks, without methanogens an obvious decreaseutilize a in highly efficient fitness it may be possible to drop-in these engineered methanogens into an existing anaero- biccentral wastewater metabolism treatment which consortium greatly to produce favors bioisoprene.the production However, of anymethane inoculated over biomass and methanogensheat. Anaerobic would treatment have to compete of wastewater for substrate result withs wild in 95% methanogens conversion in the of mixed the initial substrate microbialinto available community biogas of the anaerobicwith 5% digester. beingM. acetivoransutilizedhas for the largestmicrobial genome biomass of any [15,16]. We characterizedhypothesized methanogen that the ashighly well as efficient the widest metabo range oflism substrate of methanogens utilization, allowing may for have potential to growthproduce from high methanol, yields methyl-amines, of other value-a carbondded monoxide, products and in acetate