Available online at www.sciencedirect.com ScienceDirect Trash to treasure: production of biofuels and commodity chemicals via syngas fermenting microorganisms 1 2 2 1,2 Haythem Latif , Ahmad A Zeidan , Alex T Nielsen and Karsten Zengler Fermentation of syngas is a means through which unutilized billion gallons of biofuels by 2022 [2]. The biological organic waste streams can be converted biologically into conversion of renewable lignocellulosic biomass such as biofuels and commodity chemicals. Despite recent advances, wheat straw, spruce, switchgrass, and poplar to biofuels is several issues remain which limit implementation of industrial- expected to play a prominent role in achieving these scale syngas fermentation processes. At the cellular level, the goals. These forms of biomass address many of the con- energy conservation mechanism of syngas fermenting cerns associated with the production of first-generation microorganisms has not yet been entirely elucidated. biofuels [3,4]. However, 10–35% of lignocellulosic bio- Furthermore, there was a lack of genetic tools to study and mass is composed of lignin [5–7], which is highly resistant ultimately enhance their metabolic capabilities. Recently, to breakdown by the vast majority of microorganisms [8]. substantial progress has been made in understanding the Thus, if the EU and US cellulosic biofuel targets are intricate energy conservation mechanisms of these realized, land allocation for biofuel production will microorganisms. Given the complex relationship between increase and megatons of organic waste will be generated. energy conservation and metabolism, strain design greatly benefits from systems-level approaches. Numerous genetic This organic waste provides a significant resource of manipulation tools have also been developed, paving the way biomass that can be utilized for producing biofuels as for the use of metabolic engineering and systems biology well as commodity chemicals. Through gasification, vir- approaches. Rational strain designs can now be deployed tually any form of organic matter can be converted into a resulting in desirable phenotypic traits for large-scale mixture of carbon monoxide (CO), carbon dioxide (CO2), production. and hydrogen (H2), referred to as synthesis gas or syngas. Addresses Gasification involves high temperature (usually 600– 1 University of California San Diego, Department of Bioengineering, La 9008C) partial oxidation of biomass in the presence of a Jolla, CA 92039, United States gasifying agent (e.g. oxygen or steam) resulting in the 2 The Novo Nordisk Foundation Center for Biosustainability, Technical production of gas with significant amounts of CO and H2 University of Denmark, Kogle Alle´ 6, Hørsholm 2970, Denmark [9]. Syngas can be metabolized by certain carbon-fixing Corresponding author: Zengler, Karsten ([email protected], microorganisms and converted to valuable multi-carbon [email protected]) compounds such as acetate, ethanol, butanol, butyrate, and 2,3-butanediol [10,11]. This process, known as syngas fermentation, provides an attractive means for converting Current Opinion in Biotechnology 2014, 27:79–87 low cost organic substrates and waste streams into valu- This review comes from a themed issue on Energy biotechnology able chemicals. Syngas fermentation has numerous Edited by Arthur J Ragauskas and Korneel Rabaey advantages when compared to thermo-chemical pro- cesses such as Fischer-Tropsch synthesis. These include a higher tolerance for impurities such as sulfur com- pounds, a wider range of usable H2, CO2, and CO mix- 0958-1669/$ – see front matter, # 2013 Elsevier Ltd. All rights tures, a lower operating-temperature and -pressure, and reserved. higher product yield and uniformity. However, wide use http://dx.doi.org/10.1016/j.copbio.2013.12.001 of these syngas fermenting microorganisms as production hosts is currently hindered by several factors, including low volumetric product titers, product feedback inhi- Introduction bition, and low gas-liquid mass transfer coefficient Heightened concerns over global warming and fossil fuel (kLa) [12]. Though some of these challenges can be supply and prices have led to a paradigm shift in per- overcome, in part, through process improvements, a fun- ceived routes to chemical commodity production and damental understanding of the biology enabling syngas energy generation. The majority of the world community fermentation is needed to guide those design strategies has set challenging targets for reductions in greenhouse and to provide targets for cellular engineering. Thus, the gas emissions to be achieved in part through the de- biggest challenge facing process development for syngas velopment of sustainable routes to chemicals, fuels, fermentation may be the lack of tools and technologies and energy production. The EU has targeted a 10% share that will further our understanding of the fundamental of renewable biofuels in the transportation sector by 2020 biology behind these versatile microorganisms. This [1] while the US has mandated the production of 36 review focuses on these unique microorganisms, their www.sciencedirect.com Current Opinion in Biotechnology 2014, 27:79–87 80 Energy biotechnology metabolic and energy conservation pathways, and the known to synthesize multi-carbon organics from syngas, genetic engineering strategies that together will guide the vast majority of syngas fermenting organisms are advances in the use of syngas fermentation for the pro- acetogens (Figure 1). Acetogens are anaerobes that assim- duction of biofuels and commodity chemicals. ilate CO2 via the Wood–Ljungdahl (WL) pathway, also referred to as the reductive acetyl-CoA pathway. Though we focus here mainly on autotrophic conversion of syngas Syngas fermenting microorganisms in acetogens, the WL pathway is also active during A diverse range of microorganisms that can metabolize heterotrophic growth. syngas and produce multi-carbon compounds have been identified (Figure 1). These organisms are ubiquitous in numerous habitats such as soils, marine sediments, and The Wood–Ljungdahl pathway feces, exhibit various morphologies (e.g. rods, cocci, or The WL pathway is hypothesized to be the most ancient spirochetes), have a wide range of optimal growth tem- CO fixation pathway [16]. However, the apparent sim- peratures (psychrophilic, mesophilic, or thermophilic), 2 plicity of this linear pathway belies the complex, inter- and demonstrate different tolerance toward molecular connected energy conservation mechanisms that enable oxygen [13]. Syngas fermenting microorganisms also have growth on syngas [13,17 ]. Only a short overview of the diverse metabolic capabilities, resulting in the formation pathway will be given here since the WL pathway has of a variety of native products such as acetate, ethanol, been extensively reviewed elsewhere [13,18–20]. butanol, butyrate, formate, H2, H2S, and traces of Figure 2 shows the complete WL pathway with electron methane [10,11,13–15]. Though some methanogens are Figure 1 Genera containing acetogens Eubacterium Acetobacterium Natronincola Alkalibaculum Tindallia Eubacteriaceae Acetitomaculum Oxobacter Clostridiales Clostridiaceae Syntrophococcus Caloramator Lachnospiraceae Clostridia Marvinbryantia Clostridium Firmicutes Archaeoglobus Blautia Bacteria Archaea Euryarchaeota Acetoanaerobium Methanosarcina Thermoanaerobacter Holophaga Thermoanaerobacteraceae Moorella Halobacter- Veillonel- Treponema oidaceae laceae Thermacetogenium Sporomusa Acetohalobium Acetonema Natroniella Current Opinion in Biotechnology Syngas fermenting organisms known to produce multi-carbon organic compounds. Shown is the taxonomic classification of the genera capable of converting syngas to multi-carbon compounds based on organisms found in [10,11,13–15]. With the exception of two archaeal genera, all of the genera that produce multi-carbon organic compounds are considered acetogens. Genera are classified based on NCBI’s current taxonomic nomenclature and categorization. Current Opinion in Biotechnology 2014, 27:79–87 www.sciencedirect.com Syngas fermentation for production of organic compounds Latif et al. 81 Figure 2 Autotrophic Growth Heterotrophic Growth Wood-Ljungdahl Pathway Acetogenesis Glycolysis* Methyl Branch Carbonyl Branch Acetate Glc + CO2 CO CO CO2 Fru Fru H Acetate CH COOH CO 3 Pep Pep H+ Glc ATP Fdxox, H2O Fdx , 2 H+ red ACK FRUpts FRUpts ATP CODH CODH/ACS ADP Pyr Pyr HEX Fdx , 2 H+ Acetate-P red Fdx , H O 2- ox 2 CH3COO-PO3 ADP, H+ HS-CoA H+ HS-CoA CO2 [CO] PTA Glc-6P + Fdxred Fdxox Fdxred, H Pi CODH/ACS PFOR PGI FDH HS∼CoA Acetyl-CoA Pyr ∼ CH3-C= S-CoA Fru-1P Fru-6P Fdxox O ATP Formate Methyl-CoFeSP CO2 ATP ATP HCOO- [CH3]-CoFeS-P PYK FRUK PFK ATP,THF THF H+, ADP ADP, H+ + FTHFS MET Pep ADP, H H2O ADP, Pi CoFeSP ENO Formyl-THF Methyl-THF [CHO]-THF [CH3]-THF 2PG Fdx Fru-1,6P H+ red PGM 2 NAD+ MTHFR 3PG MTHFC FBA Fdxox ATP H2O 2 NADH, H+ + PGK NADH NAD + Methenyl-THF Methylene-THF Biomass H Pi [CH]≡THF MTHFD [CH2]=THF ADP NADPH NADP+ TPI DHAP 1,3-DPG GAPDH Gly-3P Current Opinion in Biotechnology The Wood–Ljungdahl pathway and its connection to heterotrophic metabolism. Shown is the WL pathway for Clostridium ljungdahlii as assessed in [21,22] with reduced ferredoxin, NADH, and NADPH serving as electron carriers during CO2 fixation. The left panel shows the Methyl and Carbonyl branches of the WL pathway leading to either acetate formation or assimilation of acetyl-CoA into biomass. The right panel depicts fermentation through glycolysis for fructose and glucose. The electrons