A heterogeneous microbial consortium producing short-chain fatty acids from lignocellulose

Building niches for multiple microbes Microbial conversion of heterogeneous feedstocks such as lignocellulose into one or more desired products requires assembly of pathways to both break down the inputs and produce the outputs. Shahab et al. assembled a collection of microbes that occupy different spatial niches within a bioreactor and have different metabolic capabilities. In the simplest version of their setup, an aerobic fungus adeptly breaks down cellulose into short sugar chains, an oxygen-tolerant bacterium converts these to lactic acid, and an anaerobic bacterium uses the lactic acid to synthesize the short-chain fatty acid butyric acid. Additional microbes handle limitations in the intermediate stream or divert lactic acid to longer-chain fatty acids, which are potentially more valuable. The integrated production and use of lactic acid for biosynthetic reactions could ideally serve as a platform for biosynthesis using robust, heterogeneous microbial consortia. Science, this issue p. eabb1214 Engineered microbial communities funnel carbohydrates to lactate as an intermediate in producing carboxylic acids. INTRODUCTION Consortia of microorganisms are a promising alternative to monocultures for complex biotransformations because of their inherent advantages, such as the distribution of the metabolic burden by division of labor, the ability to convert complex substrates more efficiently, and their modularity. Consortia are also inherently more stable and less susceptible to contamination than monocultures. However, achieving stable and reproducible ratios of the different strains and their performance is challenging, especially for microbial consortia where mutual beneficial interactions are missing—for example, if one strain secretes cellulolytic enzymes to provide carbohydrates as a public good. Moreover, the need to find matching fermentation conditions in well-mixed reactors limits the selection of possible consortium members and prevents the harnessing of the full power of artificial consortia. RATIONALE To tackle these challenges and to advance the understanding and engineering of synthetic microbial consortia, we attempted to mimic one of the well-known natural mechanisms for consortium stabilization: the spatial organization, along several different gradients, of abiotic factors within a biofilm. To this end, we established a membrane-aerated biofilm reactor, which allows for the creation of an oxygen-replete niche in an otherwise anoxic environment, to harbor artificial cross-kingdom microbial consortia for the consolidated bioprocessing of lignocellulose to short-chain fatty acids (SCFAs). Because many SCFA producers have a limited carbohydrate substrate spectrum, the heterogeneous lignocellulosic sugar mixture is funneled to lactate as the central intermediate. RESULTS The lactate platform consists of the aerobic fungus Trichoderma reesei, which secretes cellulolytic enzymes and thus provides soluble sugars; facultative anaerobic lactic acid bacteria (LAB); and the obligate anaerobic lactate-fermenting strain, which provides the product. To set up the consortia and to form the necessary oxygen gradient that allows the growth of all members, we applied a sequential inoculation scheme starting with the fungus, followed by LAB, and lastly the lactate fermenter. We found that the metabolic activity of T. reesei alone is not sufficient to create an environment with a redox potential suitable for obligate anaerobes; rather, it requires the additional presence of LAB. We tested the feasibility of the lactate platform to produce butyric acid using Clostridium tyrobutyricum, which co-consumes lactic acid and acetic acid in a 3:1 molar ratio, thereby valorizing this often unused component of lignocellulose and allowing for a higher carbon yield relative to direct carbohydrate conversion. We were able to produce a stable cocultivation of the three strains, and, with a mixture of cellulose and xylose as model substrate, we obtained 0.35 g of butyric acid per gram of carbohydrates—a value slightly exceeding yields achieved with soluble substrates or by addition of commercial cellulolytic enzymes. With beechwood as substrate, a yield equivalent to 196 kg of butyric acid per metric ton of feedstock was reached. Switching to Veillonella criceti, an obligate anaerobic bacterium unable to utilize carbohydrates, a mixture of propionic and acetic acid was produced from lignocellulose, underpinning the suitability of the lactate platform approach to compensate for missing metabolic capabilities of product-forming strains. Longer SCFAs such as valeric and caproic acid, which are more valuable than the C2 to C4 SCFAs, could be produced by integrating Megasphaera elsdenii, a strict anaerobic rumen bacterium able to perform chain elongation, into the lactate platform. By extending this configuration with V. criceti, the ratio of odd- and even-numbered SCFAs could be tuned, and the valeric acid concentration was higher than with the three-member community by a factor of nearly 3. To demonstrate the platform’s wide applicability, we identified several other chemicals—including alcohols (ethanol, propanol, butanol, 1,2-propanediol), polyhydroxybutyrate, and lipids—that could be produced through the lactate platform. CONCLUSION The lactate platform demonstrates several inherent benefits of synthetic consortia, such as their modularity and their ability to convert complex mixed substrates into valuable platform chemicals. The use of spatial niches in a membrane reactor—not limited to oxygen, but also for other abiotic factors such as temperature, light, or pH—expands the available toolbox for successfully engineering stable and controllable synthetic communities for numerous novel applications. Principle of the lactate platform. A membrane-aerated bioreactor featuring an oxygen gradient (depicted at the bottom) allows for the consolidated bioprocessing of lignocellulose to short-chain fatty acids by a heterogeneous consortium consisting of an aerobic, cellulolytic fungus growing as a biofilm in the oxygen-replete spatial niche, a facultative anaerobic lactic acid bacterium to funnel the carbohydrate mixture to lactate, and a lactate-consuming obligate anaerobic bacterium for product formation. Microbial consortia are a promising alternative to monocultures of genetically modified microorganisms for complex biotransformations. We developed a versatile consortium-based strategy for the direct conversion of lignocellulose to short-chain fatty acids, which included the funneling of the lignocellulosic carbohydrates to lactate as a central intermediate in engineered food chains. A spatial niche enabled in situ cellulolytic enzyme production by an aerobic fungus next to facultative anaerobic lactic acid bacteria and the product-forming anaerobes. Clostridium tyrobutyricum, Veillonella criceti, or Megasphaera elsdenii were integrated into the lactate platform to produce 196 kilograms of butyric acid per metric ton of beechwood. The lactate platform demonstrates the benefits of mixed cultures, such as their modularity and their ability to convert complex substrates into valuable biochemicals.