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Swiss Mycology Symposium 16th June 2023

Troiano, Derek

Publisher
Zenodo
DOI
10.5281/zenodo.17516206
Repository
zenodo.org
Language
en

Abstract

Derek Troiano (BFH-HAFL): Artificial microbial consortia for the conversion of lignocellulosic biomass into fuels and chemicals Microbial consortia are a promising alternative to monocultures for complex biotransformations due to their inherent advantages which include the distribution of metabolic burden by division of labor, the ability to convert complex substrates more efficiently, and their modularity. Previously, our group has engineered a biofilm reactor which enables control of spatial organization along several different gradients of abiotic factors within a biofilm. This reactor harbored an artificial cross-kingdom microbial consortia with oxygen-replete and anoxic niches and was employed in the consolidated bioprocessing of lignocellulose to valuable chemicals such as ethanol, lactic acid, or short chain fatty acids. Ongoing research in our group seeks to expand synthetic niche engineering to other types of spatial niches (e.g., light, carbon dioxide, etc.) for application in microalgal bioprocesses. Microalgal biomass represents a promising feedstock for the sustainable production of, among other things, biofuels, but its use is hindered by high water and energy requirements for growth and harvesting as well as nutrient cost. Growing microalgae in a biofilm may represent one solution to the high water and energy consumption associated with more commonly employed suspended growth approaches. Concerning nutrient cost, which is high due to the requirement for supplemental organic carbon (i.e., for mixotrophic growth) to promote sufficiently rapid microalgal growth, process economics may be improved by replacing typical sources of supplemental organic carbon (e.g., acetate) with cheap and abundant lignocellulosic biomass. While microalgae do not possess the capacity for metabolizing the complex biopolymers available in lignocellulosic biomass (e.g., cellulose), filamentous fungi are excellent at degrading components of lignocellulose into simple molecules. Here, Chlamydomonas reinhardtii was co-cultured with the cellulolytic filamentous fungus Trichoderma reesei in cellulose-based media within the biofilm bioreactor. Ultimately, the engineered symbiosis in cellulose-based media yielded significantly more algal biomass vis-a-vis anexic microalgal cultures grown in typical growth media.

Full text

SSM Annual Congress 2023 Presented by: Derek Troiano Artificial microbial consortia for the conversion of lignocellulosic biomass into fuels and chemicals School of Agricultural, Forest and Food Sciences HAFL Troiano, D. T., Hofmann, T., Brethauer, S., & Studer, M. H. (2023). Toward optimal use of biomass as carbon source for chemical bioproduction. Current Opinion in Biotechnology,81, 102942. Global consumption levels 600 EJ energy 450 Mt carbon From sustainable supply of biomass we can obtain: 100 EJ energy 2400 Mt carbon Troiano, D. T., Hofmann, T., Brethauer, S., & Studer, M. H. (2023). Toward optimal use of biomass as carbon source for chemical bioproduction. Current Opinion in Biotechnology,81, 102942. Biobased production of chemicals: Biobased production of chemicals: sugar & starch Advantages: Substrates are bulk materials No requirement foror very fast enzymatic hydrolysis Clean input Homogeneous catalysis High substrate and product concentrations Disadvantages: Food/feed price of substrate STARCH Products Liquefied starch Steam Water FERMENTATION Sugar solution Glucoamylase Microorganisms PRODUCT PURIFICATION STEAM COOKING ENZYMATIC HYDROLYSIS SUGAR Products Water Microorganisms PRODUCT PURIFICATION FERMENTATION Beer Beer a-amylase STARCH Products Liquefied starch Steam Water FERMENTATION Sugar solution Glucoamylase Microorganisms PRODUCT PURIFICATION STEAM COOKING ENZYMATIC HYDROLYSIS SUGAR Products Water Microorganisms PRODUCT PURIFICATION FERMENTATION Beer Beer a-amylase Lignocellulosic biomass as feedstock Brethauer, S., Shahab, R.L., Studer, M. 2020 Impacts of biofilms on the conversion of cellulose, Applied Microbiology and Biotechnology 104:5201–5212 https://doi.org/10.1007/s00253-020-10595-y Biochemical conversion of lignocellulose to chemicals Advantages: Most abundant organic resource 6 x 103 t/y of sustainable1 source No food v fuel dilemma Disadvantages: Bulky substrate Recalcitrant More than one sugar type Low sugar concentrations Heterogeneous catalysis ‘dirty’ material 1Troiano, D. T., Hofmann, T., Brethauer, S., & Studer, M. H. (2023). Toward optimal use of biomass as carbon source for chemical bioproduction. Current Opinion in Biotechnology,81, 102942. LIGNOCELLULOSE PRETREATMENT SOLID/LIQUID SEPARATION SOLIDS WASHING ENZYMATIC HYDROLYSIS FERMENTATION OF C6SUGARS FERMENTATION OF C5SUGARS SOLID/LIQUID SEPARATION DETOXIFICATION Beer Hydrolysate Pretreated biomass Liquid phase CaO H2SO4 Solids free beer Solids Solids Steam Lignin Enzymes Beer Recycle water PRODUCT PURIFICATION Product CBP of lignocellulose to biochemicals refers to the combining: production of saccharolytic enzymes hydrolysis of the polysaccharides fermentation of hexose sugars and fermentation of pentose sugars in one reactor Consolidated bioprocessing CBP LIGNOCELLULOSE SOLID/LIQUID SEPARATION Pretreated biomass Solids free beer Steam Lignin Beer Recycle water PRODUCT PURIFICATION Product CONSOLIDATED BIOPROCESSING PRETREATMENT +Predominantly used for biomanufacturing systems -Challenging modifications -Metabolic burden -Cytosolic or periplasmic space limitations -Competing biochemical reactions -Toxic intermediates -Low product titers and yields Consolidated bioprocessing –classical approach, based on GMO Hexoses + Pentoses Cellulose + Hemicellulose Target product Target product Hexoses + Pentoses Engineering of ecological niches: Bioreactors Shahab, R., Brethauer, S., ….Studer, M. 2020 A heterogeneous microbial consortium producing short-chain fatty acids from lignocellulose. Science 369, eabb1214 Microalgae: Background Advantages: Fast growth No requirement for arable land Easy to process Disadvantages: High water consumption Energy intensive harvesting Requires supplemental carbon source E.g., acetate Microalgae Cultivation Microalgal Biomass Methane Ethanol Acetone Butanol Bio-diesel Bio-oil Fertilizer Anaerobic digestion Fermentation Pyrolysis/liquefaction Lipid extraction Transesterification Microalgae: Membrane Photobioreactor Design: gas permeable membrane separating gas phase from bulk liquid media phase Promotes growth of biofilm on membrane surface High cell density as compared with suspended growth Higher concentration of CO2 as compared with bulk liquid may promote faster algal growth Open to atmosphere Chamber containing liquid media Gas permeable membrane Magnetic stir plate LED light CO2 Light CO2 C. reinhardtii Light intesity Membrane Photobioreactor (MBR) Using normal growth media: Chlamydomonas reinhardtii 10 days of growth comparable or slightly better biomass accumulation in membrane bioreactor vs suspended growth MBR: With and without supplemental organic carbon Biofilm: normal growth media (10 days) Biofilm: growth media without acetate (10 days) Using normal growth media: comparable or slightly better biomass accumulation in membrane bioreactor vs suspended growth When we do not supply supplemental organic carbon (acetate): Significantly less biomass Microalgae are well-attached to membrane Supplemental carbon is necessary for sufficent rate of biomass accumulation MBR: Reduced supplemental organic carbon Supplemental carbon is necessary for sufficent rate of biomass accumulation and establishment of robust biofilm Strategy: Use media containing acetate to establish biofilm, then switch to media without acetate Membrane photobioreactor produced more biomass than suspended culture following this strategy Possible evidence for advantage of growing at membrane surface, where CO2 concentrations are highest Two weeks in normal growth media Two weeks in growth media w/o acetate Lignocellulosic Carbon CO2 O2 Sugars Microalgal and filamentous fungal co-culture: Scheme Atmospheric O2 Atmospheric CO2 and light Lignocellulolytic enzymes External/added Internal/endogenous T. reesei C. reinhardtii Mycoalgal biofilm Formation of robust biofilm Largely homogenous distribution of algae and fungi mix *with microcrystalline cellulose Mycoalgal biofilm 1 cm Next Steps •Optimize for algal biomass production •Better characterize the system •Quantify lipid production •Use real biomass (e.g., pre-treated beechwood)