Microbiology has been used for centuries to produce bio-products and biochemicals from various biomass feed stocks (Buchholz and Collins, 2013). Most well-known is alcoholic fermentation by yeast, whether for beverage or fuel. Additional yeasts and bacterial systems are being developed and production of industrial chemicals such organic acids are heavily based in fungal fermentation of sugars (Grewal and Kalra, 1995; Francisco et al., 2020; Xue et al., 2021; Shikina et al., 2022; Upton et al., 2022; Chib et al., 2023).

The U. S. Department of Energy focuses on lignocellulosic biomass crops such as hybrid poplar, switchgrass, miscanthus, and other grasses and fast-growing hardwoods (Feedstock technologies). Other lignocellulosics include agricultural residues such as corn stover, sugarcane bagasse, and wheat, oat, and rice straws, forestry thinnings, and wood processing residues. Proposed feed stocks include everything from municipal solid wastes and gases to algae. Any lignocellulosic biomass can be converted given the right process and market conditions and the myriad of proposed process technologies in “dedicated” bio-refineries are usually linear and focused on optimizing yield of one or a few products from a limited feed stock input.

Food waste, manures, municipal solid waste, and sewage offer wide opportunities for conversion processes and products. As landfills increasingly reject organic wastes, alternative disposal routes are needed. Bio-refining is being proposed for many industrial food waste streams (Kumar et al., 2022) while sewage bio-solids are limited in traditional land-application for disposal (Collivignarelli et al., 2020). Expansion of municipal wastewater systems is often limited by urban sprawl so faster, more efficient options are needed (Jing et al., 2021). Manure is often concentrated by localized high volume ranching, farming, and processing operations. Some of these materials are used to generate biogas by anaerobic digestion, often in co-digestion with other ag residues, however residual digestate contains a large fraction of the original carbon and disposal is still problematic (Chiumenti et al., 2018).

Thermochemical treatment and land filling are the primary means of plastic disposal, however biological deconstruction for renewable plastic generation or other bio-products are being investigated (Bertocchini and Arias, 2023; Malik et al., 2023). Recycling and up-cycling plastics to biodegradable plastics bio-products are two approaches that can help restore the damage caused by this polymer (Kochanska et al., 2022; Morici et al., 2022). Plastics can serve as an excellent source of carbon for microbes, provided the bonds are hydrolysable. While microorganisms metabolize many natural recalcitrant compounds, they have not evolved to breaking down these recently developed man-made materials (Kim et al., 2022; Lim and Thian, 2022; Mat Yasin et al., 2022; Crystal Thew et al., 2023; Thew et al., 2023).

The grand challenge associated with biological conversion of plastics is multidimensional. Bio-catalysts that crack tough chemical bonds in plastics are limited. Enzyme engineering needs to be applied to develop enzymes for individual plastic types. Bio-conversion research on plastics has primarily focused on PET metabolism and research on other plastics is quite rudimentary. Metagenomic approaches to identify novel organisms and enzymes with plastic degrading properties must be explored to determine naturally evolving bio-catalysts from plastic enriched microbiomes, such as the landfills and oceans. Furthermore, integrated microbial and chemical approaches to deconstruct and valorize plastic carbon to bio-products will be critical to a circular bioeconomy.

Micro- and macro-algae present a massive biological resource for sequestering carbon and have long been proposed as bio-refinery feed stocks and catalysts. Algae take up CO₂ directly and often have very high productivity rates. Algae’s ability to use HCO₃ ^- directly enables 5-7-fold higher CO₂ absorption than wood (Jang et al., 2012) and biomass productivities nearly 4-fold higher than sugarcane (Adams et al., 2008). They can be grown without land or freshwater and do not compete for food production resources. In 2019, over 35 million tons of algae was harvested worldwide, with ~97% by aquaculture. Over 99.8% was macroalgae and 0.16% microalgae (Cai et al., 2021).

Microalgae are used to capture CO₂ and waste nutrients for production of bio-products and feed stock biomass. They have high neutral lipid concentrations (up to 70%), driving interest for biodiesel and biofuel production (Chisti, 2007; Sajjadi et al., 2018). Microalgae accumulate other storage compounds such as starch, which can serve for fermentative conversion to biofuels. The flexibility of growing microalgae in open ponds and enclosed photobioreactors under photoautotrophic, heterotrophic, or mixotrophic conditions make them an attractive system for bioproduct applications, though photobioreactors are generally considered too expensive and small scale for production of commodities such as biofuels.

Macroalgae (seaweeds) are starting to be recognized for applications such as waste-water treatments and natural fertilizer applications (Farghali et al., 2023). Their high carbohydrate content (over 60%), in comparison to less than 20% in microalgae (Jung et al., 2013; Jambo et al., 2016) and general lack of lignin and crystalline cellulose point towards easier biological conversion than lignocellulose, however harvest is a challenge. Their unusual polysaccharide chemistry and sometimes high protein content offer both challenges and opportunities. Alginates, carrageenan, fucoidan and laminarin found in the cell walls of macroalgae are recognized for their biological protective activities in humans, highlighting their pharmacological importance (Praveen et al., 2019). They serve as hydrocolloids or functional ingredients in the food industry. Pigments in the form of carotenoids and chlorophyll can serve as replacement for synthetic colors in the food industry (Biris-Dorhoi et al., 2020). The macroalgae industry can impact direct CO₂ removal efficiency by sequestering carbon in the form of biochar or via a bio-refinery approach (Farghali et al., 2023).

Most of the genetic engineering efforts have focused on microalgae, macroalgae are only starting to get some attention (Charrier et al., 2015; Cao et al., 2022). Significant efforts are underway to engineer microalgae for biofuel production, despite considerable scientific challenges resulting in several unsuccessful commercialization attempts. Nevertheless, the field of microalgal research has come a long way towards realizing the potential of these photosynthetic organisms. Genetic engineering in macroalgae is just developing, leaving a lot of scope to be explored. While microalgae must be engineered to improve robustness and productivity, large scale cultivation of macroalgae supported by genetic engineering efforts must be achieved for biofuel and specific bioproduct-based applications.

Cyanobacteria have properties similar to micro- and macro-algae and show promise as an environmentally friendly feed stock for production of fuels and plastics alternatives (Farrokh et al., 2019; Afreen et al., 2021). They are known to produce pigments applicable in the food and cosmetics industries, while also serving as food supplement themselves (Zahra et al., 2020). Interesting bioactivities of their metabolites have also been reported, suggesting clinical importance. With smaller genome sizes, challenges associated with improving productivity and product diversity for industrial scale deployment can be addressed effectively using synthetic biology approaches in comparison to the more-cumbersome higher algae.

Bio-generated CO₂ from fermentation and CH₄/CO₂ from anaerobic digestion represent point-source concentrated feed stocks for carbon capture, cycling, and utilization. Bio-conversion routes can generate renewable natural gas and methanol as well as ethanol which can be further upgraded to jet fuel and other products. Microbial engineering to enhance this capture and conversion is only now beginning. Syngas is already used as a feed stock for biorefining with several pilot and demonstration plants currently operating (Dahmen et al., 2019; De Bhowmick et al., 2019b; De Tissera et al., 2019; Yadav et al., 2019). LanzaTech uses flue gas bioconversion for ethanol and other bio-products and subsequent upgrading to jet fuel using the LanzaJet process.

Bio-driven GHG mitigation is likely the only viable global-scale option in the near-term and bio-refineries are primed to contributed. Biology has been capturing CO₂ since the beginning of life at a planetary scale, the energy is sunlight, and biomass represents high-density carbon. The key will be balancing economical bio-products and sequestering the low-value carbon. In contrast, Direct Air Capture using adsorbants, desorption, and underground sequestration is too energy intensive. According to the International Energy Agency, DAC CO₂ requires between 6.5 and 10 GJ/t CO₂ or 1.8 to 2.7 MWh per ton (Budinis). In simple terms, sequestering 1 GT of CO₂ using DAC would require between 1800 and 2700 TWh, roughly half of the total U.S. output in 2021 of ~4000 TWh. The largest operating DAC facility, Climeworks ORCA plant in Iceland, sequesters 4000 MT/year, necessitating 250,000 similar plants to capture 1 GT CO₂/year.

Bacteria, yeast, fungi, and other whole cell bio-catalysts dominate industrial microbiology in biofuel and biochemical production. Examples include fungal production of organic acids and lipids (Grewal and Kalra, 1995; Carvalho et al., 2019; Chib et al., 2023), alcohols and lipids in yeast (Olson et al., 2004; Hull et al., 2014; Cai et al., 2016a; Pendon et al., 2021), alcohols and biochemicals in bacteria (Cai et al., 2016b), lipids and carotenoids in microalgae (Lopes da Silva et al., 2019; Monte et al., 2020; Papachristou et al., 2021; Oh et al., 2022), and a myriad of other bio-products from a range of microbes. Advances in molecular biology have enabled rapid and targeted metabolic engineering for increased product rate and titer, biofunneling to increase yields, expanded substrate utilization, redox balancing, and other cellular redesigns. Cutting edge technologies such as high through-put sequencing and targeted genome modification tools have opened a Pandora’s box of opportunities in microbial metabolic engineering. The dawn of the -omics age has extended these opportunities even further as metabolomics, proteomics, fluxomics, genomics, transcriptomics, lipidomics, and glycomics continue to increase our understanding of cellular metabolism and pathways. Metagenomics, epigenomics, microbiomics, and secretomics have led us to the edge of engineering and directing microbial consortia in specific and targeted manner. These data-intensive techniques are tailor-made for big data applications of artificial intelligence and machine learning and as this interface of biology and data science continues to expand, we expect leaps forward in our understanding and manipulation of these cellular processes.

Enzymatic bioconversion of lignocellulosic biomass in the past 40+ years has evolved from relatively simple models of fungal cellulases such as Cel7A from Trichoderma reesei ( Taylor et al., 2018), to a broader and more comprehensive understating of mesoscale deconstruction mechanisms employed by multifunctional bacterial enzymes such as CelA from Caldicellulosiruptor bescii (Brunecky et al., 2017), synthetic multifunctional cellulases (Brunecky et al., 2020), and megaDalton sized cellulosomal complexes utilized by CBP organisms like Clostridium thermocellum (Hirano et al., 2016). Moreover, the critical debranching roles of accessory enzymes acting on xylan and other hemicelluloses (Moon et al., 2011; Barr et al., 2012; Pryor et al., 2012; Lagaert et al., 2014; Cao et al., 2015; Goncalves et al., 2015; Hu et al., 2015; Laothanachareon et al., 2015; Sun et al., 2015; Yang et al., 2018; Ogunyewo et al., 2021) and the discovery of Lytic Polysaccharide Mono-Oxygenase enzymes have also been critical in the development of modern commercial cellulases (Agger et al., 2014; Muller et al., 2015; Bernardi et al., 2020; Cheng et al., 2020; Calderaro et al., 2021).

Two primary challenges will be understanding deconstruction of lignocellulosic materials by novel enzymes and enzyme classes and leveraging that understanding to develop advanced commercial enzymes. Future enzyme cocktails will apply these deconstruction strategies to minimally pretreated lignocellulosic feed stocks in a cost-effective manner. Facile, robust, and low-cost production of enzymes must be developed significantly beyond the current state of the art to be applicable at global commodity scale.

Bio-based fuels and chemicals rely on living microbial cells, presenting challenges in engineering and optimizing metabolic pathways for these compounds. Mass transfer and pathway optimization are constrained by cell membranes and intracellular processes (Lu, 2017; Wilding et al., 2018) and much of the carbon ends up in cell biomass, reducing overall carbon efficiency. A high carbon efficiency bioeconomy could benefit from a move toward highly efficient cell-free synthetic biology (Sheldon and Woodley, 2018; Wilding et al., 2018). Cell-free synthetic biology is an emerging interdisciplinary approach utilizing enzymes and cofactor components that are engineered and optimized without the use of living cells, allowing direct control of transcription, translation, and metabolism in an open environment (Lu, 2017).

The primary advantages of cell free enzyme systems are facile manipulation of substrate ratios, and careful adjustment of high energy flux ratios that are either difficult or impossible to control in microbial systems. Enzyme activity and temperature optima are tuned through careful enzyme selection. In contrast, living systems issues include metabolite competition, generation of side products, suboptimal enzyme ratios, and variable temperature optima for the cell (Bergquist et al., 2020) and devotes significant energy and effort in keeping itself alive and reproducing. Unfortunately, key problems for cell free systems remain, largely related to robust and facile protein expression, where post translational modifications are key. Recycling energy carriers such as NADH/NADPH or ATP is also an problem, however there are some approaches using whole cell lysates or redox balancing reactions (Ullah et al., 2016). Examples of possible reactions are too many to list, but two common substrates, glucose and glycerol can be utilized to produce a variety of products (Bergquist et al., 2020).