Biodiversity Improves Life Cycle Sustainability Metrics in Algal Biofuel Production.

Algal biofuel has yet to realize its potential as a commercial and sustainable bioenergy source, largely due to the challenge of maximizing and sustaining biomass production with respect to energetic and material inputs in large-scale cultivation. Experimental studies have shown that multispecies algal polycultures can be designed to enhance biomass production, stability, and nutrient recycling compared to monocultures. Yet, it remains unclear whether these impacts of biodiversity make polycultures more sustainable than monocultures. Here, we present results of a comparative life cycle assessment (LCA) for algal biorefineries to compare the sustainability metrics of monocultures and polycultures of six fresh-water algal species. Our results showed that when algae were grown in outdoor experimental ponds, certain bicultures improved the energy return on investment (EROI) and greenhouse gas emissions (GHGs) by 20% and 16%, respectively, compared to the best monoculture. Bicultures outperformed monocultures by performing multiple functions simultaneously (e.g., improved stability, nutrient efficiency, biocrude characteristics), which outweighed the higher productivity attainable by a monoculture. Our results demonstrate that algal polycultures with optimized multifunctionality lead to enhanced life cycle metrics, highlighting the significant potential of ecological engineering for enabling future environmentally sustainable algal biorefineries.

Microbial production of high octane and high sensitivity olefinic ester biofuels.

BACKGROUND: Advanced spark ignition engines require high performance fuels with improved resistance to autoignition. Biologically derived olefinic alcohols have arisen as promising blendstock candidates due to favorable octane numbers and synergistic blending characteristics. However, production and downstream separation of these alcohols are limited by their intrinsic toxicity and high aqueous solubility, respectively. Bioproduction of carboxylate esters of alcohols can improve partitioning and reduce toxicity, but in practice has been limited to saturated esters with characteristically low octane sensitivity. If olefinic esters retain the synergistic blending characteristics of their alcohol counterparts, they could improve the bioblendstock combustion performance while also retaining the production advantages of the ester moiety. RESULTS: Optimization of Escherichia coli isoprenoid pathways has led to high titers of isoprenol and prenol, which are not only excellent standalone biofuel and blend candidates, but also novel targets for esterification. Here, a selection of olefinic esters enhanced blendstock performance according to their degree of unsaturation and branching. E. coli strains harboring optimized mevalonate pathways, thioester pathways, and heterologous alcohol acyltransferases (ATF1, ATF2, and SAAT) were engineered for the bioproduction of four novel olefinic esters. Although prenyl and isoprenyl lactate titers were limited to 1.48 ± 0.41 mg/L and 5.57 ± 1.36 mg/L, strains engineered for prenyl and isoprenyl acetate attained titers of 176.3 ± 16.0 mg/L and 3.08 ± 0.27 g/L, respectively. Furthermore, prenyl acetate (20% bRON = 125.8) and isoprenyl acetate (20% bRON = 108.4) exhibited blend properties comparable to ethanol and significantly better than any saturated ester. By further scaling cultures to a 2-L bioreactor under fed-batch conditions, 15.0 ± 0.9 g/L isoprenyl acetate was achieved on minimal medium. Metabolic engineering of acetate pathway flux further improved titer to attain an unprecedented 28.0 ± 1.0 g/L isoprenyl acetate, accounting for 75.7% theoretical yield from glucose. CONCLUSION: Our study demonstrated novel bioproduction of four isoprenoid oxygenates for fuel blending. Our optimized E. coli production strain generated an unprecedented titer of isoprenyl acetate and when paired with its favorable blend properties, may enable rapid scale-up of olefinic alcohol esters for use as a fuel blend additive or as a precursor for longer-chain biofuels and biochemicals.

Translating advances in microbial bioproduction to sustainable biotechnology.

Advances in synthetic biology have radically changed our ability to rewire microorganisms and significantly improved the scalable production of a vast array of drop-in biopolymers and biofuels. The success of a drop-in bioproduct is contingent on market competition with petrochemical analogues and weighted upon relative economic and environmental metrics. While the quantification of comparative trade-offs is critical for accurate process-level decision making, the translation of industrial ecology to synthetic biology is often ambiguous and assessment accuracy has proven challenging. In this review, we explore strategies for evaluating industrial biotechnology through life cycle and techno-economic assessment, then contextualize how recent developments in synthetic biology have improved process viability by expanding feedstock availability and the productivity of microbes. By juxtaposing biological and industrial constraints, we highlight major obstacles between the disparate disciplines that hinder accurate process evaluation. The convergence of these disciplines is crucial in shifting towards carbon neutrality and a circular bioeconomy.

Diversifying Isoprenoid Platforms via Atypical Carbon Substrates and Non-model Microorganisms.

Isoprenoid compounds are biologically ubiquitous, and their characteristic modularity has afforded products ranging from pharmaceuticals to biofuels. Isoprenoid production has been largely successful in Escherichia coli and Saccharomyces cerevisiae with metabolic engineering of the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways coupled with the expression of heterologous terpene synthases. Yet conventional microbial chassis pose several major obstacles to successful commercialization including the affordability of sugar substrates at scale, precursor flux limitations, and intermediate feedback-inhibition. Now, recent studies have challenged typical isoprenoid paradigms by expanding the boundaries of terpene biosynthesis and using non-model organisms including those capable of metabolizing atypical C1 substrates. Conversely, investigations of non-model organisms have historically informed optimization in conventional microbes by tuning heterologous gene expression. Here, we review advances in isoprenoid biosynthesis with specific focus on the synergy between model and non-model organisms that may elevate the commercial viability of isoprenoid platforms by addressing the dichotomy between high titer production and inexpensive substrates.

Optimized gene expression from bacterial chromosome by high-throughput integration and screening.

Chromosomal integration of recombinant genes is desirable compared with expression from plasmids due to increased stability, reduced cell-to-cell variability, and elimination of the need for antibiotics for plasmid maintenance. Here, we present a new approach for tuning pathway gene expression levels via random integration and high-throughput screening. We demonstrate multiplexed gene integration and expression-level optimization for isobutanol production in Escherichia coli The integrated strains could, with far lower expression levels than plasmid-based expression, produce high titers (10.0 ± 0.9 g/liter isobutanol in 48 hours) and yields (69% of the theoretical maximum). Close examination of pathway expression in the top-performing, as well as other isolates, reveals the complexity of cellular metabolism and regulation, underscoring the need for precise optimization while integrating pathway genes into the chromosome. We expect this method for pathway integration and optimization can be readily extended to a wide range of pathways and chassis to create robust and efficient production strains.

Random Chromosomal Integration and Screening Yields E. coli K-12 Derivatives Capable of Efficient Sucrose Utilization.

Chromosomal expression of heterologous genes offers stability and maintenance advantages over episomal expression, yet remains difficult to optimize through site-specific integration. The challenge has in large part been due to the variability of chromosomal gene expression, which has only recently been shown to be affected by multiple factors, including the local genomic context. In this work we utilize Tn5 transposase to randomly integrate a three-gene csc operon encoding nonphosphotransferase sucrose catabolism into the E. coli K-12 chromosome. Isolates from the transposon library yielded a range of growth rates on sucrose as the sole carbon source, including some that were comparable to that of E. coli K-12 on glucose (µmax = 0.70 ± 0.03 h-1). Narrowness of the growth rate distributions and faster growth compared to plasmids indicate that efficient csc expression is attainable. Furthermore, enhanced growth rate upon transduction into strains that underwent adaptive laboratory evolution indicate that sucrose catabolism is not limiting to cellular growth. We also show that transduction of a csc fast-growth locus into an isobutanol production strain yields high titer (7.56 ± 0.25 g/L) on sucrose as the sole carbon source. Our results demonstrate that random integration is an effective strategy for optimizing heterologous expression within the context of cellular metabolism for both fast growth and biochemical production phenotypes.

Demonstration of transgressive overyielding of algal mixed cultures in microdroplets.

Algae are ubiquitous in natural ecosystems and have been studied extensively for biofuel production due to their unique metabolic capabilities. Most studies to date have approached biofuel optimization through synthetic biology and process engineering with few industrial scale projects considering algal community interactions. Such interactions can potentially lead to increased productivity and reduced community invasability, both important characteristics for scalable algal biofuel production. It is estimated that over a million species of algae exist such that elucidating the interactions that might be beneficial for biofuel production remains extremely resource and time intensive. Here we describe a strategy for rapid, high-throughput screening of algal community combinations using a microfluidic platform to generate millions of parallel, nanoliter-scale algal mixed cultures for estimation of biomass accumulation. Model communities were first studied in a bench scale flask experiment and then examined using microfluidic droplets. These experiments showed consistent results for both positively interacting algal bicultures that increase biomass when together, and negatively interacting bicultures that decrease biomass. Specifically, these included enhanced performance of two bicultures, Ankistrodesmus falcatus and Chlorella sorokiniana, Chlorella sorokiniana and Selenastrum minutum, and reduced performance of a biculture consisting of Selenastrum capricornutum and Scenedesmus ecornis. While the ultimate techno-economic feasibility of algal bioproducts hinges on an amalgamation of scientific fields, rapid screening of algal communities will prove imperative for efficiently discovering community interactions.