RuBisCO activity assays: a simplified biochemical redox approach for in vitro quantification and an RNA sensor approach for in vivo monitoring.

BACKGROUND: Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the most abundant soluble protein in nature. Extensive studies have been conducted for improving its activity in photosynthesis through approaches like protein engineering. Concurrently, multiple biochemical and radiolabeling assays have been developed for determining its activity. Although these existing assays yield reliable results, they require addition of multiple external components, rendering them less convenient and expensive. Therefore, in this study, we have developed two relatively cheaper, convenient, and easily reproducible assays for quantitative and qualitative estimation of RuBisCO activity. RESULTS: We simplified a contemporary NADH based spectrophotometric RuBisCO assay by using cyanobacterial cell lysate as the source for Calvin cycle enzymes. We analyzed the influence of inorganic carbon substrates, CO2 and NaHCO3, and varying protein concentrations on RuBisCO activity. Ribulose-1,5-bisphosphate (RuBP) consumption rates for the cultures grown under 5% CO2 were 5-7 times higher than the ones grown with 20 mM NaHCO3, at different protein concentrations. The difference could be due to the impaired activity of carbonic anhydrase in the cell lysate, which is required for the conversion of HCO3- to CO2. The highest RuBisCO activity of 2.13 nmol of NAD+/ µg of Chl-a/ min was observed with 50 µg of protein and 5% CO2. Additionally, we developed a novel RNA-sensor based fluorescence assay that is based on the principle of tracking the kinetics of ATP hydrolysis to ADP during the conversion of 3-phosphoglycerate (3-PG) to 1,3-bisphosphoglycerate (1,3-BPG) in the Calvin cycle. Under in vitro conditions, the fluorometric assay exhibited  ~ 3.4-fold slower reaction rate (0.37 min-1) than the biochemical assay when using 5% CO2. We also confirmed the in vivo application of this assay, where increase in the fluorescence was observed with the recombinant strain of Synechocystis sp. PCC 6803 (SSL142) expressing the ADP-specific RNA sensor, compared to the WT. In addition, SSL142 exhibited three-fold higher fluorescence when supplemented with 20 mM NaHCO3 as compared to the cells that were grown without NaHCO3 supplementation. CONCLUSIONS: Overall, we have developed a simplified biochemical assay for monitoring RuBisCO activity and demonstrated that it can provide reliable results as compared to the prior literature. Furthermore, the biochemical assay using 5% CO2 (100% relative activity) provided faster RuBP consumption rate compared to the biochemical assay utilizing 20 mM NaHCO3 (30.70% relative activity) and the in vitro fluorometric assay using 5% CO2 (29.64% relative activity). Therefore, the absorbance-based biochemical assay using 5% CO2 or higher would be suitable for in vitro quantification of the RuBisCO activity. On the other hand, the RNA-sensor based in vivo fluorometric assay can be applied for qualitative analysis and be used for high-throughput screening of RuBisCO variants. As RuBisCO is an enzyme shared amongst all the photoautotrophs, the assays developed in this study can easily be extended for analyzing the RuBisCO activities even in microalgae and higher plants.

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.

Advances in engineering algae for biofuel production.

While algae demonstrate potential as a sustainable fuel source, low productivities limit the economic realization of algal biofuels. High-throughput strain engineering, omics-informed genome-scale modeling, and microbiome engineering are key technologies for enabling algal biofuels. High-throughput strain engineering efforts generate improved traits, including high biomass productivity and lipid content, in diverse algal species. Genome-scale models, constructed with the aid of omics data, provide insight into metabolic limitations and guide rational algal strain engineering efforts. As outdoor cultivation systems introduce exogenous organisms, microbiome engineering seeks to eliminate harmful organisms and introduce beneficial species. Optimizing algal biomass production and lipid content using these technologies may overcome the productivity barrier for the commercialization of algal biofuels.

Corynebacterium glutamicum as an Efficient Omnivorous Microbial Host for the Bioconversion of Lignocellulosic Biomass.

Corynebacterium glutamicum has been successfully employed for the industrial production of amino acids and other bioproducts, partially due to its native ability to utilize a wide range of carbon substrates. We demonstrated C. glutamicum as an efficient microbial host for utilizing diverse carbon substrates present in biomass hydrolysates, such as glucose, arabinose, and xylose, in addition to its natural ability to assimilate lignin-derived aromatics. As a case study to demonstrate its bioproduction capabilities, L-lactate was chosen as the primary fermentation end product along with acetate and succinate. C. glutamicum was found to grow well in different aromatics (benzoic acid, cinnamic acid, vanillic acid, and p-coumaric acid) up to a concentration of 40 mM. Besides, 13C-fingerprinting confirmed that carbon from aromatics enter the primary metabolism via TCA cycle confirming the presence of ß-ketoadipate pathway in C. glutamicum. 13C-fingerprinting in the presence of both glucose and aromatics also revealed coumarate to be the most preferred aromatic by C. glutamicum contributing 74 and 59% of its carbon for the synthesis of glutamate and aspartate respectively. 13C-fingerprinting also confirmed the activity of ortho-cleavage pathway, anaplerotic pathway, and cataplerotic pathways. Finally, the engineered C. glutamicum strain grew well in biomass hydrolysate containing pentose and hexose sugars and produced L-lactate at a concentration of 47.9 g/L and a yield of 0.639 g/g from sugars with simultaneous utilization of aromatics. Succinate and acetate co-products were produced at concentrations of 8.9 g/L and 3.2 g/L, respectively. Our findings open the door to valorize all the major carbon components of biomass hydrolysate by using C. glutamicum as a microbial host for biomanufacturing.

Techno-economic assessment for the production of algal fuels and value-added products: opportunities for high-protein microalgae conversion.

BACKGROUND: Microalgae possess numerous advantages for use as a feedstock in producing renewable fuels and products, with techno-economic analysis (TEA) frequently used to highlight the economic potential and technical challenges of utilizing this biomass in a biorefinery context. However, many historical TEA studies have focused on the conversion of biomass with elevated levels of carbohydrates and lipids and lower levels of protein, incurring substantial burdens on the ability to achieve high cultivation productivity rates relative to nutrient-replete, high-protein biomass. Given a strong dependence of algal biomass production costs on cultivation productivity, further TEA assessment is needed to understand the economic potential for utilizing potentially lower-cost but lower-quality, high-protein microalgae for biorefinery conversion. RESULTS: In this work, we conduct rigorous TEA modeling to assess the economic viability of two conceptual technology pathways for processing proteinaceous algae into a suite of fuels and products. One approach, termed mild oxidative treatment and upgrading (MOTU), makes use of a series of thermo-catalytic operations to upgrade solubilized proteins and carbohydrates to hydrocarbon fuels, while another alternative focuses on the biological conversion of those substrates to oxygenated fuels in the form of mixed alcohols (MA). Both pathways rely on the production of polyurethanes from unsaturated fatty acids and valorization of unconverted solids for use as a material for synthesizing bioplastics. The assessment found similar, albeit slightly higher fuel yields and lower costs for the MA pathway, translating to a residual solids selling price of $899/ton for MA versus $1033/ton for MOTU as would be required to support a $2.50/gallon gasoline equivalent (GGE) fuel selling price. A variation of the MA pathway including subsequent upgrading of the mixed alcohols to hydrocarbon fuels (MAU) reflected a required solids selling price of $975/ton. CONCLUSION: The slight advantages observed for the MA pathway are partially attributed to a boundary that stops at oxygenated fuels versus fungible drop-in hydrocarbon fuels through a more complex MOTU configuration, with more comparable results obtained for the MAU scenario. In either case, it was shown that an integrated algal biorefinery can be economical through optimal strategies to utilize and valorize all fractions of the biomass.

Corrigendum: Corynebacterium glutamicum as an efficient omnivorous microbial host for the bioconversion of lignocellulosic biomass.

[This corrects the article DOI: 10.3389/fbioe.2022.827386.].

Application of attached algae flow-ways for coupling biomass production with the utilization of dilute non-point source nutrients in the Upper Laguna Madre, TX.

The purpose of this study is to determine the potential for an attached algae flow-way system to efficiently produce algal biomass in estuarine surface waters by utilizing dilute non-point source nutrients from local urban, industrial, and agricultural discharges into the Upper Laguna Madre, Corpus Christi, Texas. The study was conducted over the course of two years to establish seasonal base-line biomass productivity and composition for bioproducts applications, and to identify key environmental factors and flow-way cohorts impacting biomass production. For the entire cultivation period, continuous ash-free biomass production at 4 to 10 g/m2/day (corresponding to nutrient recovery at 300 to 500 mg of nitrogen/m2/day and 15 to 30 mg of phosphorus/m2/day) was successfully achieved without system restart. Upon start-up, a latency period was observed which indicates roles for species succession from relatively low productivity, high ash content pioneer periphytic culture composed primarily of benthic diatoms from the source waters to higher productivity, reduced ash content, and more resilient culture mainly composed of filamentous chlorophyta, Ulva lactuca. Principal Component Analysis (PCA) was used to identify environmental factors driving biomass production, and machine learning (ML) models were constructed to assess the predictive capability of the data set for system performance using the local multi-season environmental variations. Environmental datasets were segregated for ML training, validation, and testing using three methods: regression tree, ensemble regression, and Gaussian process regression (GPR). The predicted ash-free biomass productivity using ML models resulted in root-squared-mean-errors (RSME) from 1.78 to 1.86 g/m2/day, and R2 values from 0.67 to 0.75 using different methods. The greatest contributor to net productivity was total solar irradiation, followed by air temperature, salinity, and pH. The results of the study should be useful as a decision-making tool to application of attached algae flow-ways for biomass production while preventing algal blooms in the environment.

Conversion of Distiller's Grains to Renewable Fuels and High Value Protein: Integrated Techno-Economic and Life Cycle Assessment.

Distiller's grains are a byproduct of corn ethanol production and provide an opportunity for increasing the economic viability and sustainability of the overall grain-to-fuels process. Typically, these grains are dried and sold as a ruminant feed adjunct. This study considers utilization of the residuals in a novel supplementary fermentation process to produce two products, enriched protein and fusel alcohols. The value-added proposition and environmental impact of this second fermentation step for distiller's grains are evaluated by considering three different processing scenarios. Techno-economic results show the minimum protein selling price, assuming fusel alcohol products are valued at $0.79 per liter gasoline equivalent, ranges between $1.65-$2.48 kg protein-1 for the different cases. Environmental impacts of the systems were evaluated through life cycle assessment. Results show a baseline emission results of 17 g CO2-eq (MJ fuel)-1 for the fuel product and 10.3 kg CO2-eq kg protein-1 for the protein product. Sensitivity to allocation methods show a dramatic impact with results ranging between -8 to 140 g CO2-eq (MJ fuel)-1 for the fuel product and -0.3 to 6.4 kg CO2-eq kg protein-1 for the protein product. The discussion is focused on the potential impact of the technology on corn ethanol production economics and sustainability.

Development of a closed-loop process for fusel alcohol production and nutrient recycling from microalgae biomass.

Improving the economic feasibility is necessary for algae-based processes to achieve commercial scales for biofuels and bioproducts production. A closed-loop system for fusel alcohol production from microalgae biomass with integrated nutrient recycling was developed, which enables the reuse of nitrogen and phosphorus for downstream application and thus reduces the operational requirement for external major nutrients. Mixed fusel alcohols, primarily isobutanol and isopentanol were produced from Microchloropsis salina hydrolysates by an engineered E. coli co-culture. During the process, cellular nitrogen from microalgae biomass was converted into ammonium, whereas cellular phosphorus was liberated by an osmotic shock treatment. The formation of struvite from the liberated ammonium and phosphate, and the subsequent utilization of struvite to support M. salina cultivation was demonstrated. The closed loop system established here should help overcome one of the identified economic barriers to scale-up of microalgae production, and enhance the sustainability of microalgae-based chemical commodities production.

Monoterpene production by the carotenogenic yeast Rhodosporidium toruloides.

BACKGROUND: Due to their high energy density and compatible physical properties, several monoterpenes have been investigated as potential renewable transportation fuels, either as blendstocks with petroleum or as drop-in replacements for use in vehicles (both heavy and light-weight) or in aviation. Sustainable microbial production of these biofuels requires the ability to utilize cheap and readily available feedstocks such as lignocellulosic biomass, which can be depolymerized into fermentable carbon sources such as glucose and xylose. However, common microbial production platforms such as the yeast Saccharomyces cerevisiae are not naturally capable of utilizing xylose, hence requiring extensive strain engineering and optimization to efficiently utilize lignocellulosic feedstocks. In contrast, the oleaginous red yeast Rhodosporidium toruloides is capable of efficiently metabolizing both xylose and glucose, suggesting that it may be a suitable host for the production of lignocellulosic bioproducts. In addition, R. toruloides naturally produces several carotenoids (C40 terpenoids), indicating that it may have a naturally high carbon flux through its mevalonate (MVA) pathway, providing pools of intermediates for the production of a wide range of heterologous terpene-based biofuels and bioproducts from lignocellulose. RESULTS: Sixteen terpene synthases (TS) originating from plants, bacteria and fungi were evaluated for their ability to produce a total of nine different monoterpenes in R. toruloides. Eight of these TS were functional and produced several different monoterpenes, either as individual compounds or as mixtures, with 1,8-cineole, sabinene, ocimene, pinene, limonene, and carene being produced at the highest levels. The 1,8-cineole synthase HYP3 from Hypoxylon sp. E74060B produced the highest titer of 14.94 ± 1.84 mg/L 1,8-cineole in YPD medium and was selected for further optimization and fuel properties study. Production of 1,8-cineole from lignocellulose was also demonstrated in a 2L batch fermentation, and cineole production titers reached 34.6 mg/L in DMR-EH (Deacetylated, Mechanically Refined, Enzymatically Hydorlized) hydrolysate. Finally, the fuel properties of 1,8-cineole were examined, and indicate that it may be a suitable petroleum blend stock or drop-in replacement fuel for spark ignition engines. CONCLUSION: Our results demonstrate that Rhodosporidium toruloides is a suitable microbial platform for the production of non-native monoterpenes with biofuel applications from lignocellulosic biomass.

IRMOF-74(n)-Mg: a novel catalyst series for hydrogen activation and hydrogenolysis of C-O bonds.

Metal-Organic Frameworks (MOFs) that catalyze hydrogenolysis reactions are rare and there is little understanding of how the MOF, hydrogen, and substrate molecules interact. In this regard, the isoreticular IRMOF-74 series, two of which are known catalysts for hydrogenolysis of aromatic C-O bonds, provides an unusual opportunity for systematic probing of these reactions. The diameter of the 1D open channels can be varied within a common topology owing to the common secondary building unit (SBU) and controllable length of the hydroxy-carboxylate struts. We show that the first four members of the IRMOF-74(Mg) series are inherently catalytic for aromatic C-O bond hydrogenolysis and that the conversion varies non-monotonically with pore size. These catalysts are recyclable and reusable, retaining their crystallinity and framework structure after the hydrogenolysis reaction. The hydrogenolysis conversion of phenylethylphenyl ether (PPE), benzylphenyl ether (BPE), and diphenyl ether (DPE) varies as PPE > BPE > DPE, consistent with the strength of the C-O bond. Counterintuitively, however, the conversion also follows the trend IRMOF-74(III) > IRMOF-74(IV) > IRMOF-74(II) > IRMOF-74(I), with little variation in the corresponding selectivity. DFT calculations suggest the unexpected behavior is due to much stronger ether and phenol binding to the Mg(ii) open metal sites (OMS) of IRMOF-74(III), resulting from a structural distortion that moves the Mg2+ ions toward the interior of the pore. Solid-state 25Mg NMR data indicate that both H2 and ether molecules interact with the Mg(ii) OMS and hydrogen-deuterium exchange reactions show that these MOFs activate dihydrogen bonds. The results suggest that both confinement and the presence of reactive metals are essential for achieving the high catalytic activity, but that subtle variations in pore structure can significantly affect the catalysis. Moreover, they challenge the notion that simply increasing MOF pore size within a constant topology will lead to higher conversions.

Short-chain ketone production by engineered polyketide synthases in Streptomyces albus.

Microbial production of fuels and commodity chemicals has been performed primarily using natural or slightly modified enzymes, which inherently limits the types of molecules that can be produced. Type I modular polyketide synthases (PKSs) are multi-domain enzymes that can produce unique and diverse molecular structures by combining particular types of catalytic domains in a specific order. This catalytic mechanism offers a wealth of engineering opportunities. Here we report engineered microbes that produce various short-chain (C5-C7) ketones using hybrid PKSs. Introduction of the genes into the chromosome of Streptomyces albus enables it to produce >1 g · l-1 of C6 and C7 ethyl ketones and several hundred mg · l-1 of C5 and C6 methyl ketones from plant biomass hydrolysates. Engine tests indicate these short-chain ketones can be added to gasoline as oxygenates to increase the octane of gasoline. Together, it demonstrates the efficient and renewable microbial production of biogasolines by hybrid enzymes.

Hybrid phenolic-inducible promoters towards construction of self-inducible systems for microbial lignin valorization.

BACKGROUND: Engineering strategies to create promoters that are both higher strength and tunable in the presence of inexpensive compounds are of high importance to develop metabolic engineering technologies that can be commercialized. Lignocellulosic biomass stands out as the most abundant renewable feedstock for the production of biofuels and chemicals. However, lignin a major polymeric component of the biomass is made up of aromatic units and remains as an untapped resource. Novel synthetic biology tools for the expression of heterologous proteins are critical for the effective engineering of a microbe to valorize lignin. This study demonstrates the first successful attempt in the creation of engineered promoters that can be induced by aromatics present in lignocellulosic hydrolysates to increase heterologous protein production. RESULTS: A hybrid promoter engineering approach was utilized for the construction of phenolic-inducible promoters of higher strength. The hybrid promoters were constructed by replacing the spacer region of an endogenous promoter, PemrR present in E. coli that was naturally inducible by phenolics. In the presence of vanillin, the engineered promoters Pvtac, Pvtrc, and Pvtic increased protein expression by 4.6-, 3.0-, and 1.5-fold, respectively, in comparison with a native promoter, PemrR. In the presence of vanillic acid, Pvtac, Pvtrc, and Pvtic improved protein expression by 9.5-, 6.8-, and 2.1-fold, respectively, in comparison with PemrR. Among the cells induced with vanillin, the emergence of a sub-population constituting the healthy and dividing cells using flow cytometry was observed. The analysis also revealed this smaller sub-population to be the primary contributor for the increased expression that was observed with the engineered promoters. CONCLUSIONS: This study demonstrates the first successful attempt in the creation of engineered promoters that can be induced by aromatics to increase heterologous protein production. Employing promoters inducible by phenolics will provide the following advantages: (1) develop substrate inducible systems; (2) lower operating costs by replacing expensive IPTG currently used for induction; (3) develop dynamic regulatory systems; and (4) provide flexibility in operating conditions. The flow cytometry findings strongly suggest the need for novel approaches to maintain a healthy cell population in the presence of phenolics to achieve increased heterologous protein expression and, thereby, valorize lignin efficiently.

Engineering Escherichia coli for the production of terpene mixture enriched in caryophyllene and caryophyllene alcohol as potential aviation fuel compounds.

Recent studies have revealed that caryophyllene and its stereoisomers not only exhibit multiple biological activities but also have desired properties as renewable candidates for ground transportation and jet fuel applications. This study presents the first significant production of caryophyllene and caryolan-1-ol by an engineered E. coli with heterologous expression of mevalonate pathway genes with a caryophyllene synthase and a caryolan-1-ol synthase. By optimizing metabolic flux and fermentation parameters, the engineered strains yielded 449 mg/L of total terpene, including 406 mg/L sesquiterpene with 100 mg/L caryophyllene and 10 mg/L caryolan-1-ol. Furthermore, a marine microalgae hydrolysate was used as the sole carbon source for the production of caryophyllene and other terpene compounds. Under the optimal fermentation conditions, 360 mg/L of total terpene, 322 mg/L of sesquiterpene, and 75 mg/L caryophyllene were obtained from the pretreated algae hydrolysates. The highest yields achieved on the biomass basis were 48 mg total terpene/g algae and 10 mg caryophyllene/g algae and the caryophyllene yield is approximately ten times higher than that from plant tissues by solvent extraction. The study provides a sustainable alternative for production of caryophyllene and its alcohol from microalgae biomass as potential candidates for next generation aviation fuels.

Impact of Flue Gas Compounds on Microalgae and Mechanisms for Carbon Assimilation and Utilization.

To shift the world to a more sustainable future, it is necessary to phase out the use of fossil fuels and focus on the development of low-carbon alternatives. However, this transition has been slow, so there is still a large dependence on fossil-derived power, and therefore, carbon dioxide is released continuously. Owing to the potential for assimilating and utilizing carbon dioxide to generate carbon-neutral products, such as biodiesel, the application of microalgae technology to capture CO2 from flue gases has gained significant attention over the past decade. Microalgae offer a more sustainable source of biomass, which can be converted into energy, over conventional fuel crops because they grow more quickly and do not adversely affect the food supply. This review focuses on the technical feasibility of combined carbon fixation and microalgae cultivation for carbon reuse. A range of different carbon metabolisms and the impact of flue gas compounds on microalgae are appraised. Fixation of flue gas carbon dioxide is dependent on the selected microalgae strain and on flue gas compounds/concentrations. Additionally, current pilot-scale demonstrations of microalgae technology for carbon dioxide capture are assessed and its future prospects are discussed. Practical implementation of this technology at an industrial scale still requires significant research, which necessitates multidisciplinary research and development to demonstrate its viability for carbon dioxide capture from flue gases at the commercial level.

Bioconversion of distillers' grains hydrolysates to advanced biofuels by an Escherichia coli co-culture.

BACKGROUND: First generation bioethanol production utilizes the starch fraction of maize, which accounts for approximately 60% of the ash-free dry weight of the grain. Scale-up of this technology for fuels applications has resulted in a massive supply of distillers' grains with solubles (DGS) coproduct, which is rich in cellulosic polysaccharides and protein. It was surmised that DGS would be rapidly adopted for animal feed applications, however, this has not been observed based on inconsistency of the product stream and other logistics-related risks, especially toxigenic contaminants. Therefore, efficient valorization of DGS for production of petroleum displacing products will significantly improve the techno-economic feasibility and net energy return of the established starch bioethanol process. In this study, we demonstrate 'one-pot' bioconversion of the protein and carbohydrate fractions of a DGS hydrolysate into C4 and C5 fusel alcohols through development of a microbial consortium incorporating two engineered Escherichia coli biocatalyst strains. RESULTS: The carbohydrate conversion strain E. coli BLF2 was constructed from the wild type E. coli strain B and showed improved capability to produce fusel alcohols from hexose and pentose sugars. Up to 12 g/L fusel alcohols was produced from glucose or xylose synthetic medium by E. coli BLF2. The second strain, E. coli AY3, was dedicated for utilization of proteins in the hydrolysates to produce mixed C4 and C5 alcohols. To maximize conversion yield by the co-culture, the inoculation ratio between the two strains was optimized. The co-culture with an inoculation ratio of 1:1.5 of E. coli BLF2 and AY3 achieved the highest total fusel alcohol titer of up to 10.3 g/L from DGS hydrolysates. The engineered E. coli co-culture system was shown to be similarly applicable for biofuel production from other biomass sources, including algae hydrolysates. Furthermore, the co-culture population dynamics revealed by quantitative PCR analysis indicated that despite the growth rate difference between the two strains, co-culturing didn't compromise the growth of each strain. The q-PCR analysis also demonstrated that fermentation with an appropriate initial inoculation ratio of the two strains was important to achieve a balanced co-culture population which resulted in higher total fuel titer. CONCLUSIONS: The efficient conversion of DGS hydrolysates into fusel alcohols will significantly improve the feasibility of the first generation bioethanol process. The integrated carbohydrate and protein conversion platform developed here is applicable for the bioconversion of a variety of biomass feedstocks rich in sugars and proteins.

Characterization of four endophytic fungi as potential consolidated bioprocessing hosts for conversion of lignocellulose into advanced biofuels.

Recently, several endophytic fungi have been demonstrated to produce volatile organic compounds (VOCs) with properties similar to fossil fuels, called "mycodiesel," while growing on lignocellulosic plant and agricultural residues. The fact that endophytes are plant symbionts suggests that some may be able to produce lignocellulolytic enzymes, making them capable of both deconstructing lignocellulose and converting it into mycodiesel, two properties that indicate that these strains may be useful consolidated bioprocessing (CBP) hosts for the biofuel production. In this study, four endophytes Hypoxylon sp. CI4A, Hypoxylon sp. EC38, Hypoxylon sp. CO27, and Daldinia eschscholzii EC12 were selected and evaluated for their CBP potential. Analysis of their genomes indicates that these endophytes have a rich reservoir of biomass-deconstructing carbohydrate-active enzymes (CAZys), which includes enzymes active on both polysaccharides and lignin, as well as terpene synthases (TPSs), enzymes that may produce fuel-like molecules, suggesting that they do indeed have CBP potential. GC-MS analyses of their VOCs when grown on four representative lignocellulosic feedstocks revealed that these endophytes produce a wide spectrum of hydrocarbons, the majority of which are monoterpenes and sesquiterpenes, including some known biofuel candidates. Analysis of their cellulase activity when grown under the same conditions revealed that these endophytes actively produce endoglucanases, exoglucanases, and ß-glucosidases. The richness of CAZymes as well as terpene synthases identified in these four endophytic fungi suggests that they are great candidates to pursue for development into platform CBP organisms.

Growth of mono- and mixed cultures of Nannochloropsis salina and Phaeodactylum tricornutum on struvite as a nutrient source.

The suitability of crude and purified struvite (MgNH4PO4), a major precipitate in wastewater streams, was investigated for renewable replacement of conventional nitrogen and phosphate resources for cultivation of microalgae. Bovine effluent wastewater stone, the source of crude struvite, was characterized for soluble N/P, trace metals, and biochemical components and compared to the purified mineral. Cultivation trials using struvite as a major nutrient source were conducted using two microalgae production strains, Nannochloropsis salina and Phaeodactylum tricornutum, in both lab and outdoor pilot-scale raceways in a variety of seasonal conditions. Both crude and purified struvite-based media were found to result in biomass productivities at least as high as established media formulations (maximum outdoor co-culture yield ∼20±4gAFDW/m(2)/day). Analysis of nutrient uptake by the alga suggest that struvite provides increased nutrient utilization efficiency, and that crude struvite satisfies the trace metals requirement and results in increased pigment productivity for both microalgae strains.

The role of photo-osmotic adaptation in semi-continuous culture and lipid particle release from Dunaliella viridis.

Although great efforts have been made to elucidate the phenotypic responses of alga to varying levels of nutrients, osmotic environments, and photosynthetically active radiation intensities, the role of interactions among these variables is largely nebulous. Here, we describe a general method for establishing and maintaining semi-continuous cultures of the halophilic microalgal production strain, Dunaliella viridis, that is independent of variations in salinity and illumination intensity. Using this method, the cultures were evaluated to elucidate the overlapping roles of photosynthetic and osmotic adaptation on the accumulation and compositional variation of the biomass, photosynthetic productivity, and physiological biomarkers, as well as spectroscopic and morphological details at the single-cell level. Correlation matrices defining the relationships among the observables and based on variation of the illumination intensity and salinity were constructed for predicting bioproduct yields for varying culture conditions. Following maintenance of stable cultures for 6-week intervals, phenotypic responses to photo-osmotic drift were explored using a combination of single-cell hyperspectral fluorescence imaging and flow cytometry. In addition to morphological changes, release of lipid microparticles from the cells that is disproportionate to cell lysis was observed under hypotonic drift, indicating the existence of a reversible membrane permeation mechanism in Dunaliella. This phenomenon introduces the potential for low-cost strategies for recovering lipids and pigments from the microalgae by minimizing the requirement for energy intensive harvesting and dewatering of the biomass. The results should be applicable to outdoor culture, where seasonal changes resulting in variable solar flux and precipitation and evaporation rates are anticipated.

Multiplex fluorometric assessment of nutrient limitation as a strategy for enhanced lipid enrichment and harvesting of Neochloris oleoabundans.

Detailed in this study are the results of fluorometric assays used to assess the impact of gradual nutrient limitation versus punctuated nitrate limitation on the lipid content and morphology of Neochloris oleoabundans cells in batch culture. Punctuated nitrate limitation was imposed during pre-log, log, late-log, stationary, and senescent growth phases, and the cells were analyzed by bulk fluorescence emission, flow cytometry, and hyperspectral fluorescence imaging. In addition to intrinsic spectroscopic signatures provided by scatter and endogenous fluorescence, Nile Red staining was employed to monitor relative changes in lipid concentration. Analysis of the fluorescence images and temporal data sets was performed using multivariate curve resolution and fitting to logistic growth models to extract parameters of interest. The spectral components independently isolated from the image and temporal data sets showed close agreement with one another, especially relating to chlorophylls and Nile Red in polar and neutral lipid fractions, respectively. The fastest accumulation and highest total neutral lipid per cell and per chlorophyll were obtained with punctuated nitrate limitation during log phase growth on day 4 of culture. The presence of unbound chlorophyll in the resulting lipid bodies supports a membrane recycling TAG accumulation mechanism mediated by chloropolast-ER lipid exchange. Furthermore, an increase in cell size, indicated by forward scatter, was also found to correlate with increased neutral lipid, providing a size selection mechanism for passive harvest of algal cells at peak lipid enrichment.