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All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Data for MALDI-MS Screening of Microbial Colonies With Isomer Resolution to Select Fatty Acid Desaturase Variants

Creating controlled lipid unsaturation locations in oleochemicals can be a key to many bioengineered products. However, evaluating the effects of modifications to the acyl-ACP desaturase on lipid unsaturation is not currently amenable to high-throughput assays, limiting the scale of redesign efforts to <200 variants. Here, we report a rapid mass spectrometry (MS) assay for profiling the positions of double bonds on membrane lipids produced by Escherichia coli colonies after treatment with ozone gas. By MS measurement of the ozonolysis products of Δ6 and Δ8 isomers of membrane lipids from colonies expressing recombinant Thunbergia alata desaturase, we screened a randomly mutagenized library of the desaturase gene at 5 s per sample. Two variants with altered regiospecificity were isolated, indicated by an increase in 16:1 Δ8 proportion. We also demonstrated the ability of these desaturase variants to influence the membrane composition and fatty acid distribution of E. coli strains deficient in the native acyl-ACP desaturase gene, fabA. Finally, we used the fabA deficient chassis to concomitantly express a non-native acyl-ACP desaturase and a medium-chain thioesterase from Umbellularia californica, demonstrating production of only saturated free fatty acids.

Conversion↗
DOE OSTI2022

Metabolic engineering strategies to produce medium-chain oleochemicals via acyl-ACP:CoA transacylase activity

Abstract Microbial lipid metabolism is an attractive route for producing oleochemicals. The predominant strategy centers on heterologous thioesterases to synthesize desired chain-length fatty acids. To convert acids to oleochemicals (e.g., fatty alcohols, ketones), the narrowed fatty acid pool needs to be reactivated as coenzyme A thioesters at cost of one ATP per reactivation - an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. Here, we demonstrate such an alternative acyl-transferase strategy by heterologous expression of PhaG, an enzyme first identified in Pseudomonads , that transfers 3-hydroxy acyl-chains between acyl-carrier protein and coenzyme A thioester forms for creating polyhydroxyalkanoate monomers. We use it to create a pool of acyl-CoA’s that can be redirected to oleochemical products. Through bioprospecting, mutagenesis, and metabolic engineering, we develop three strains of Escherichia coli capable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Pfleger-Lab/Metabolic-Modeling---Yield-Analysis-of-PhaG

Microbial lipid metabolism is an attractive route for producing oleochemicals. The predominant strategy centers on heterologous thioesterases to synthesize desired chain-length fatty acids. To convert acids to oleochemicals (e.g., fatty alcohols, ketones), the narrowed fatty acid pool needs to be reactivated as coenzyme A thioesters at cost of one ATP per reactivation – an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. Here, we demonstrate such an alternative acyl-transferase strategy by heterologous expression of PhaG, an enzyme first identified in Pseudomonads, that transfers 3-hydroxy acyl-chains between acyl-carrier protein and coenzyme A thioester forms for creating polyhydroxyalkanoate monomers. We use it to create a pool of acyl-CoA’s that can be redirected to oleochemical products. Through bioprospecting, mutagenesis, and metabolic engineering, we develop three strains of Escherichia coli capable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones.

Yan, Qiang↗
DOE OSTI2021

EnZymClass: Substrate specificity prediction tool of plant acyl-ACP thioesterases based on ensemble learning

Characterizing the functional properties of plant acyl-ACP thioesterases (TEs), a key enzyme class used in the production of renewable oleochemicals in microbial hosts, experimentally, can be an expensive and time consuming process since it requires manual screening of thousands of candidates in a database. Using amino acid sequence to computationally predict an enzyme’s function might accelerate this process; however obtaining the necessary amount of information on previously characterized enzymes and their respective sequences required by standard Machine Learning (ML) based approaches to accurately infer sequence-function relationships can be prohibitive, especially with a low-throughput testing cycle. Experimental noise, unbalanced dataset where high sequence similarity does not always imply identical functional properties will further prevent robust prediction performance. Herein we present a ML method, Ensemble method for enZyme Classification (EnZymClass), that is specifically designed to address these issues. We used EnZymClass to classify TEs into short, long and mixed free fatty acid substrate specificity categories. While general guidelines for inferring substrate specificity have been proposed before, prediction of chain-length preference from primary sequence has remained elusive for plant acyl-ACP TEs. By applying EnZymClass to a subset of TEs in the ThYme database, we identified two medium chain TEs, ClFatB3 and CwFatB2, with previously uncharacterized activity in E. coli fatty acid production hosts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

deeprob/ThioesteraseEnzymeSpecificity: EnZymClass-first-release

Characterizing the functional properties of plant acyl-ACP thioesterases (TEs), a key enzyme class used in the production of renewable oleochemicals in microbial hosts, experimentally, can be an expensive and time consuming process since it requires manual screening of thousands of candidates in a database. Using amino acid sequence to computationally predict an enzyme’s function might accelerate this process; however obtaining the necessary amount of information on previously characterized enzymes and their respective sequences required by standard Machine Learning (ML) based approaches to accurately infer sequence-function relationships can be prohibitive, especially with a low-throughput testing cycle. Experimental noise, unbalanced dataset where high sequence similarity does not always imply identical functional properties will further prevent robust prediction performance. Herein we present a ML method, Ensemble method for enZyme Classification (EnZymClass), that is specifically designed to address these issues. We used EnZymClass to classify TEs into short, long and mixed free fatty acid substrate specificity categories. While general guidelines for inferring substrate specificity have been proposed before, prediction of chain-length preference from primary sequence has remained elusive for plant acyl-ACP TEs. By applying EnZymClass to a subset of TEs in the ThYme database, we identified two medium chain TEs, ClFatB3 and CwFatB2, with previously uncharacterized activity in E. coli fatty acid production hosts.

Banerjee, Deepro↗
DOE OSTI2022

Different feeding patterns affect meat quality of Tibetan pigs associated with intestinal microbiota alterations

This study aimed to investigate the effects of different feeding patterns on meat quality, gut microbiota and its metabolites of Tibetan pigs. Tibetan pigs with similar body weight were fed the high energy diets ( HEP , 20 pigs) and the regular diets ( RFP , 20 pigs), and free-ranging Tibetan pigs ( FRP , 20 pigs) were selected as the reference. After 6 weeks of experiment, meat quality indexes of semitendinosus muscle ( SM ) and cecal microbiota were measured. The results of meat quality demonstrated that the shear force of pig SM in FRP group was higher than that in HEP and RFP groups ( p < 0.001); the pH-value of SM in HEP pigs was higher at 45 min ( p < 0.05) and lower at 24 h ( p < 0.01) after slaughter than that in FRP and RFP groups; the SM lightness ( L* value) of FRP pigs increased compared with RFP and HEP groups ( p < 0.001), while the SM redness ( a* value) of FRP pigs was higher than that of RFP group ( p < 0.05). The free fatty acid ( FA ) profile exhibited that the total FAs and unsaturated FAs of pig SM in HEP and RFP groups were higher than those in FRP group ( p < 0.05); the RFP pigs had more reasonable FA composition with higher n-3 polyunsaturated FAs ( PUFAs ) and lower n-6/n-3 PUFA ratio than HEP pigs ( p < 0.05). Based on that, we observed that Tibetan pigs fed high energy diets (HEP) had lower microbial α-diversity in cecum ( p < 0.05), and distinct feeding patterns exhibited a different microbial cluster. Simultaneously, the short-chain FA levels in cecum of FRP and RFP pigs were higher compared with HEP pigs ( p < 0.05). A total of 11 genera related to muscle lipid metabolism or meat quality, including Alistipes , Anaerovibrio , Acetitomaculun , etc., were identified under different feeding patterns ( p < 0.05). Spearman correlation analysis demonstrated that alterations of free FAs in SM were affected by the genera Prevotellaceae_NK3B31_group , Prevotellaceae UCG-003 and Christensenellaceae_R-7_group ( p < 0.05). Taken together, distinct feeding patterns affected meat quality of Tibetan pigs related to gut microbiota alterations.

Zhu, Yanbin↗
DOE OSTI2023

Coconut oil derived five‐component synthetic oviposition deterrent for oriental fruit fly, Bactrocera dorsalis

Abstract BACKGROUND Bactrocera dorsalis, oriental fruit fly (OFF), is one of the most destructive agricultural pests. Although bait sprays can effectively control OFF,resistance development has been a concern. We evaluated the oviposition deterrent activity of coconut free fatty acids (CFFA), a mixture of eight coconut oil‐derived fatty acids known to repel hematophagous insects and deter their feeding and oviposition, against OFF females. RESULTS In laboratory 72‐h two‐choice assays using guava‐juice infused‐agar as an oviposition substrate, CFFA deterred OFF oviposition in a dose‐dependent manner with the greatest reduction of 87% at 20 mg dose compared to the control. When the eight CFFA components were tested individually, four compounds (caprylic, capric, oleic, and linoleic acids) significantly reduced OFF oviposition (‘negative‐compounds’), two (lauric and myristic acids) had no effect (‘neutral‐compounds’), and two (palmitic and stearic acids) stimulated OFF oviposition (‘positive‐compounds’). In two‐choice tests, the ‘negative‐compounds’ blend failed to elicit the same level of oviposition reduction as CFFA at equivalent concentrations found in CFFA. Adding the two ‘neutral‐compounds’ recovered the oviposition deterrence similar to CFFA. Subsequent subtraction tests showed that four ‘negative‐compounds’ plus lauric acid was as effective as CFFA in reducing OFF oviposition in guava‐juice agar. This five‐component key‐deterrent blend also reduced OFF oviposition by 95 and 72% on papaya and tomato fruit, respectively. CONCLUSION CFFA acts as an oviposition deterrent for OFF. Given that CFFA compounds are generally regarded as safe for humans and the environment, CFFA and its bioactive components have potential use in behavioral control strategies against OFF. © 2023 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Agriculture↗
DOE OSTI2021

Hydrothermal pretreatment for valorization of genetically engineered bioenergy crop for lipid and cellulosic sugar recovery

Lipids accumulated in the vegetative tissues of cellulosic feedstocks can be a potential raw material for biodiesel and bioethanol production. In this work, bagasse of genetically engineered sorghum was subjected to liquid hot-water pretreatment at 170, 180, and 190°C for different reaction time. Under the optimal pretreatment condition (170°C, 20 min), the residue was enriched in glucan (57.39 ± 2.63 % w/w) and xylan (13.38 ± 0.49 % w/w). The total lipid content of the pretreated residue was 6.81% w/w, similar to that observed in untreated bagasse (6.30% w/w). Pretreatment improved the enzymatic digestibility of bagasse, allowing a recovery of 79% w/w and 86% w/w of glucose and xylose, respectively. The pretreatment and enzymatic saccharification resulted in a 2-fold increase in total lipid in enzymatic residue compared to the original bagasse. Furthermore, pretreatment and enzymatic hydrolysis enabled high sugar recovery while concentrating triglycerides and free fatty acids in the residue.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Renewable diesel and bio-aromatics production from waste cooking oil using ethanol as a hydrogen donor in deoxygenation reaction

Biofuels offer a promising solution in the fight against climate change. With a global increase in waste cooking oil, this research investigated the production of bio-hydrogenated diesel (BHD) from waste cooking oil, using ethanol as a hydrogen donor in the deoxygenation process. A hydrolyzed waste cooking oil model compound served as the feedstock, and the deoxygenation was performed at 300–400 °C. The catalysts used in the experiments were 2.6 wt% Ni and 7.8 wt% Mo (2.6Ni-7.8Mo) and 10 wt% Ni and 5 wt% Mo (10Ni-5Mo) on γ-Al 2 O 3 . The results showed that ethanol is an effective hydrogen donor for biofuel production without the need for external hydrogen at an elevated pressure. The increasing temperature enhanced the free fatty acid (FFA) conversion and n-alkane selectivity in the oil product, with the highest FFA conversion and alkane selectivity of 100 % and 46 %, respectively, observed at 400 °C for the sulfided 10Ni-5Mo catalyst. On the other hand, 2.6Ni-7.8Mo offers 100 % FFA conversion with a lower n-alkane selectivity of 35 % at identical temperatures. The total acid number (TAN) of the oil products decreased from 174.03 mg KOH/g of feedstock to 9.43 and 8.67 mg KOH/g with the sulfided 2.6Ni-7.8Mo and 10Ni-5Mo catalysts, respectively. Both the catalysts achieved similar heating values (~43 MJ/kg) at 400 °C. This is a significant improvement to the HHV of the feedstock, which was 36.02 MJ/kg. Additionally, aromatic compounds, mainly BTXE (benzene, toluene, xylene, and ethylbenzene), were also produced. Compared to glycerol as a hydrogen donor, ethanol more effectively increased n-alkane selectivity due to its higher effective hydrogen-to-carbon ratio (H/C eff ). Conversely, glycerol was more advantageous for achieving greater selectivity towards BTXE compounds due to its lower H/C eff , which potentially leads to coke formation. Since aromatic compounds are intermediates in coke production, glycerol provides higher aromatic selectivity than ethanol. Finally, this study presents an alternative pathway for producing diesel fuel from waste cooking oil using ethanol as a hydrogen donor.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Cooling and dietary crude protein affected milk production on heat-stressed dairy cows

Environmental factors, such as temperature and humidity, exert dramatic effects on the production, health, and well-being of lactating dairy cows. Heat-stressed cows undergo a series of homeorhetic adaptations that alters cellular homeostasis and nutrient utilization. As such, the objective of this study was to determine the effects of cooling and dietary crude protein (CP) content on fermentability of rumen inoculum, milk production, and health of heat-stressed cows. It was hypothesized that cooled but not heat-stressed cows would support productivity in response to an increase in dietary CP content. Thirty-six multiparous Holstein cows were randomly assigned to 1 of 4 treatments in a 2 × 2 factorial arrangement (9 cows/treatment). Treatments consisted of cooling (CO) or moderate long-term heat stress (HS) and diets containing low CP content (LCP; 12.5% CP) or moderate CP content (MCP; 16.1% CP) of dry matter. The CO treatment provided heat abatement with sprinklers and fans that came on at > 20.0°C, whereas the HS treatment did not provide sprinklers and fans. Cows were housed in pens in a freestall barn, and treatments were imposed for 21 d during July and August. Compared with the HS treatment, CO decreased afternoon rectal and vaginal temperatures (0.60 and 0.70°C) and afternoon respiration rate (27.6 breaths/min). Treatment interactions showed that compared with the LCP treatment, MCP increased milk fat yield (23%) in CO but not in HS cows. Compared with the LCP treatment, MCP decreased milk protein percent (0.14 units) in CO but increased milk protein percent (0.09 units) in HS cows. Rumen inoculum collected on d 21 showed a ~3-fold increase in total gas production in vitro in CO compared with the HS treatment. Relative to the LCP treatment, MCP increased plasma glucose and insulin and decreased total free fatty acids concentrations. In summary, under the conditions of this study CO but not HS cows responded to a CP stimulus by increasing productivity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Using molecular simulation to understand the skin barrier

Skin's effectiveness as a barrier to permeation of water and other chemicals rests almost entirely in the outermost layer of the epidermis, the stratum corneum (SC), which consists of layers of corneocytes surrounded by highly organized lipid lamellae. As the only continuous path through the SC, transdermal permeation necessarily involves diffusion through these lipid layers. The role of the SC as a protective barrier is supported by its exceptional lipid composition consisting of ceramides (CERs), cholesterol (CHOL), and free fatty acids (FFAs) and the complete absence of phospholipids, which are present in most biological membranes. Molecular simulation, which provides molecular level detail of lipid configurations that can be connected with barrier function, has become a popular tool for studying SC lipid systems. We review this ever-increasing body of literature with the goals of (1) enabling the experimental skin community to understand, interpret and use the information generated from the simulations, (2) providing simulation experts with a solid background in the chemistry of SC lipids including the composition, structure and organization, and barrier function, and (3) presenting a state of the art picture of the field of SC lipid simulations, highlighting the difficulties and best practices for studying these systems, to encourage the generation of robust reproducible studies in the future. This review describes molecular simulation methodology and then critically examines results derived from simulations using atomistic and then coarse-grained models.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Disruption of acyl-acyl carrier protein (acyl-ACP) synthetase in cyanobacteria impairs lipid remodeling as revealed by acyl-ACP measurements

Free fatty acid (FFA) production in bacteria is a key target for metabolic engineering. The knockout of the acyl-ACP synthetase (AAS) prevents reincorporation of FFA into the fatty acid biosynthetic cycle and is widely used to enhance their secretion. However, the role of AAS in membrane lipid remodeling under environmental stress, such as altered temperature, remains poorly understood. In cyanobacteria, temperature shifts are known to affect fatty acid desaturation and membrane fluidity, yet it is unclear whether AAS contributes to these adaptive responses through re-esterification of membrane-released acyl chains. We elucidated unique aspects of fatty acid metabolism in response to temperature changes in biotechnologically relevant microbes with the development of an efficient method for quantifying acyl-ACP intermediates using anion exchange chromatography (AEX). In Escherichia coli, which performs desaturation during fatty acid biosynthesis, we detected saturated and unsaturated acyl-ACPs that confirm biosynthetic pathway operation. In the cyanobacteria, Picosynechococcus sp. PCC 7002 and the Δaas strain, changes between two temperatures were interpreted with support from proteomic and lipidomic analyses and indicated that the AAS is tied to membrane lipid remodeling. Further, polyunsaturated acyl-ACPs were detected in the Δaas strain, which was unexpected because fatty acid synthesis does not produce polyunsaturates in cyanobacteria, suggesting the presence of alternative acyl-activating enzymes or unknown acyl-ACP desaturases. This study highlights the possible link between acyl chain recycling and lipid remodeling in cyanobacteria and demonstrates the utility of AEX-based acyl-ACP profiling in dissecting fatty acid metabolism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A Fast-Pass, Desorption Electrospray Ionization Mass Spectrometry Strategy for Untargeted Metabolic Phenotyping

Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) provides direct analytical readouts of small molecules that can be used to characterize the metabolic phenotypes of genetically engineered bacteria. In an effort to accelerate the time frame associated with the screening of mutant libraries, we have developed a high-throughput DESI-MSI analytical workflow implementing a single raster line-scan strategy that facilitates the collection of location-resolved molecular information from engineered strains on a subminute time scale. Evaluation of this “Fast-Pass” DESI-MSI phenotyping workflow on analytical standards demonstrated the capability of acquiring full metabolic profiling information with a throughput of ~40 s per sample. This Fast-Pass strategy was implemented in the analysis of genetically edited Escherichia coli strains that have been engineered to produce various free-fatty acids (FFAs) for applications relevant to biofuels. Due to the untargeted nature of DESI-MSI, the investigation of these strains yielded molecular information for both global metabolites and targeted detection of accumulated bioproducts, allowing simultaneous readouts of strain-specific chemical profiles and comparative measurements of FFA production levels.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Design and synthesis of biobased superhydrophobic biochar catalyst derived from Citrus sinensis for biodiesel production using inedible oil feedstocks

In an one-pot in situ trans/esterification of low-grade feedstocks, the simultaneous presence of triglycerides (TAGs) and free fatty acids (FFAs) presents a dual catalytic challenge. While TAGs undergo transesterification to form biodiesel and glycerol, FFAs present in the feedstocks are esterified simultaneously, producing water as a by-product. This water can deactivate acid catalysts by interacting with their active sites, reducing catalytic efficiency and reusability. To overcome this, we developed a hydrophobic sulfonic acid-functionalized biochar catalyst derived from Citrus sinensis (orange peel) via sulfonation with H 2 SO 4 and subsequent silylation using hexamethyldisilazane (HMDS). The optimized catalyst exhibited a high surface area (355.09 m 2 g −1 ), sulfur content (5.33 wt%), and strong hydrophobicity (water contact angle: 154°). Compared to non-hydrophobic analogs, it showed enhanced activity and reusability (up to 10 cycles). Using response surface methodology with central composite design, a biodiesel yield of 99.1 ± 0.4% was achieved under optimal conditions. Life cycle assessment was performed to evaluate the environmental impacts of biodiesel production utilising the synthesized catalyst, considering 1000 kg of biodiesel produced as 1 functional unit. In conclusion, the recorded results showed the cumulative abiotic depletion of fossil resources over the entire biodiesel production process as 87 243.423 MJ and global warming potential as 4103.494 kg CO 2 equivalent.

Biochar↗
DOE OSTI2024

The human plasma lipidome response to exertional heat tolerance testing

The year of 2023 displayed the highest average global temperatures recorded in history— the duration and severity of extreme heat are projected to increase. Rising global temperatures represent a major public health threat, especially to occupations exposed to hot environments, such as construction and agricultural workers, and first responders. Despite efforts of the scientific community, there is still a need to characterize the pathophysiological processes leading to heat related illness and develop biomarkers that can predict its onset. Here, we performed a plasma lipidomic analysis on male and female subjects who underwent heat tolerance testing (HTT), consisting of a 2-h treadmill walk at 5 km/h with 2% inclination at a controlled temperature of 40oC. We identified 995 lipids from 27 classes, with nearly half of all detected lipids being responsive to HTT. Lipid classes related to substrate utilization were predominantly affected by HTT, with a downregulation of triacylglycerols and upregulation of free fatty acids and acyl-carnitines. We additionally examined correlations between changes in plasma lipids by using the even chain acyl-carnitines is physiological strain index (PSI). Here, even chain acyl-carnitines, bi-products of incomplete beta oxidation, and diacylglycerols displayed the highest correlation to PSI. PSI did not correlate with plasma lactate levels, which suggests that correlations with related to metabolic efficiency versus physical exertion. Overall, our results show that HTT has a strong impact on the plasma lipidome and that incomplete beta oxidation may underlie heat intolerance.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Data for Hydrothermal Pretreatment for Valorization of Genetically Engineered Bioenergy Crop for Lipid and Cellulosic Sugar Recovery

Lipids accumulated in the vegetative tissues of cellulosic feedstocks can be a potential raw material for biodiesel and bioethanol production. In this work, bagasse of genetically engineered sorghum was subjected to liquid hot-water pretreatment at 170, 180, and 190 °C for different reaction time. Under the optimal pretreatment condition (170 °C, 20 min), the residue was enriched in glucan (57.39 ± 2.63 % w/w) and xylan (13.38 ± 0.49 % w/w). The total lipid content of the pretreated residue was 6.81% w/w, similar to that observed in untreated bagasse (6.30% w/w). Pretreatment improved the enzymatic digestibility of bagasse, allowing a recovery of 79% w/w and 86% w/w of glucose and xylose, respectively. The pretreatment and enzymatic saccharification resulted in a 2-fold increase in total lipid in enzymatic residue compared to the original bagasse. Thus, pretreatment and enzymatic hydrolysis enabled high sugar recovery while concentrating triglycerides and free fatty acids in the residue.

Conversion↗
DOE OSTI2022

Data for Coprocessing Corn Germ Meal for Oil Recovery and Ethanol Production, a Process Model for Lipid-Producing Energy Crops

Efforts to engineer high-productivity crops to accumulate oils in their vegetative tissue present the possibility of expanding biodiesel production. However, processing the new crops for lipid recovery and ethanol production from cell wall saccharides is challenging and expensive. In a previous study using corn germ meal as a model substrate, we reported that liquid hot water (LHW) pretreatment enriched the lipid concentration by 2.2 to 4.2 fold. This study investigated combining oil recovery with ethanol production by extracting oil following LHW and simultaneous saccharification and co-fermentation (SSCF) of the biomass. Corn germ meal was again used to model the oil-bearing energy crops. Pretreated germ meal hydrolysate or solids (160 °C and 180 °C for 10 minutes) were fermented, and lipids were extracted from both the spent fermentation whole broth and fermentation solids, which were recovered by centrifugation and convective drying. Lipid contents in spent fermentation solids increased 3.7 to 5.7 fold compared to the beginning germ meal. The highest lipid yield achieved after fermentation was 36.0 mg lipid g−1 raw biomass; the maximum relative amount of triacylglycerol (TAG) was 50.9% of extracted oil. Although the fermentation step increased the lipid concentration of the recovered solids, it did not improve the lipid yields of pretreated biomass and detrimentally affected oil compositions by increasing the relative concentrations of free fatty acids.

Conversion↗
DOE OSTI2020

Conceptual Basis and Techno-Economic Modeling for Integrated Algal Biorefinery Conversion of Microalgae to Fuels and Products (2019 NREL TEA Update: Highlighting Paths to Future Cost Goals via a New Pathway for Combined Algal Processing)

The report documents the conceptual basis for a new potential Combined Algal Processing design strategy which may allow more flexibility in accommodating different algal biomass feedstock compositions, by enabling upgrading of both protein and carbohydrates in a single step, without a strict requirement for either component to be in soluble or monomeric form, while maintaining effective wet lipid extraction techniques to enable high lipid recoveries. In light of previously-established constraints around algal biomass costs (which are significantly higher than lignocellulosic terrestrial biomass), the present CAP processing strategy reflects an integrated biorefinery concept producing both fuels and value-added chemical coproducts as a means to improve profitability and generate coproduct revenues to help drive down the minimum fuel selling price (MFSP) towards economically viable levels. Namely, this report highlights an integrated CAP biorefinery process and associated technical targets that would be required to achieve U.S. Department of Energy target MFSP goals of $2.5/gallon gasoline equivalent by 2030. This is accomplished by a process involving low-cost seasonal storage of algal biomass during high-growth seasons, rapid flash hydrolysis pretreatment of the biomass, solvent extraction of pretreated biomass, cleanup and fractionation of lipids into triglyceride and free fatty acid fractions, and a series of thermochemical conversion steps to upgrade carbohydrates and protein to hydrocarbon fuels. These steps include mild oxidative treatment (MOT), a process originally investigated at NREL for upgrading lignin, followed by catalytic ketonization and hydrotreating of MOT products to fuels. Isolated triglycerides are sent to a coproduct train, with the base case focused on upgrading to polyurethane foams as a high-value, high-market-volume coproduct.

09 BIOMASS FUELS↗