Engineering topics

Cheng, Ming-Hsun

Publications and source records attributed to Cheng, Ming-Hsun.

At least 19 records

DOE OSTI2026

Data for Engineering and Evolution of Yarrowia lipolytica for Producing Lipids from Lignocellulosic Hydrolysates

Yarrowia lipolytica , an oleaginous yeast, shows promise for industrial fermentation due to its robust acetyl-CoA flux and well-developed genetic engineering tools. However, its lack of an active xylose metabolism restricts the conversion of cellulosic sugars to valuable products. To address this, metabolic engineering, and adaptive laboratory evolution (ALE) were applied to the Y. lipolytica PO1f strain, resulting in an efficient xylose-assimilating strain (XEV). Whole-genome sequencing (WGS) of the XEV followed by reverse engineering revealed that the amplification of the heterologous oxidoreductase pathway and a mutation in the GTPase-activating protein gene (YALI0B12100g) might be the primary reasons for improved xylose assimilation in the XEV strain. When a sorghum hydrolysate was used, the XEV strain showed superior xylose consumption and lipid production compared to its parental strain (X123). This study advances our understanding of xylose metabolism in Y. lipolytica and proposes effective metabolic engineering strategies for optimizing lignocellulosic hydrolysates.

Hydrolysate↗

DOE OSTI2024

Engineering and evolution of Yarrowia lipolytica for producing lipids from lignocellulosic hydrolysates

Yarrowia lipolytica, an oleaginous yeast, shows promise for industrial fermentation due to its robust acetyl-CoA flux and well-developed genetic engineering tools. However, its lack of an active xylose metabolism restricts the conversion of cellulosic sugars to valuable products. To address this, metabolic engineering, and adaptive laboratory evolution (ALE) were applied to the Y. lipolytica PO1f strain, resulting in an efficient xylose-assimilating strain (XEV). Whole-genome sequencing (WGS) of the XEV followed by reverse engineering revealed that the amplification of the heterologous oxidoreductase pathway and a mutation in the GTPase-activating protein gene (YALI0B12100g) might be the primary reasons for improved xylose assimilation in the XEV strain. When a sorghum hydrolysate was used, the XEV strain showed superior xylose consumption and lipid production compared to its parental strain (X123). This study advances our understanding of xylose metabolism in Y. lipolytica and proposes effective metabolic engineering strategies for optimizing lignocellulosic hydrolysates.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2024

Data for Sustainable Co-Production of Plant Lipids and Cellulosic Sugars from Transgenic Energycane at an Industrially Relevant Scale: A Proof of Concept for Alternative Feedstocks

Development of sustainable and scalable technologies to convert lignocellulosic biomass to biofuels is critical to achieving carbon neutrality. The potential of transgenic bioenergy crops as a renewable source of sugars and lipids has been demonstrated at bench-scale. However, scaling up these processes is important for holistic analysis. Here proof-of-concept for chemical-free hydrothermal pretreatment of transgenic energycane-oilcane line L13 at an industrially relevant scale to recover vegetative lipids along with cellulosic sugars is presented. Pilot-scale processing of 97 kg of transgenic energycane-oilcane L13 stems and high solids pretreatment of bagasse enhanced the recovery of cellulosic glucose and xylose by 5-fold as compared to untreated bagasse and helped in the enrichment of vegetative lipids in the biomass residues which allowed its recovery at the end of the bioprocess. Palmitic and oleic acids were the predominant fatty acids (FAs) extracted from stems and leaves. The processing did not affect lipid composition. The efficiency of lipid recovery from untreated biomass was 75.9% which improved to 88.7% upon pretreatment. The vegetative tissues of transgenic energycane-oilcane L13 contained 0.42 metric tons/hectare of lipids. Processing vegetative tissues yielded 0.38 metric tons/hectare of lipids. This approaches an oil yield similar to soybean (global average 0.44 metric tons/hectare) and is almost twice as high as the oil yield from sugarcane engineered to hyperaccumulate lipids (0.20 metric tons/hectare). The study suggests that further optimization by state-of-the-art metabolic engineering and biomass processing can establish transgenic bioenergy crops for commercial drop-in fuel production.

Biomass Analytics↗

DOE OSTI2024

Sustainable co-production of plant lipids and cellulosic sugars from transgenic energycane at an industrially relevant scale: A proof of concept for alternative feedstocks

Development of sustainable and scalable technologies to convert lignocellulosic biomass to biofuels is critical to achieving carbon neutrality. The potential of transgenic bioenergy crops as a renewable source of sugars and lipids has been demonstrated at bench-scale. However, scaling up these processes is important for holistic analysis. Here proof-of-concept for chemical-free hydrothermal pretreatment of transgenic energycane-oilcane line L13 at an industrially relevant scale to recover vegetative lipids along with cellulosic sugars is presented. Here, pilot-scale processing of 97 kg of transgenic energycane-oilcane L13 stems and high solids pretreatment of bagasse enhanced the recovery of cellulosic glucose and xylose by 5-fold as compared to untreated bagasse and helped in the enrichment of vegetative lipids in the biomass residues which allowed its recovery at the end of the bioprocess. Palmitic and oleic acids were the predominant fatty acids (FAs) extracted from stems and leaves. The processing did not affect lipid composition. The efficiency of lipid recovery from untreated biomass was 75.9% which improved to 88.7% upon pretreatment. The vegetative tissues of transgenic energycane-oilcane L13 contained 0.42 metric tons/hectare of lipids. Processing vegetative tissues yielded 0.38 metric tons/hectare of lipids. This approaches an oil yield similar to soybean (global average 0.44 metric tons/hectare) and is almost twice as high as the oil yield from sugarcane engineered to hyperaccumulate lipids (0.20 metric tons/hectare). The study suggests that further optimization by state-of-the-art metabolic engineering and biomass processing can establish transgenic bioenergy crops for commercial drop-in fuel production.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Waste-to-Energy Pipeline through Consolidated Fermentation–Microbial Fuel Cell (MFC) System

The rise in population, urbanization, and industrial developments have led to a substantial increase in waste generation and energy demand, posing significant challenges for waste management as well as energy conservation and production. Bioenergy conversions have been merged as advanced, sustainable, and integrated solutions for these issues, encompassing energy generation and waste upcycling of different types of organic waste. Municipal solid waste (MSW) and agricultural residues (AR) are two main resources for bioenergy conversions. Bioenergy production involves feedstock deconstruction and the conversion of platform chemicals to energy products. This review provides a detailed overview of waste sources, biofuel, and bioelectricity production from fermentation and microbial fuel cell (MFC) technology, and their economic and environmental perspectives. Fermentation plays a critical role in liquid biofuel production, while MFCs demonstrate promising potential for simultaneous production of electricity and hydrogen. Fermentation and MFCs hold a significant potential to be integrated into a single pipeline, enabling the conversion of organic matter, including a variety of waste material and effluent, into diverse forms of bioenergy via microbial cultures under mild conditions. Furthermore, MFCs are deemed a promising technology for pollutant remediation, reducing COD levels while producing bioenergy. Importantly, the consolidated fermentation–MFC system is projected to produce approximately 7.17 trillion L of bioethanol and 6.12 × 10 4 MW/m 2 of bioelectricity from MSW and AR annually, contributing over USD 465 billion to the global energy market. Such an integrated system has the potential to initiate a circular economy, foster waste reduction, and improve waste management practices. This advancement could play a crucial role in promoting sustainability across the environmental and energy sectors.

09 BIOMASS FUELS↗

DOE OSTI2023

Data for Production of Designer Xylose-Acetic Acid Enriched Hydrolysate from Bioenergy Sorghum, Oilcane, and Energycane Bagasses

Xylan accounts for up to 40% of the structural carbohydrates in lignocellulosic feedstocks. Along with xylan, acetic acid in sources of hemicellulose can be recovered and marketed as a commodity chemical. Through vibrant bioprocessing innovations, converting xylose and acetic acid into high-value bioproducts via microbial cultures improves the feasibility of lignocellulosic biorefineries. Enzymatic hydrolysis using xylanase supplemented with acetylxylan esterase (AXE) was applied to prepare xylose-acetic acid enriched hydrolysates from bioenergy sorghum, oilcane, or energycane using sequential hydrothermal-mechanical pretreatment. Various biomass solids contents (15 to 25%, w/v) and xylanase loadings (140 to 280 FXU/g biomass) were tested to maximize xylose and acetic acid titers. The xylose and acetic acid yields were significantly improved by supplementing with AXE. The optimal yields of xylose and acetic acid were 92.29% and 62.26% obtained from hydrolyzing energycane and oilcane at 25% and 15% w/v biomass solids using 280 FXU xylanase/g biomass and AXE, respectively.

Biomass Analytics↗

DOE OSTI2023

Production of Designer Xylose-Acetic Acid Enriched Hydrolysate from Bioenergy Sorghum, Oilcane, and Energycane Bagasses

09 BIOMASS FUELS↗

DOE OSTI2022

The effects of sequential hydrothermal-mechanical refining pretreatment on cellulose structure changes and sugar recoveries

The recalcitrance of lignocellulosic biomass necessitates an efficient pretreatment protocol for operating a successful cellulosic biorefinery. It is critical to improve cellulose accessibility for hydrolysis and fermentation by altering the plant cell wall’s physical structure and chemical composition. Sequential hydrothermal-mechanical refining pretreatment (HMR) allows efficient recovery of cellulosic sugars without utilizing any hazardous chemicals. HMR has been successfully applied to Liberty switchgrass, a bioenergy cultivar released by the USDA, and now it is being applied to oilcane, a recently developed transgenic sugarcane variety engineered to accumulate lipids in its vegetative tissues. Sugar yields of oilcane bagasse (OCB) and switchgrass (SG) treated with HMR are 96.4% and 75.4%, respectively. This study sought to correlate cellulosic sugar yields with structural changes within the cell wall caused by HMR on two distinct bioenergy crops. Simon’s staining technique for the specific surface area analysis showed that HMR increased the specific surface area of pretreated biomass residues by 80–112%. In addition, ATR-FTIR was performed to determine the effects of HMR on physical structures based on the total crystallinity index (TCI) and hydrogen bonding intensity (HBI). Irrespective of biomass type, HMR decreased the initial crystalline cellulose contents of untreated biomass residues by 3.5% and reduced TCI and HBI by 7–13%. Here, the study found that sugar yields were negatively correlated to reducing values of hydrogen bonding intensity, crystalline cellulose content, and total crystallinity index.

09 BIOMASS FUELS↗

DOE OSTI2022

Data for The Effects of Sequential Hydrothermal-Mechanical Refining Pretreatment on Cellulose Structure Changes and Sugar Recoveries

The recalcitrance of lignocellulosic biomass necessitates an efficient pretreatment protocol for operating a successful cellulosic biorefinery. It is critical to improve cellulose accessibility for hydrolysis and fermentation by altering the plant cell wall’s physical structure and chemical composition. Sequential hydrothermal-mechanical refining pretreatment (HMR) allows efficient recovery of cellulosic sugars without utilizing any hazardous chemicals. HMR has been successfully applied to Liberty switchgrass, a bioenergy cultivar released by the USDA, and now it is being applied to oilcane, a recently developed transgenic sugarcane variety engineered to accumulate lipids in its vegetative tissues. Sugar yields of oilcane bagasse (OCB) and switchgrass (SG) treated with HMR are 96.4% and 75.4%, respectively. This study sought to correlate cellulosic sugar yields with structural changes within the cell wall caused by HMR on two distinct bioenergy crops. Simon’s staining technique for the specific surface area analysis showed that HMR increased the specific surface area of pretreated biomass residues by 80-112%. In addition, ATR-FTIR was performed to determine the effects of HMR on physical structures based on the total crystallinity index (TCI) and hydrogen bonding intensity (HBI). Irrespective of biomass type, HMR decreased the initial crystalline cellulose contents of untreated biomass residues by 3.5% and reduced TCI and HBI by 7-13%. The study found that sugar yields were negatively correlated to reducing values of hydrogen bonding intensity, crystalline cellulose content, and total crystallinity index.

Biomass Analytics↗

DOE OSTI2022

Cas9-Based Metabolic Engineering of Issatchenkia orientalis for Enhanced Utilization of Cellulosic Hydrolysates

Issatchenkia orientalis, exhibiting high tolerance against harsh environmental conditions, is a promising metabolic engineering host for producing fuels and chemicals from cellulosic hydrolysates containing fermentation inhibitors under acidic conditions. Although genetic tools for I. orientalis exist, they require auxotrophic mutants so that the selection of a host strain is limited. We developed a drug resistance gene (cloNAT)-based genome-editing method for engineering any I. orientalis strains and engineered I. orientalis strains isolated from various sources for xylose fermentation. Specifically, xylose reductase, xylitol dehydrogenase, and xylulokinase from Scheffersomyces stipitis were integrated into an intended chromosomal locus in four I. orientalis strains (SD108, IO21, IO45, and IO46) through Cas9-based genome editing. Furthermore, the resulting strains (SD108X, IO21X, IO45X, and IO46X) efficiently produced ethanol from cellulosic and hemicellulosic hydrolysates even though the pH adjustment and nitrogen source were not provided. As they presented different fermenting capacities, selection of a host I. orientalis strain was crucial for producing fuels and chemicals using cellulosic hydrolysates.

09 BIOMASS FUELS↗

DOE OSTI2021

Conversion of High-Solids Hydrothermally Pretreated Bioenergy Sorghum to Lipids and Ethanol Using Yeast Cultures

Glucose and xylose are the major sugars in cellulosic hydrolysates. The cellulosic sugars can be used for the production of biofuels and value-added bioproducts. In this study, lipid and ethanol were produced from bioenergy sorghum syrups using engineered yeasts. Here, bioenergy sorghum was hydrothermally pretreated at 50% solids loading in a continuous reactor system and mechanically refined sequentially using a burr mill to improve biomass accessibility for hydrolysis. Fed-batch enzymatic hydrolysis was conducted with 50% w/v solids loading to achieve 230 g/L sugar concentration. Different strains of Rhodosporidium toruloides were used to ferment sugars into lipids, and the highest lipid yield of 9.2 g/L was observed. The lipid yield was improved to 19.0 g/l by implementing a two-stage culture where once the sugars were exhausted in the first stage, the yeast was introduced into fresh hydrolysate without adding nitrogen. For ethanol production, the engineered Saccharomyces cerevisiae SR8ΔADH6 was utilized to coferment glucose and xylose. Additionally, the effects of nutrient media (YP, YNB/urea, and urea), cellulosic sugar concentration, and sulfite addition were investigated to optimize the ethanol yield from sorghum syrups. The optimal ethanol yield at 73.3% was obtained from the YNB/urea culture consisting of 34 g glucose/L and 17 g xylose/L.

09 BIOMASS FUELS↗

DOE OSTI2021

Data for Conversion of High-Solids Hydrothermally Pretreated Bioenergy Sorghum to Lipids and Ethanol Using Yeast Cultures

Glucose and xylose are the major sugars present in cellulosic hydrolysates. The cellulosic sugars can be used for the production of platform chemicals. In this study, productions of lipid and ethanol by yeasts were compared for concentrated bioenergy sorghum syrup. Bioenergy sorghum was hydrothermally pretreated at 50% w/w solids in a continuous industrial reactor and sequentially mechanically refined using a burr mill to improve biomass accessibility for hydrolysis. Fed-batch enzymatic hydrolysis was conducted with 50% w/v solids loading and cellulase cocktail (50 FPU/g biomass) to achieve 230 g/L sugar concentration. Various strains of Rhodosporidium toruloides were evaluated for converting sugars into lipids, and strain Y-6987 had the highest lipid titer (9.2 g/L). The lipid titer was improved to 19.0 g/L by implementing a two-stage culture scheme, where the first stage was optimized for yeast growth and the second for lipid production. For ethanol production, the engineered Saccharomyces cerevisiae SR8ΔADH6 was used to coferment glucose and xylose. Ethanol fermentation was optimized for media nutrients (YP, YNB/urea, and urea), cellulosic sugar concentration, and sulfite conditioning to maximize the ethanol concentration from sorghum syrups. Fermentation of 70% v/v concentrated hydrolysate conditioned with sulfite produces 50.1 g/L ethanol from 141 g/L of sugars.

Conversion↗

DOE OSTI2021

Data from Economic Perspective of Ethanol and Biodiesel Coproduction from Industrial Hemp

In this study, the economics of producing biofuels from an industrial hemp (Cannabis sativa) genotype – 19m96136 was investigated. A lignocellulosic biofuel plant, hourly consuming 85 metric tons of hemp biomass was modeled in SuperPro Designer®. The integrated bioenergy plant produced hemp biodiesel and bioethanol from lipids and carbohydrates, respectively. The structural composition of the industrial hemp plant was analyzed in a previous study. The data obtained was used to simulate feedstock composition in SuperPro Designer®. The simulation results indicated that Hemp containing 2% lipids can yield up to 3.95 million gallons of biodiesel annually. On improving biomass lipid content to 5 and 10%, biodiesel production increased to 9.88 and 19.91 million gallons, respectively. The breakeven unit production cost of hemp biodiesel with 2, 5, and 10% lipid containing hemp was $18.49, $7.87, and $4.13/gallon, respectively. The biodiesel unit production cost when utilizing 10% lipid-containing hemp was comparable to soybean biodiesel at $4.13/gallon. Furthermore, sensitivity analysis revealed the possibility of a 7.80% reduction in unit production cost upon a 10% reduction in hemp feedstock cost. Furthermore, industrial hemp was capable of producing between 307.80 and 325.82 gallons of total biofuels per hectare of agricultural land than soybean.

Conversion↗

DOE OSTI2021

Economic perspective of ethanol and biodiesel coproduction from industrial hemp

Herein, the economics of producing biofuels from an industrial hemp (Cannabis sativa) genotype e 19m96136 was investigated. A lignocellulosic biofuel plant, hourly consuming 85 metric tons of hemp biomass was modeled in SuperPro Designer®. The integrated bioenergy plant produced hemp biodiesel and bioethanol from lipids and carbohydrates, respectively. The structural composition of the industrial hemp plant was analyzed in a previous study. The data obtained was used to simulate feedstock composition in SuperPro Designer®. The simulation results indicated that Hemp containing 2% lipids can yield up to 3.95 million gallons of biodiesel annually. On improving biomass lipid content to 5 and 10%, biodiesel production increased to 9.88 and 19.91 million gallons, respectively. The breakeven unit production cost of hemp biodiesel with 2, 5, and 10% lipid containing hemp was $18.49, $7.87, and $4.13/gallon, respectively. The biodiesel unit production cost when utilizing 10% lipid-containing hemp was comparable to soybean biodiesel at $4.13/ gallon. Furthermore, sensitivity analysis revealed the possibility of a 7.80% reduction in unit production cost upon a 10% reduction in hemp feedstock cost. Furthermore, industrial hemp was capable of pro- ducing between 307.80 and 325.82 gallons of total biofuels per hectare of agricultural land than soybean.

09 BIOMASS FUELS↗

DOE OSTI2020

Variability in structural carbohydrates, lipid composition, and cellulosic sugar production from industrial hemp varieties

The aim of this study was to determine carbohydrate recovery from hemp for ethanol production and quantify biodiesel from TAG (Triacylglycerol) present in hemp. The structural composition of five different hemp varieties (Seward County-SC, York County-YC, Loup County-LC, 19 m96136-19 m, and CBD Hemp-CBD) were analyzed. Concentration of glucan and xylan ranged between 32.63 to 44.52% and 10.62 to 15.48% respectively. The biomass was then pretreated with Liquid hot water followed by disk milling and then hydrolyzed enzymatically to yield monomeric sugars. High glucose (63-85%) and xylose (73-88%) recovery was achieved. Lipids were extracted from hemp using hexane and isopropanol and then transesterified to produce biodiesel. Approximately, 50% of total fatty acids in SC, LC, and CBD hemp were linoleic acid. Palmitic acid was present between 32 to 50% in varieties YC and 19 m. Highest TAG concentration at 25% of total lipids was observed in CBD hemp. In conclusion, the analysis on lipid composition and high sugar recovery demonstrates hemp as a potential bioenergy crop for ethanol and biodiesel coproduction.

09 BIOMASS FUELS↗

DOE OSTI2020

Data for Variability in Structural Carbohydrates, Lipid Composition, and Cellulosic Sugar Production from Industrial Hemp Varieties

The aim of this study was to determine carbohydrate recovery from hemp for ethanol production and quantify biodiesel from TAG (triacylglycerol) present in hemp. The structural composition of five different hemp varieties (Seward County-SC, York County-YC, Loup County-LC, 19 m96136-19 m, and CBD Hemp-CBD) were analyzed. Concentration of glucan and xylan ranged between 32.63 to 44.52% and 10.62 to 15.48% respectively. The biomass was then pretreated with Liquid hot water followed by disk milling and then hydrolyzed enzymatically to yield monomeric sugars. High glucose (63-85%) and xylose (73-88%) recovery was achieved. Lipids were extracted from hemp using hexane and isopropanol and then transesterified to produce biodiesel. Approximately, 50% of total fatty acids in SC, LC, and CBD hemp were linoleic acid. Palmitic acid was present between 32 to 50% in varieties YC and 19 m. Highest TAG concentration at 25% of total lipids was observed in CBD hemp. The analysis on lipid composition and high sugar recovery demonstrates hemp as a potential bioenergy crop for ethanol and biodiesel coproduction.

Biomass Analytics↗

DOE OSTI2020

High solids loading biorefinery for the production of cellulosic sugars from bioenergy sorghum

A novel process applying high solids loading in chemical-free pretreatment and enzymatic hydrolysis was developed to produce sugars from bioenergy sorghum. Hydrothermal pretreatment with 50% solids loading was performed in a pilot scale continuous reactor followed by disc refining. Sugars were extracted from the enzymatic hydrolysis at 10% to 50% solids content using fed-batch operations. Here, three surfactants (Tween 80, PEG 4000, and PEG 6000) were evaluated to increase sugar yields. Hydrolysis using 2% PEG 4000 had the highest sugar yields. Glucose concentrations of 105, 130, and 147 g/L were obtained from the reaction at 30%, 40%, and 50% solids content, respectively. The maximum sugar concentration of the hydrolysate, including glucose and xylose, obtained was 232 g/L. Additionally, the glucose recovery (73.14%) was increased compared to that of the batch reaction (52.74%) by using two- stage enzymatic hydrolysis combined with fed-batch operation at 50% w/v solids content.

09 BIOMASS FUELS↗

DOE OSTI2020

Data from High Solids Loading Biorefinery for the Production of Cellulosic Sugars from Bioenergy Sorghum

A novel process applying high solids loading in chemical-free pretreatment and enzymatic hydrolysis was developed to produce sugars from bioenergy sorghum. Hydrothermal pretreatment with 50% solids loading was performed in a pilot scale continuous reactor followed by disc refining. Sugars were extracted from the enzymatic hydrolysis at 10% to 50% solids content using fed-batch operations. Three surfactants (Tween 80, PEG 4000, and PEG 6000) were evaluated to increase sugar yields. Hydrolysis using 2% PEG 4000 had the highest sugar yields. Glucose concentrations of 105, 130, and 147 g/L were obtained from the reaction at 30%, 40%, and 50% solids content, respectively. The maximum sugar concentration of the hydrolysate, including glucose and xylose, obtained was 232 g/L. Additionally, the glucose recovery (73.14%) was increased compared to that of the batch reaction (52.74%) by using two-stage enzymatic hydrolysis combined with fed-batch operation at 50% w/v solids content.

Conversion↗