Source: OHIO STATE UNIVERSITY submitted to
DEVELOPMENT OF BIO-PLATFORMS FOR EFFICIENT CONVERSION OF LIGNOCELLULOSIC BIOMASS AND GREENHOUSE GAS TO FUELS AND CHEMICALS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1020980
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 15, 2019
Project End Date
Sep 30, 2024
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Project Director
Ezeji, TH.
Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691
Performing Department
Animal Sciences
Non Technical Summary
Numerous processes have been developed or are currently in development for the bioconversion of plant carbohydrates (sugars), especially lignocellulosic biomass (LB), to fuels and chemicals. Due to the diverse nature of LB, this feedstock has yet to be economically converted into fuels and commodity chemicals. The proposed research would focus on the biosynthesis of 2,3-butanediol (2,3-BD), hydrogen (H2), acetone, ethanol and butanol using compatible bacteria and waste products - anaerobic digestion effluents, biodiesel-derived glycerol and LB. Butanol and 2,3-BD yields from LB (e.g. glucose, xylose, starch) conversion is not optimal because a significant amount of the biomass is converted by these microorganisms into un-captured CO2 and H2. We, therefore, also propose to develop a viable strategy to capture released CO2 and H2 and convert CO2 to acetone, ethanol and butanol; and improve the usable energy yield from LB. In parallel, the generated CO2 may be used in the waste and water treatment plants as part of wastes treatment - biofuel production integrated process. Meanwhile, while H2 is an excellent fuel with no carbon footprint upon combustion, 2,3-BD can be used as a precursor in the manufacture of a range of chemical products such as perfumes, printing inks, moistening and softening agents, fumigants, explosives, plasticizers, and octane isomers. The 2,3-BD is also an essential feedstock chemical for the synthesis of 1,3-butadiene (1,3-BD), the monomer of synthetic rubber, currently produced by cracking petroleum. The majority of hyper-2,3-BD producing microorganisms are pathogens, and this may have considerable effects on why most ongoing research in this area is being conducted overseas (probably due to regulatory and liability issues in the US). The bacteria we proposed to use for the production of 2,3-BD is non-pathogenic. The downstream products of 2,3-BD is estimated to have a global market for around 32 million tons annually, valued at approximately $43 billion (US) in revenue. The other product of interest, butanol, currently manufactured with petroleum feedstocks, is also an important chemical with many applications in the production of solvents, plasticizers, butylamines, amino resins, butyl acetates, etc. Global butanol consumption is expected to reach 13 million tons by 2024. The current market value of butanol is about $6.4 billion and its downstream products is valued at well over $40 billion. With increasing efforts to develop LB-based biorefineries to produce fuels and chemicals on a commercial-scale, residual wastes such as fermentation effluents are expected to markedly increase. Consequently, the relatively large nitrogen (N) concentrations of fermentation wastes may pose a serious hazard to human health and biodiversity because wastewater disposal may contribute significantly to environmental N concentrations. Anaerobic digestion (AD) is an attractive strategy for reducing the risks associated with large amounts of carbon and N in water bodies. Treatment (digestion) of protein-rich wastes, however, leads to the production of ammonia, which frequently compromises or abolishes the bio-digester reactions (stemming from multifaceted ammonia toxicity to the microorganisms involved in AD); a major economic challenge for the waste treatment industry. Our goal in this project, therefore, is to develop a viable strategy for generating butanol, 2,3-BD and H2 from agro-based wastes - LB, biodiesel-derived glycerol, and anaerobic digestion effluents along with development of effective N removal from solid and liquid wastes. This task has some challenges because biosynthesis reactions that result in the production of valuable fuels and chemicals do not generate compounds in amounts that are economically feasible for large-scale production because most fermentation processes are product limiting due to feedback inhibition and product toxicity to the fermentation microorganisms. We plan to develop or retrofit existing real-time product recovery technologies and adapt them for real-recovery of 2,3-BD, H2, acetone, ethanol and butanol during fermentation. Overall, we expect to develop platforms, which have the potential of becoming a part of the rubric of "green chemical" approaches that allow for conversion of LB and glycerol to valuable fuels and chemicals such as ethanol, acetone, H2, butanol and 2,3-BD.
Animal Health Component
60%
Research Effort Categories
Basic
30%
Applied
60%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
4024099202030%
4030650200040%
4031629104030%
Knowledge Area
403 - Waste Disposal, Recycling, and Reuse; 402 - Engineering Systems and Equipment;

Subject Of Investigation
4099 - Microorganisms, general/other; 0650 - Wood and wood products; 1629 - Perennial grasses, other;

Field Of Science
2020 - Engineering; 1040 - Molecular biology; 2000 - Chemistry;
Goals / Objectives
Numerous processes have been developed or are currently in development for the bioconversion of plant carbohydrates (sugars), especially lignocellulosic biomass (LB), to fuels and chemicals. Due to the complex heterogeneous nature of LB, this feedstock has yet to be economically converted into fuels and commodity chemicals. The proposed research would focus on the biosynthesis of 2,3-butanediol (2,3-BD), hydrogen (H2) and butanol through microbial-assisted interdependent utilization of two different waste products - biodiesel-derived glycerol and LB. Butanol and 2,3-BD yields from LB (e.g. glucose, xylose, starch) conversion is not optimal because a significant amount of the biomass is converted by these microorganisms into un-captured CO2 and H2. We, therefore, also propose to develop a viable strategy to capture released CO2 and H2 and convert CO2 to fuels and chemicals; and improve the usable energy yield from LB. The overarchinggoal of the proposed study is to use synthetic biology and functional genomics techniques to potentiate C. beijerinckii (Cb), C. carboxidivorans (Cc), and P. polymyxa (Pp) with mechanisms to counter the adverse consequences of LDMICs and enhance fermentation of LBH to butanol, 2,3-BD and H2. Additionally, we will incorporate, as needed, Saccharomyces cerevisiae and Pseudomonas putida to our plan to facilitate valorization of wastewaters to fuels and chemicals. Our overall objectives are:?1) increase NADPH generation by enhancing glycerol metabolism by overexpression of glycerol dehydrogenase (GDH) and dihydroxyacetone kinase (DHAK) genes2) develop LDMIC tolerant Cb and Cc strains with an improved capacity to convert LBH (switchgrass, Miscanthus, and corn stover) and CO2 to H2, acetone and butanol, and improve the yield and economics of production of these compounds 3) elucidate a process of degeneration of Pp and increase 2,3-BD production from LBH4) develop a bioprocess that converts industrial and agricultural wastewaters to fuels and chemicals5) develop an efficient bioreactor system for butanol fermentation and in situ real-time product recovery
Project Methods
MethodsThe DNA sequences of genes proposed to clone or delete to improve the function of the biocatalyst will be obtained from the EMBL-European Bioinformatics Institute. Nested PCR and splicing by overlap extension (SOE) PCR procedures will be used to amplify target genes from native sources. This will be followed by cloning as single and fused gene constructs into the Clostridium-E. coli shuttle plasmids pWUR459 and pWUR460. Each insert will be spliced into pWUR460 or pWUR459, to permit transcription by a constitutive thiolase promoter or inducible acetoacetate decarboxylase promoter (from C. acetobutylicum), respectively. Following selection of transformants, generated recombinant strains will be used in batch fermentation with and without pH control to evaluate acetone butanol ethanol (ABE) and 2,3-BD production. As substrates, we will use: (i) glucose, (ii) glucose + glycerol mixtures, (iii) dilute acid-pretreated Miscanthus giganteus/switchgrass/corn stover hydrolysates (LBH), and (iv) LBH + glycerol mixture. For groups ii and iv, we will use pure and crude glycerol, with the latter already obtained from JatroDiesel, a biodiesel company in Ohio.Microbial activity data collection: C. beijerinckii, C. carboxidivorans, P. polymyxa, Pseudomonas putida, and Saccharomyces cerevisiae growth rate will be determined using spectrophotometer and plate counts (colony forming unit [cfu]), and the substrate utilization rate will be evaluated using HPLC to determine glucose, xylose, cellobiose, arabinose (MH constituents), and glycerol content.System operations data collection: Concentrations of acetone, ethanol, and 2,3-butanediol will be determined with gas chromatography. Butanol, ethanol, and 2,3-butanediol yield and productivity, will be measured using the PI analytical protocol.Evaluation: We will assess the following outcomes: (i) rate of ammonia, phosphorus, benzene, phenol, xylene, toluene, naphthalene, furfural, and hydroxybenzaldehyde utilization (e.g. 70 mg/L/h exceeds the threshold for scale up_20-Liters studies); (ii) butanol- and 2,3-butanediol producing capacity and stability of generated strains, and (iii), the substrate utilizing capacity of generated strains. Documentation and analysis: Data for the experimental design and all bench experiments will be archived as hard copies in individual laboratory notebooks and backed up with electronic copies where possible. As deemed appropriate, experiments will be repeated at least three times. All metabolic engineering experiments employed to develop improved cultures of C. beijerinckii, C. carboxidivorans, P. polymyxa, Pseudomonas putida, and Saccharomyces cerevisiae, and imaging data and statistical analysis will also be stored as electronic copies. For fermentations, indices pertaining to observed phenotypes, such as ethanol-butanol and 2,3-butanediol formation, growth of C. beijerinckii, C. carboxidivorans, P. polymyxa, Pseudomonas putida, and Saccharomyces cerevisiae, and indices pertaining to ethanol, acetone-butanol, and 2,3-butanediol concentration, yield, and productivity, will be subjected to one-way ANOVA analysis where applicable, followed by Tukey's multiple-range tests. All data will be validated using statistical software (SPSS v.17, Chicago, IL) and will be reported at a 95% confidence interval.

Progress 10/15/19 to 09/30/20

Outputs
Target Audience:Target audiences include USDA/NIFA AFRI, City wastes and water treatment plants, Ohio State University (OSU)/ Ohio Agricultural Research and Development Center (OARDC, Agricultural engineers, Bioprocess engineers, Systems analysts, Microbiologist, Graduate and Undergraduate students, Research associates and Postdoctoral research associates from various OSU and OARDC programs, Technicians, Farmers, Biorefineries, and government agencies. A portion of the results have been presented at the American Society for Microbiology Annual Conference, Chicago, IL, USA (Virtual Conference), June 22 - August 17, 2020. Some results were also presented at the 2020 International E-Conference on New Horizons in Biochemistry, Microbiology and Food Technology meeting jointly organized by Yogi Vemana University India and Universiti Malaysia Kelantan Malaysia, October 12 - 13, 2020. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Ms. Tinuola Olorunsogbon (PhD student), Mr. Ifeanyi Iloba (PhD student), Mr. Suranny Jiménez Chacón (MS student), Mr. Yinka Adesanya (MS student), Mr. Brennen Rush (undergraduate student), Ms. Grace McCrea (undergraduate student), and Drs. Christopher Okonkwo and Xiao Sun (Postdocs) received training in waste water treatment and bioconversion of LBH to butanol, ethanol, and 2,3-butanediol. Mr. Brennen Rush, Ms. Grace McCrea, Mr. Ifeanyi Iloba, and Dr. Christopher Okonkwo received training in wastewater recovery and conversion to value-added products. Mr. Suranny Jiménez Chacón, Mr. Yinka Adesanya and Dr. Xiao Sun received training in process engineering for improved utilization and conversion of LBH to butanol. Ms. Tinuola Olorunsogbon, Mr. Ifeanyi Iloba and Dr. Okonkwo received training in the metabolic engineering of C. beijerinckii, P. polymyxa and S. saccharomyces for improved tolerance of LDMIC, and improved conversion of LBH to butanol, 2,3-BD and ethanol. Dr. Okonkwo is currently receiving Postdoctoral training under this project. Activities emanating from this project have improved Dr. Thaddeus Ezeji's knowledge and skills in biomass conversion and utilization, and 2,3-BD fermentation. Dr. Ezeji has participated in conferences and seminars where he presented and discussed wastewater processing, and bioconversion of lignocellulosic biomass to value-added products, challenges and ways to overcoming them. How have the results been disseminated to communities of interest?On October 12 - 13 2020, Dr. Ezeji was a keynote speaker at the 2020 International E-Conference on New Horizons in Biochemistry, Microbiology and Food Technology jointly organized by Yogi Vemana University India and Universiti Malaysia Kelantan Malaysia. Dr. Ezeji spoke about "Past, Present, and Future of Bioenergy: An Example of Butanol" to over 750 mixed audiences that comprised graduate students, postdocs, research professionals, Professors, Industry executives and entrepreneurs, government representatives, and representatives from different non-governmental organizations, from over 35 countries including the United States. Drs. Christopher Okonkwo, Victor Ujor, and Thaddeus Ezeji presented a research poster titled "Biotransformation of 5-hydroxymethyl furfural (HMF) and furfural during 2,3-butanediol fermentation by Paenibacillus polymyxa DSM 365" at the 2020 American Society for Microbiology Annual Conference, Chicago, IL, USA (Virtual Conference), June 22 - August 17, 2020. Further, Ms. Tinuola Olorunsogbon presented a research poster titled "Overexpression of aldo-keto reductase and short-chain dehydrogenase genes in Clostridium beijerinckii leads to enhanced butanol production from switchgrass hydrolysates" at the 2020 American Society for Microbiology Annual Conference, Chicago, IL, USA (Virtual Conference), June 22 - August 17, 2020 What do you plan to do during the next reporting period to accomplish the goals? Plan to work on the development of LDMIC tolerant solventogenic Clostridium species and C. carboxidivorans (Cc) strains with an improved capacity to convert LBH (switchgrass, Miscanthus, and corn stover) and CO2 to H2, acetone, ethanol and butanol. Plan to work on the development of an efficient bioreactor system for butanol fermentation and in situ real-time product recovery Fine-tune the bioprocess that converts industrial and agricultural wastewaters to fuels and chemicals. Write up previously obtained results and submit manuscripts for publication.

Impacts
What was accomplished under these goals? Objective 2: Development of lignocellulose derived microbial inhibitory compounds (LDMIC) tolerant solventogenic Clostridium species and C. carboxidivorans (Cc) strains with an improved capacity to convert lignocellulosic biomass hydrolysates (LBH) and CO2 to H2, acetone and butanol, and improve the yield and economics of production of these compounds. In this objective, we evaluated the feasibility of using Clostridium saccharoperbutylacetonicum DSM 14923 to ferment sugarcane derived hemicellulose hydrolysates and sugarcane molasses to butanol. While C. saccharoperbutylacetonicum was not able to ferment sugarcane hemicellulose hydrolysates supplemented with either laboratory-grade nutrients or sugarcane molasses due to the presence of high levels of LDMIC, the culture was able to co-ferment sugarcane molasses-to-sugarcane hemicellulose hydrolysates volume ratio of 3:1 under intermittent feeding regimen that started after 24 h of growth. Butanol yield (0.31 g/g) was remarkable, and butanol titer was 10.0 g/L after 72 h of fermentation. This research demonstrated that sugarcane molasses, a byproduct of the sugar industry rich in sucrose and nutrients, can be an efficient feedstock for enabling the production of butanol from sugarcane bagasse hemicellulose hydrolysate. Objective 3: Elucidating the process of degeneration of Paenibacillus polymyxa and increase 2,3-BD production from LBH. Exopolysaccharide (EPS) production during 2,3-butanediol (2,3-BD) fermentation constitutes a problem during downstream processing. Specifically, EPS negatively impacts 2,3-BD separation from the fermentation broth, thereby increasing the overall cost of 2,3-BD production. Therefore, to introduce this objective, we successfully used homologous recombination to disable the EPS biosynthetic pathway in P. polymyxa, which led to the disruption of levansucrase-the major enzyme responsible for EPS biosynthesis in the microorganism. This was followed by the characterization of the levansucrase null mutant for genetic stability, substrate utilization, EPS and 2,3-BD production. Indeed, evaluation of the genetic stability of the levansucrase null mutant showed that it remained genetically stable over fifty generations, with no observable decrease in growth or 2,3-BD formation. Although more work is needed to unravel the process of degeneration in P. polymyxa, our preliminary results show that P. polymyxa has potential for use as an industrial biocatalyst for a cost-effective large-scale 2,3-BD fermentation process devoid of EPS-related challenges. Objective 4: Development of a bioprocess that converts industrial and agricultural wastewaters to fuels and chemicals. Under this objective, we evaluated the feasibility of concomitant nutrient removal, cleaner water recovery, and ethanol production with liquid fraction of anaerobic digestate (ADE) by Saccharomyces cerevisiae. Indeed, different concentrations ranging from 0% to 100% ADE were evaluated for ethanol production by S. cerevisiae. Interestingly, 25%, 50%, and 100% (v/v) ADE supported the growth of S. cerevisiae, glucose utilization (~100 g/L) and ethanol production (up to 50.4 ± 6.4 g/L). After a 144 h fermentation in the 50% ADE, the concentrations of ammonia, total nitrogen, phosphate, and total phosphorus in ADE decreased 1000-, 104.43-, 1.94-, and 2.20-fold, respectively. Notably, only 0.40 ± 0.61 mg/L ammonia was detected in the 50% ADE post-fermentation. Similarly, the concentrations of aluminum, copper, magnesium, manganese, molybdenum, potassium, sodium, iron, sulfur, zinc, chloride, and sulfate decreased significantly in the ADE. Further analysis suggests that the nitrogen (ammonia and protein), phosphate, and the metal contents of the digestate work in tandem to promote the growth of S. cerevisiae and ethanol production. Among these, ammonia and protein appear to exert considerable effects on S. cerevisiae. These results represent a significant first step towards repurposing ADE as a resource in bio-production of fuels and chemicals, whilst generating effluent that is economically treatable by conventional wastewater treatment technologies.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Chac�n JS, Matias G, Vieira CFD, Ezeji TC, Filho RM, and AP Mariano (2020) Enabling butanol production from crude sugarcane bagasse hemicellulose hydrolysate by batch-feeding it into molasses fermentation. Industrial Crops and Products 155: 112837
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Ujor VC, Christopher Okonkwo CC, Rush BB, McCrea GE and T C Ezeji (2020) Harnessing the residual nutrients in anaerobic digestate for ethanol fermentation and digestate remediation using Saccharomyces cerevisiae. Fermentation 6:52 doi:10.3390/fermentation 6020052
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Okonkwo CC, Ujor, V, Cornish K, and TC Ezeji (2020) Inactivation of the Levansucrase Gene in Paenibacillus polymyxa DSM 365 Diminishes Exopolysaccharide Biosynthesis during 2,3-Butanediol Fermentation. Applied and Environmental Microbiology 86 (9): e00196-20. doi: 10.1128/AEM.00196-20
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Sun X, Atiyeh HK, Adesanya Y, Zhang H, Okonkwo C and T. Ezeji (2020) Feasibility of using biochar as buffer and mineral nutrients replacement for acetone-butanol-ethanol production from non-detoxified switchgrass hydrolysate. Bioresource Technology 298: 122569
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Ezeji TC (2020) Keynote speaker on Past, Present, and Future of Bioenergy: An Example of Butanol. 2020 International E-Conference on New Horizons in Biochemistry, Microbiology and Food Technology jointly organized by Yogi Vemana University India and Universiti Malaysia Kelantan Malaysia, October 12  13
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Okonkwo, CC, Ujor, V, Ezeji, TC (2020) Biotransformation of 5-hydroxymethyl furfural (HMF) and furfural during 2,3-butanediol fermentation by Paenibacillus polymyxa DSM 365. American Society for Microbiology Annual Conference, Chicago, IL, USA (Virtual Conference), June 22 - August 17, 2020.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Olorunsogbon, T, Okonkwo, C, Iloba, I, Atiyeh, H, Adesanya, Y, Ujor, V, Ezeji, T (2020) Overexpression of aldo-keto reductase and short-chain dehydrogenase genes in Clostridium beijerinckii leads to enhanced butanol production from switchgrass hydrolysates. American Society for Microbiology Annual Conference, Chicago, IL, USA (Virtual Conference), June 22 - August 17, 2020.