Development of a separation system for efficient fermentation of mixed substrates and in situ recovery of hydrophobic bulk chemical

DEVELOPMENT OF A SEPARATION SYSTEM FOR EFFICIENT FERMENTATION OF MIXED SUBSTRATES AND IN SITU RECOVERY OF HYDROPHOBIC BULK CHEMICAL

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

COMPLETE

Funding Source

AFRI COMPETITIVE GRANT

Reporting Frequency

Annual

Accession No.

1020065

Grant No.

2019-67021-29944

Cumulative Award Amt.

$467,122.00

Proposal No.

2018-08109

Multistate No.

(N/A)

Project Start Date

Aug 1, 2019

Project End Date

Jul 31, 2024

Grant Year

2019

Program Code

[A1531]- Biorefining and Biomanufacturing

Recipient Organization
OHIO STATE UNIVERSITY
1680 MADISON AVENUE
WOOSTER,OH 44691

Performing Department
Animal Sciences

Non Technical Summary
Often, biosynthesis reactions that produce valuable fuels and chemicals do not generate compounds at levels 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; thus, culminating in low concentrations in the bioreactor. A case in point is butanol, a four-carbon alcohol. Even though butanol can be produced renewably via acetone butanol ethanol (ABE) fermentation and has excellent fuel attributes, such as high energy content, low vapor pressure, good blending with gasoline, compatibility with oil infrastructure and automobiles without engine modification, butanol is not used as a biofuel because of its high production cost. However, fossil-derived butanol is currently used in numerous applications in the production of solvents, plasticizers, butylamines, amino resins, butyl acetates, and lacquers.Despite numerous merits, ABE fermentation suffers from a number of limitations (e.g. low titer, productivity, and poor production economics) due to butanol toxicity to microbial cells and high substrate cost. To address these, we have developed real time in situ recovery methods, namely gas stripping and vacuum-assisted gas stripping. However, these technologies are fraught with high level of water removal from the bioreactor requiring the need to replenish water, and at the same time excessive energy requirement for butanol separation from a highly diluted recovered butanol stream. Although these previous efforts have led to improvements in ABE fermentation, there is need to further resolve inter-twined issues related to substrate compatibility, butanol toxicity to microorganisms and excessive water removal from the bioreactor. To resolve these issues, we propose a novel lignocellulose compatible separation technology comprised of a water-butanol separation unit with water repelling properties that: (1) removes fuels and bioproducts in real-time from the bioreactor/fermenter during fermentation to prevent product toxicity to the fermentation microorganisms and allow fermentation to go on unimpeded, and (2) eliminates or minimizes water escaping the bioreactor with butanol.The overall goal of the proposed project, therefore, is to develop a novel fermentation technology with real-time product recovery that incorporates five key attributes namely: (I) compatibility with cheap fibrous, colloidal, and impure substrates; (II) support of the growth of fermentation microorganisms; (III) simplicity of operation; (IV) reduction of water losses from the bioreactor/fermenter and generation of high concentration of butanol; and (V) technically feasible and cost effective system. It is our expectation that accomplishing these attributes will result in a robust commercializable technology for fermentative production of hydrophobic bulk chemicals such as butanol.

Animal Health Component

60%

Research Effort Categories

Basic

20%

Applied

60%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
403	0650	2020	50%
402	2299	2000	30%
402	0699	1000	20%

Knowledge Area
403 - Waste Disposal, Recycling, and Reuse; 402 - Engineering Systems and Equipment;

Subject Of Investigation
2299 - Miscellaneous and new crops, general/other; 0650 - Wood and wood products; 0699 - Trees, forests, and forest products, general;

Field Of Science
2020 - Engineering; 1000 - Biochemistry and biophysics; 2000 - Chemistry;

Keywords

biofuel

butanol

downstream processing

lignocellulosic biomass

Goals / Objectives
Developing a sustainable process for the production of fuels and chemicals will require a technology that is amenable to lignocellulosic biomass-to-biofuels/chemicals fermentation, is easy to operate and maintain, and accepts heterogeneous fibrous and colloidal substrates such as lignocellulosic biomass and glycerol. We plan to use engineering-centered approaches to develop a membraneless and hybrid of membrane and sieve vacuum-assisted gas stripping process that overrides product-mediated feedback inhibition during fermentation, generates high concentrations of the target product, and reduces the need for added anoxic sterile water. The overall goal is to develop a scalable and economically feasible technology that is capable of both accepting mixed fibrous/colloidal substrates for fermentation and facilitating real-time removal of products of fermentation from the bioreactor so that the fermentation microorganisms are not subject to product inhibition or toxicity. The specific objectives, which are designed to accomplish this goal, are to: (i) fabricate a water-butanol plate separator using superhydrophobic principles to retain water in the reactor, and superoleophilic properties to allow butanol to pass through for recovery; (ii) integrate this technology and optimize the system for a culture-friendly, vacuum-assisted gas stripping process for in situ butanol recovery during fermentation; and (iii) evaluate the techno-economic feasibility of scaling up the system for commercial biobutanol production.

Project Methods
We have developed a simultaneous butanol fermentation and recovery by vacuum-assisted gas stripping technology that is microbe-friendly and does not require a membrane, sparger, or agitation. For this project, a compatible water-butanol separation systems (1- or 2- plates or 1-plate & 1-membrane) will be constructed and installed in the bioreactor, and the technology will be optimized. Optimization of the bioreactor process conditions will include:Microbial activity data collection: C. beijerinckii 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: Butanol concentration in the bioreactor and recovered stream, butanol yield and productivity, water loss from the bioreactor, butanol selectivity from fermentations involving different water-butanol separation systems, will be measured.Evaluation: We will assess the following outcomes: (i) water-repelling and oleophilic compound-attracting capacity of the coated steel wire mesh, as revealed by spraying a mixture of water and butanol on the mesh. (ii) assess the efficiency of the hydrophobic separation unit, as revealed by an average butanol selectivity >14. We will also assess the efficiency of fermentation of Miscanthus hydrolysates (MH) and co-fermentation of MH and glycerol to butanol.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 water-butanol separation unit design and imaging data and statistical analysis will also be stored as electronic copies. For the fermentations, indices pertaining to observed phenotypes, such as acetone-butanol formation, growth of C. beijerinckii, transformation/metabolism of lignocellulosic-derived sugars (Miscanthus giganteus hydrolysates-MH) and glycerol, and indices pertaining to acetone-butanol in situ recovery, such as rate of recovery, concentration of butanol in the recovered stream (condensate) and reduction in water losses, 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 08/01/19 to 07/31/24

Outputs
Target Audience:Our outreach efforts targeted a wide range of professionals, both locally and globally. The diverse audience included agricultural engineers, bioprocess engineers, systems analysts, microbiologists, graduate and undergraduate students, research associates, postdoctoral fellows, lecturers, professors, farmers, company executives, and government agencies. We also engaged the Ohio State University Technology Commercialization Office (TCO) and discussed the prospect of developinga patent from the work. Our aim was to engage professionals from various fields and sectors, ensuring that our findings and information reached a broad spectrum of stakeholders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students (one PhD and one MS) received comprehensive training in upstream and downstream processing techniques for producing biofuels and chemicals from lignocellulosic biomass. Additionally, two PhD students received partial training focused on lignocellulosic biomass pretreatment, enzymatic hydrolysis, fermentation, and downstream processing methods. Two research associates with PhDs received training and contributed to the project by applying their expertise in downstream processing techniques and assisting in training two undergraduate students. A postdoctoral researcher was also trained in conducting a techno-economic analysis related to the project. Overall, in terms of full-time equivalents (FTEs) for six months or 1-3 years period, the project provided training to four staff members with PhD degrees, five PhD students, and three technical staff members. How have the results been disseminated to communities of interest?Some results from this project were part of an invited talk at an international conference listed below. USDA/NIFA support was acknowledged in the presentation. Okonkwo, CC, Duduyemi, A, Ujor, VC, and Ezeji TC (2023) Development of nonmembrane separation system for real-time recovery of acetone-butanol-ethanol (ABE) during fermentation. American Society for Microbiology Annual Conference, George R. Brown Convention Center, Houston, Texas, USA, June 15 - 19, 2023 Ezeji TC (2023) Keynote Speaker on "Green chemistry: A case for sustainable production of biofuels from non-food substrates. 2023 International Joint Conference on Environmental Engineering and Biotechnology (CoEEB 2023) held in Malmo, Sweden on May 19-21, 2023. Duduyemi, AS, Okezie, E, Udegbe, F, and Ezeji TC (2023) Improving Product Yield and Reducing Carbon Footprints in Biomanufacturing. The Ohio State University Biomanufacturing Opportunities Workshop, Columbus, Ohio, USA, April 4 - 5, 2023 Duduyemi, A, Okonkwo, CC, Ujor, VC, and Ezeji TC (2023) In-situ recovery of C5 - C8 alcohols from bioreactor by vacuum-assisted gas stripping. 2023 CFAES Annual Research Forum, Wooster, OH, USA, March 27-28, 2023. Ezeji TC (2022) Keynote Speaker on "Green Chemistry-A case for the production of 2,3-butanediol and butanol from biomass". 75th Chemical Engineering Congress (CHEMCON 2022) Annual Meeting. Sustainability in Chemical Processes through Digitization, Artificial Intelligence, and Green Chemistry, Harcourt Butler Technical University, HBTU East Campus, HBIT East Campus, Nawabganj, Kanpur, Uttar Pradesh 208002, India, December 27 - 30, 2022 Ezeji TC (2022) Invited speaker on "Biofuels production: Downstream processing, challenges, and potential mitigations. Department of Food Technology, University of Ibadan, Oyo State, Nigeria. November 03, 2022 Ezeji TC and Ujor V (2022) Invited talk on "Bioprocessing of energy crops to fuels and chemicals: influence of simultaneous product recovery". Program of International Conference between Ohio State University and Ukraine. Theme: Growing Energy Crops by Recycling Biosolids on Marginal Lands. Sponsored by the U.S. CRDF Global and Ukrainian Ministry of Education and Science, The Ohio State university, Columbus, September 22, 2022. Ezeji TC. 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,2020, October 12 - 13 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Reduced water loss from the bioreactor during integrated fermentation with in situ product recovery is an essential part of the technology in development. To achieve our goal of significantly reducing water loss from bioreactor, we proposed using curved shaped superhydrophobic mesh. The curved surface facilitated the rapid sliding of water vapor back into the vessel, thus reducing the amount of water carried along with the ABE vapor during condensation. Consequently, the application of vacuum-assisted gas stripping (VAGS) at 50 mbar using a PDMS/PTFE-coated superhydrophobic mesh with a pore size of 180 μm successfully resulted in the recovery of a highly concentrated ABE stream. This process successfully retained up to 51% of the water in the bioreactor, which effectively met our target for water retention within the bioreactor. Secondly, we conducted analysis of the thermodynamics of high molecular weight alcohols, including pentanol (C5), hexanol (C6), heptanol (C7), and octanol (C8), both individually and as mixtures. Our goal was to gain insights into their recovery during fermentation and in situ recovery processes. To achieve this, we employed the UNIFAC property package of DWSIM™, an open-source software used for process simulation, modeling, and optimization. Using DWSIM™, we simulated the behavior of binary mixtures of water with C5 - C8 alcohols to determine the azeotropic tendencies of these mixtures. The simulation took into account the key thermodynamic variables for vapor-liquid equilibrium (VLE), namely pressure, temperature, and the concentration (mass fraction) of the alcohols. Based on the simulation data, we obtained temperature and concentration (mass fraction) profiles for the alcohols in the binary mixtures. The temperature varied within the range of boiling points of water and the respective alcohol in each mixture. Similarly, the mass fraction ranged from a pure water solution (100 wt.% water and 0 wt.% alcohol) to a pure alcohol solution (0 wt.% water and 100 wt.% alcohol). By plotting temperature against the mass fraction of alcohol, we constructed binary phase envelopes for each pressure level, which allowed us to determine the azeotropic characteristics. These characteristics represent the temperature and mass fraction at which the vapor and liquid phases of the mixture reach equilibrium. Likewise, the simulation data enabled us to predict the mass fraction of each alcohol present in the vapor phase at the simulated pressure, based on the initial concentration (mass fraction) of that alcohol in the aqueous solution. The VLE data helped us understand the behavior of the liquid and vapor phases in the binary mixtures, while the LLE data allowed us to identify the region of immiscibility, where the mixture exists as two distinct phases, in the equilibrium phase. Overall, through the use of DWSIM™ and the analysis of VLE and LLE data, we gained valuable insights into the thermodynamic properties and azeotropic characteristics of binary mixtures of water and high molecular weight alcohols (C5 - C8). Thirdly, the findings from our simulation studies contributed to our understanding of the recovery processes of high molecular weight (C5 - C8) alcohols during fermentation and in situ recovery, and we conducted laboratory experiments to corroborate the simulation data. The laboratory testing involved the preparation of different concentrations (0.2 - 2.0 g/L) of pentanol (C5), hexanol (C6), heptanol (C7), and octanol (C8), which are typical of the values obtained during batch fermentation in the respective media and cultures. The concentrations were increased (2.0 - 10.0 g/L) to evaluate the effect of increased concentrations on the thermodynamic properties of the high molecular weight alcohols and recovery. Subsequently, model solutions of C5 - C8 alcohols were supplemented with ethanol, butanol, acetic acid, and butyric acid in the ratio of 4: 2.25:1: 14: 4.25 typical to C. carboxydivorans P7 fermentation products. The supplementation of the C5 - C8 alcohols with short chain fatty acids was crucial to evaluate the selectivity and recovery of the C5 - C8 alcohols in the presence of other fermentation byproducts. Using response surface methodology and artificial neural network modeling, the optimal process conditions for recovering C5-C8 alcohols from a model solution were identified as a vacuum pressure of 43 mbar and a temperature of 37.5 °C. Under these conditions, VAGS proved highly effective for recovering C5-C8 alcohols, even at a low concentration of 1.85 g/L in the bioreactor. Overall, this study highlights the efficiency of VAGS for in-situ recovery of C5-C8 alcohols, presenting a promising strategy for the sustainable bioproduction of these alcohols. Fourthly, we conducted experiments to determine the surface tension of binary aqueous solutions of pentanol, hexanol, heptanol, and octanol. The investigation is necessary since measuring the surface tension of a binary aqueous solution can give additional information about the evaporation of molecules from the interface for enhanced product recovery during fermentation. It was found that as the concentrations of the respective alcohols in the aqueous solution increased from 0.2 -10.0 g/L, the surface tension of the aqueous solutions of pentanol, hexanol, heptanol, and octanol decreased from 64.28 to 27.83, 60.18 to 27.40, 50.65 to 25.78, and 41.88 to 26.80 mN/m, respectively. The decrease in surface tension of the binary aqueous solution of C5 - C8 alcohols demonstrates the increase in the evaporation rate of the molecules of the binary solution as the concentration of alcohol increases. Although the evaporation of molecules in a solution relies on other thermodynamic factors such as temperature and pressure, the surface tension determined in this work provides additional information regarding the effect of increased concentrations of the C5 - C8 alcohols on the evaporation and condensation for enhanced product recovery. Fifthly, dilute acid pretreatment of lignocellulosic biomass (switchgrass) was conducted, followed by enzymatic hydrolysis to produce lignocellulosic biomass hydrolysates (LBH) containing a mixture of sugars, including cellobiose, glucose, xylose, and arabinose. However, lignocellulose-derived microbial inhibitory compounds (LDMICs), which interfere with the growth and fermentation efficiency of biocatalysts such as C. beijerinckii, were also generated during the process. When the LBH was subjected to acetone-butanol-ethanol (ABE) fermentation using C. beijerinckii, no growth or ABE production was observed. Interestingly, co-fermentation of LBH with glycerol increased the intracellular NADH/NAD? ratio in C. beijerinckii, which enabled the microorganism to detoxify LDMICs and successfully produce ABE. Sixthly, we developed techno-economic analysis (TEA) model for evaluating butanol production using different Clostridium species with and without the use of the vacuum-assisted gas stripping (VAGS) separation system. We performed TEA of the butanol production system integrating the separation unit featuring superhydrophobic mesh. The TEA incorporated experimental data generated from butanol production in the presence of the separation unit. The integration of the bioreactor with the superhydrophobic separation module resulted in a 20% reduction in the total annual operating cost compared to the system without a superhydrophobic water/butanol separation module. The enhanced system with superhydrophobic water/butanol separation module exhibited significantly reduced net butanol production cost of $0.69/L compared to the $1.04/L production cost without the superhydrophobic water/butanol separation module. The study indicated that this strategic modification not only reduces operational costs, but also optimizes the recovery and purification processes, making it a promising approach for sustainable biobutanol production.

Publications

Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Okonkwo, CC, Duduyemi, A, Ujor, VC, Qureshi N, and Ezeji TC (2024) Developing a separation system to enable real-time recovery of acetone-butanol during fermentation. Appl Microbiol Biotechnol 108: 506. https://doi.org/10.1007/s00253-024-13340-x
Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Okezie E, Rozina, and Ezeji TC (2024) Utilization of biomass-based resources for biofuel production: A mitigating approach towards zero emission. Sustainable Chemistry one World 2: 100007. https://doi.org/10.1016/j.scowo.2024.100007
Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Duduyemi, AS, Okezie, E, Udegbe, F, and Ezeji TC (2023) Improving Product Yield and Reducing Carbon Footprints in Biomanufacturing. The Ohio State University Biomanufacturing Opportunities Workshop, Columbus, Ohio, USA, April 4 5, 2023
Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Duduyemi, A, Okonkwo, CC, Ujor, VC, and Ezeji TC (2023) In-situ recovery of C5 C8 alcohols from bioreactor by vacuum-assisted gas stripping. 2023 CFAES Annual Research Forum, Wooster, OH, USA, March 27-28, 2023.
Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Ezeji TC (2022) Keynote Speaker on "Green Chemistry-A case for the production of 2,3-butanediol and butanol from biomass". 75th Chemical Engineering Congress (CHEMCON 2022) Annual Meeting. Sustainability in Chemical Processes through Digitization, Artificial Intelligence, and Green Chemistry, Harcourt Butler Technical University, HBTU East Campus, HBIT East Campus, Nawabganj, Kanpur, Uttar Pradesh 208002, India, December 27 - 30, 2022
Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Ezeji TC (2022) Invited speaker on "Biofuels production: Downstream processing, challenges, and potential mitigations. Department of Food Technology, University of Ibadan, Oyo State, Nigeria. November 03, 2022
Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Ezeji TC and Ujor V (2022) Invited talk on "Bioprocessing of energy crops to fuels and chemicals: influence of simultaneous product recovery". Program of International Conference between Ohio State University and Ukraine. Theme: Growing Energy Crops by Recycling Biosolids on Marginal Lands. Sponsored by the U.S. CRDF Global and Ukrainian Ministry of Education and Science, The Ohio State university, Columbus, September 22, 2022.
Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Rozina, Okezie E, and Ezeji TC (2024) Exploring the synergy of nanomaterials and microbial cell factories during biohydrogen and biobutanol production from different carbon sources. Sustainable Chemistry for the Environment 6: 100098. https://doi.org/10.1016/j.scenv.2024.100098
Type: Peer Reviewed Journal Articles Status: Published Year Published: 2023 Citation: Ezeji TC, Atiyeh H, Mariano AP and Rakshit SK (2023) Innovative bioconversion of non-food substrates to fuels. Front. Bioeng. Biotechnol. 11:1163513. doi: 10.3389/fbioe.2023.1163513
Type: Book Chapters Status: Published Year Published: 2022 Citation: Panahi Shariat HK, Dehhaghi M, Guillemin GJ, Okonkwo CC, Kinder, JE, and Ezeji TC (2022) Biobutanol production: scope, significance, and applications. Chapter in: Advances and Developments in Biobutanol Production. (Eds: Segovia-Hernandez et al.) Woodhead Publishing; 1st edition (December 9, 2022), Elsevier BV, Amsterdam, The Netherlands
Type: Theses/Dissertations Status: Awaiting Publication Year Published: 2024 Citation: Duduyemi Ademola (2024) Development of a nonmembrane superhydrophobic separation system for efficient in situ recovery during acetone-butanol-ethanol fermentation. PhD Dissertation, The Ohio State University.
Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Okonkwo, CC, Duduyemi, A, Ujor, VC, and Ezeji TC (2023) Development of nonmembrane separation system for real-time recovery of acetone-butanol-ethanol (ABE) during fermentation. American Society for Microbiology Annual Conference, George R. Brown Convention Center, Houston, Texas, USA, June 15 - 19, 2023
Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Ezeji TC (2023) Keynote Speaker on "Green chemistry: A case for sustainable production of biofuels from non-food substrates. 2023 International Joint Conference on Environmental Engineering and Biotechnology (CoEEB 2023) held in Malmo, Sweden on May 19-21, 2023.
Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Ezeji TC. 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,2020, October 12 - 13
Type: Other Journal Articles Status: Under Review Year Published: 2025 Citation: Duduyemi, A and Ezeji TC (202x) Development of real-time in-situ recovery of C5 C8 alcohols from bioreactor. Chemical Engineering Journal (In preparation)
Type: Other Journal Articles Status: Other Year Published: 2025 Citation: Duduyemi, A, Okonkwo, CC, Shah, A and Ezeji TC (202x) Incorporation of superhydrophobic separation module to the bioreactor and impact on the technoeconomic analysis of butanol fermentation. Bioresource Technol (in preparation)
Type: Other Journal Articles Status: Other Year Published: 2025 Citation: Duduyemi, A, Okonkwo, CC, Ujor, VC, and Ezeji TC (202x) Impact of superhydrophobic water-butanol separation module on butanol recovery during acetone-butanol-ethanol fermentation by Clostridium beijerinckii. Bioresource Technol (in preparation)

Progress 08/01/22 to 07/31/23

Outputs
Target Audience:During this reporting period, our outreach efforts targeted a wide range of professionals, both locally and globally. The diverse audience included agricultural engineers, bioprocess engineers, systems analysts, microbiologists, graduate and undergraduate students, research associates, postdoctoral fellows, lecturers, professors, farmers, company executives, and government agencies. Our aim was to engage professionals from various fields and sectors, ensuring that our findings and information reached a broad spectrum of stakeholders. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A graduate student is currently undergoing comprehensive training in both upstream and downstream processing techniques for biofuels and chemicals derived from lignocellulosic biomass. To expedite the investigation on process development aspect of this project, a new research associate has been recently recruited to complement the efforts of the graduate student. This addition to the team became necessary as the previous individual accepted a faculty position and consequently left the project. A postdoctoral researcher is currently being trained in conducting technoeconomic analysis of the proposed system. How have the results been disseminated to communities of interest?Some results from this project were part of an invited talk at an international conference listed below. USDA/NIFA support was acknowledged in the presentation. Ezeji TC (2023) Keynote Speaker on "Green chemistry: A case for sustainable production of biofuels from non-food substrates. 2023 International Joint Conference on Environmental Engineering and Biotechnology (CoEEB 2023) held in Malmo, Sweden on May 19-21, 2023. Ezeji TC (2022) Keynote Speaker on "Green Chemistry-A case for the production of 2,3-butanediol and butanol from biomass". 75th Chemical Engineering Congress (CHEMCON 2022) Annual Meeting. Sustainability in Chemical Processes through Digitization, Artificial Intelligence, and Green Chemistry, Harcourt Butler Technical University, HBTU East Campus, HBIT East Campus, Nawabganj, Kanpur, Uttar Pradesh 208002, India, December 27 - 30, 2022 Ezeji TC and Ujor V (2022) Invited talk on "Bioprocessing of energy crops to fuels and chemicals: influence of simultaneous product recovery". Program of International Conference between Ohio State University and Ukraine. Theme: Growing Energy Crops by Recycling Biosolids on Marginal Lands. Sponsored by the U.S. CRDF Global and Ukrainian Ministry of Education and Science, The Ohio State university, Columbus, September 22, 2022. What do you plan to do during the next reporting period to accomplish the goals?The current scope of work encompasses three types of separation units: a unit featuring a superhydrophobic mesh, a unit consisting of two superhydrophobic meshes, and a hybrid separation unit combining a superhydrophobic mesh with a membrane. Our team has successfully completed the development of the unit with a single superhydrophobic mesh and conducted extensive characterization of the thermodynamics of the target products in both their individual and mixed forms. The data obtained from this unit will be utilized to assess the performance of the unit with two superhydrophobic meshes. Additionally, for the hybrid separation unit, we will synthesize and evaluate membranes based on polydimethylsiloxane (PDMS) to create a hybrid coated mesh-membrane system for water-butanol separation, as proposed in our project. By incorporating a PDMS-based membrane into the separation process, we anticipate achieving a higher efficiency level than what can be accomplished using only the coated superhydrophobic mesh. One significant challenge in membrane-based separation processes is fouling caused by microorganisms or impure substrates. To address this issue, we have designed the hybrid system in a way that minimizes fouling by preventing direct contact between these contaminants and the membrane. The coated superhydrophobic mesh will serve as a protective barrier, reducing fouling and enhancing the durability and efficiency of the membrane during the separation process. This integrated approach aims to overcome the limitations associated with membrane fouling and optimize the overall performance of the separation system. We will conduct a thorough techno-economic analysis of ABE fermentation and production of high molecule alcohols, specifically focusing on the integration of single and double layer of superhydrophobic meshes and hybrid separation units for in situ product recovery. We will also assess the technical feasibility and economic viability of employing vacuum-assisted gas stripping technology within this system.

Impacts
What was accomplished under these goals? Reduced water loss from the bioreactor during fermentation and in situ product recovery is an essential part of the technology in development. After critically comparing the surface area and volumes of various headplates for pressure vessels, a hemispherical dished head was chosen, and a stainless steel headplate was successfully fabricated to retrofit the existing 5 L New-Brunswick Bioflo bioreactor. This choice was made based on the need for a large volume (resulting in weaker intermolecular forces) at the headspace and zinc oxide (ZnO)/polydimethylsiloxane (PDMS)-coated superhydrophobic and super- oleophilic mesh of sizes 0.18 - 0.425 mm. These meshes were incorporated into the 5 L bioreactor and tested for their ability to reduce water losses in the bioreactor with and without microbial cells. Although our results showed that the use of coated mesh significantly enhanced the retention (>10 - 25%) of water in the bioreactor and facilitated the recovery of acetone-butanol-ethanol (ABE) during the process, our target goal for water retention in the bioreactor of > 20 - 45% was not achieved. To achieve our goal, we proposed changing the shape of the superhydrophobic mesh from flat to curved. The curved surface would facilitate the rapid sliding of water vapor back into the vessel, thus reducing the amount of water carried along with the ABE vapor during condensation. Secondly, we conducted analysis of the thermodynamics of high molecular weight alcohols, including pentanol (C5), hexanol (C6), heptanol (C7), and octanol (C8), both individually and as mixtures. Our goal was to gain insights into their recovery during fermentation and in situ recovery processes. To achieve this, we employed the UNIFAC (Universal Functional-group Activity Coefficients) property package of DWSIM™ (v8.3.4; Medeiros, 2021), an open-source software used for process simulation, modeling, and optimization. Using DWSIM™, we simulated the behavior of binary mixtures of water with C5 - C8 alcohols to determine the azeotropic tendencies of these mixtures. The simulation took into account the key thermodynamic variables for vapor-liquid equilibrium (VLE), namely pressure, temperature, and the concentration (mass fraction) of the alcohols. We conducted VLE and liquid-liquid equilibrium (LLE) simulations at four different pressure levels: vacuum pressures (30, 40, and 50 mbar) for the vacuum-assisted gas stripping (VAGS) process, and atmospheric pressure (1013.25 mbar). Based on the simulation data, we obtained temperature and concentration (mass fraction) profiles for the alcohols in the binary mixtures. The temperature varied within the range of boiling points of water and the respective alcohol in each mixture. Similarly, the mass fraction ranged from a pure water solution (100 wt.% water and 0 wt.% alcohol) to a pure alcohol solution (0 wt.% water and 100 wt.% alcohol). By plotting temperature against the mass fraction of alcohol, we constructed binary phase envelopes for each pressure level, which allowed us to determine the azeotropic characteristics. These characteristics represent the temperature and mass fraction at which the vapor and liquid phases of the mixture reach equilibrium. Likewise, the simulation data enabled us to predict the mass fraction of each alcohol present in the vapor phase at the simulated pressure, based on the initial concentration (mass fraction) of that alcohol in the aqueous solution. The VLE data helped us understand the behavior of the liquid and vapor phases in the binary mixtures, while the LLE data allowed us to identify the region of immiscibility, where the mixture exists as two distinct phases, in the equilibrium phase. Overall, through the use of DWSIM™ and the analysis of VLE and LLE data, we gained valuable insights into the thermodynamic properties and azeotropic characteristics of binary mixtures of water and high molecular weight alcohols (C5 - C8). Thirdly, the findings from our simulation studies contributed to our understanding of the recovery processes of high molecular weight alcohols during fermentation and in situ recovery, and we conducted laboratory experiments to corroborate the simulation data. The laboratory testing involved the preparation of different concentrations (0.2 - 2.0 g/L) of pentanol, hexanol, heptanol, and octanol, which are typical of the values obtained during batch fermentation in the respective media and cultures. The concentrations were increased (2.0 - 10.0 g/L) to evaluate the effect of increased concentrations on the thermodynamic properties of the high molecular weight alcohols and recovery. Subsequently, model solutions of C5 - C8 alcohols were supplemented with ethanol, butanol, acetic acid, and butyric acid in the ratio of 4: 2.25:1: 14: 4.25 typical to C. carboxydivorans P7 fermentation products. The supplementation of the C5 - C8 alcohols with short chain fatty acids was crucial to evaluate the selectivity and recovery of the C5 - C8 alcohols in the presence of other fermentation byproducts. We are happy to report that our laboratory data are compatible with our simulation data. Fourthly, we conducted experiments to determine the surface tension of binary aqueous solutions of pentanol, hexanol, heptanol, and octanol. The investigation is necessary since measuring the surface tension of a binary aqueous solution can give additional information about the evaporation of molecules from the interface for enhanced product recovery during fermentation. It was found that as the concentrations of the respective alcohols in the aqueous solution increased from 0.2 - 10.0 g/L, the surface tension of the aqueous solutions of pentanol, hexanol, heptanol, and octanol decreased from 64.28 to 27.83, 60.18 to 27.40, 50.65 to 25.78, and 41.88 to 26.80 mN/m, respectively. The decrease in surface tension of the binary aqueous solution of C5 - C8 alcohols demonstrates the increase in the evaporation rate of the molecules of the binary solution as the concentration of alcohol increases. Although the evaporation of molecules in a solution relies on other thermodynamic factors such as temperature and pressure, the surface tension determined in this work provides additional information regarding the effect of increased concentrations of the C5 - C8 alcohols on the evaporation and condensation for enhanced product recovery. Fifthly, we developed a baseline techno-economic analysis (TEA) model for evaluating butanol production using different Clostridium species without the use of the separation system. We are currently performing TEA of the butanol production system that integrates the separation unit featuring superhydrophobic mesh. The TEA incorporates experimental data generated from the butanol production including the separation unit. The TEA will provide techno-economic estimates for both batch-fed and semi-continuous processes for butanol production. Using process input data like process volume, temperature, pressure, cycles, and product yields, the main outcome of this analysis will be estimates of materials, energy usage, resources required, and overall costs. Specifically, the TEA will evaluate the impact of the integration of separation unit on the water recovery, butanol productivity and yield, energy use and overall costs. In addition, the viable methods for waste valorization will also be incorporated. Finally, we will divulge cost and material parameters for a feasible production of butanol via the fermentation route of biomass using the proposed separation systems.

Publications

Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Duduyemi, A., Okonkwo, C. C., Ujor, V. C. and Ezeji, T. C. In-situ recovery of C5 C8 alcohols from bioreactor by vacuum-assisted gas stripping. Chinese Journal of Catalysis (Under review).
Type: Journal Articles Status: Other Year Published: 2023 Citation: Okonkwo, C. C., Duduyemi, A., Ujor, V. C. and Ezeji, T. C. Development of nonmembrane separation system for real-time recovery of acetone-butanol-ethanol (ABE) during fermentation. Applied Energy (in preparation).
Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Okonkwo, C. C., Duduyemi, A., Ujor, V. C. and Ezeji, T. C. Development of nonmembrane separation system for real-time recovery of acetone-butanol-ethanol (ABE) during fermentation. American Society for Microbiology Annual General Meeting, Houston, TX. June, 2023.

Progress 08/01/21 to 07/31/22

Outputs
Target Audience:The target audiences for this reporting period included Agricultural engineers, Bioprocess engineers, Systems analysts, Microbiologist, graduate and undergraduate students, research associates and Postdocs, technicians of The Ohio State University (OSU), Farmers, Company executives, and government agencies. Changes/Problems:1. The greatest challenge we are facing is the inability of the lead graduate student working on the project to take classes and conduct laboratory research. In fact, the graduate student is struggling in the PhD program, and co-PIs and I are considering increasing the post-doc time on the project. Of course, we will seek approval from your office if we decide to go through that route. What opportunities for training and professional development has the project provided?One graduate student is receiving training in upstream and downstream processing of biofuels and chemicals from lignocellulosic biomass, and a postdoctoral visiting scientist is currently being trained in techno-economic analysis of the proposed system. How have the results been disseminated to communities of interest?Some results from this project were part of an invited talk at an international conference listed below. USDA/NIFA support was acknowledged in the presentation. Ezeji TC (2022) Bioprocessing of energy crops to fuels and chemicals: influence of simultaneous product recovery. International Conference on Growing Energy Crops by Recycling Biosolids on Marginal Lands. The Ohio State university, Columbus, September 22, 2022. Ezeji TC (2022) Invited speaker on "Biofuels production: Downstream processing, challenges, and potential mitigations. Department of Food Technology, University of Ibadan, Oyo State, Nigeria. November 03, 2022 What do you plan to do during the next reporting period to accomplish the goals?We will produce meshes with water contact angles in superhydrophobic range (>160°). We will separately install superhydrophobic one-mesh and two-mesh units in the headplate of the 5-L bioreactor and characterize the unit for butanol, pentanol, and hexanol recovery as well as water retention in the bioreactor. It is our expectation that the improved separation unit will cut down water losses in the bioreactor by more than 50% during acetone butanol ethanol (ABE) fermentation. We will evaluate a detailed techno-economic analysis of the ABE fermentation including the improved separation process and in situ product recovery by vacuum-assisted gas stripping technology.

Impacts
What was accomplished under these goals? The overarching goal of this project is to develop a scalable and economically feasible technology that can accept mixed fibrous/colloidal substrates for fermentation and facilitating real-time removal of products of fermentation from the bioreactor so that the fermentation microorganisms are not subject to product inhibition or toxicity. Reduced water loss from the bioreactor during fermentation and in situ product recovery is an essential part of the technology in development. After critically comparing the surface area and volumes of various headplates for pressure vessels, a hemispherical dished head was chosen, and a stainless steel headplate was successfully fabricated to retrofit the existing 5 L New-Brunswick Bioflo bioreactor. This choice was made based on the need for a large volume (resulting in weaker intermolecular forces) at the headspace that allows for optimal condensation of the acetone-butanol-ethanol (ABE) vapor during recovery from the fermentation broth. Other design considerations included headplate material and thickness, inner diameter, pressure, temperature, maximum allowable stress, weld efficiency, and corrosion efficiency. Secondly, we embarked on refining the zinc oxide (ZnO)/polydimethylsiloxane (PDMS)-coated superhydrophobic and super-oleophilic mesh of sizes 0.18 - 0.425 mm and tested their ability to reduce water losses in the bioreactor with and without microbial cells. While the coated 0.18 mm mesh retained 37.3 - 67.7% more water in the bioreactor than the uncoated mesh in the absence of microbial cells, the coated mesh retained only 13.7% - 39.2% more water in the bioreactor than the uncoated mesh in the presence of microbial cells. All the adjustments made in the coating techniques did not improve the water retention in the bioreactor, which led us to suspect that the coating materials and the process may not be producing superhydrophobic mesh or surfaces. Consequently, a contact angle goniometer (Ossila Inc., UK) was purchased and used to measure the water contact angle of coated and uncoated stainless steel meshes. The average water contact angles for the uncoated 0.18, 0.30, and 0.425 mm stainless steel meshes were 111.8 ± 2.68°, 88.47 ± 3.24°, and 80.31 ± 2.09°, respectively, while the average water contact angles for the ZnO/PDMS coated 0.18, 0.30, and 0.425 mm stainless steel meshes were 127.78 ± 1.80°, 120.54 ± 3.19°, and 112.52 ± 3.45°, respectively. Although the contact angle of 127.78 ± 1.80° is in the hydrophobic range, it is not in the superhydrophobic range (> 160°). Currently, we are testing different surface modifying compounds silver nitrate, zeolites (e.g., benzimidazole), and silanes with lower surface energy (e.g., hexadecyltrimethoxysilane) to alter the bulk properties of the coating polymer (oxide (ZnO)/PDMS). Additionally, we are testing spray coating method as it is expected to result in more uniform thin film coating than the dip coating method. A techno-economic analysis (TEA) is currently in progress that incorporates experimental data generated from the retrofitted, improved separation system described above for a bioreactor. The experiments using lignocellulosic biomass and crude glycerol will provide baseline data for butanol production process improvement. The TEA will provide techno-economic estimates for both batch-fed and semi-continuous process butanol production. Using process input data like total material solid, volume, temperature, pressure, cycles, etc., the main outcome of this analysis will be estimates of materials, energy usage, resources required, and overall costs. With revenue estimates of butanol as the main product, and acetone, ethanol, and hydrogen as by-products, the analyses will elucidate data on the viability of the work in progress. In addition, the viable methods for waste valorization will also be incorporated. Finally, as expected outcomes from the TEA, we hope to divulge cost and material parameters for a feasible production of butanol via the fermentation route of biomass using the proposed separation system.

Publications

Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Ezeji TC (2022) Bioprocessing of energy crops to fuels and chemicals: influence of simultaneous product recovery. International Conference on Growing Energy Crops by Recycling Biosolids on Marginal Lands. The Ohio State university, Columbus, September 22, 2022.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Ezeji TC (2022) Invited speaker on Biofuels production: Downstream processing, challenges, and potential mitigations. Department of Food Technology, University of Ibadan, Oyo State, Nigeria. November 03, 2022

Progress 08/01/20 to 07/31/21

Outputs
Target Audience:The target audiences for this reporting period included Agricultural engineers, Bioprocess engineers, Systems analysts, Microbiologist, graduate and undergraduate students, research associates and Postdocs, technicians of The Ohio State University (OSU), Farmers, Company executives, and government agencies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student is receiving training in upstream and downstream processing of biofuels and chemicals from lignocellulosic biomass, and one postdoc is currently being trained in techno-economic analysis of the proposed system. How have the results been disseminated to communities of interest?Some results from this project were part of an invited talk at an international conference listed below. USDA/NIFA support was acknowledged in the presentation. TC Ezeji. 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,2020, October 12 - 13 What do you plan to do during the next reporting period to accomplish the goals?We will refine the zinc oxide (ZnO)/polydimethylsiloxane (PDMS)-coated superhydrophobic and super-oleophilic mesh, and PDMS based membranes, based on obtained results from model solution containing microbial cells. The refined mesh will be used in the fabrication of new superhydrophobic and super-oleophilic plates for the separation of butanol and higher alcohols, and fermentation parameters will be determined. We will build upon our preliminary baseline techno-economic analysis model to develop a detailed model for techno-economic analysis of ABE fermentation with in situ product recovery by vacuum-assisted gas stripping technology.

Impacts
What was accomplished under these goals? Theoverarching goal of this project is to develop a scalable and economically feasible technology that can accept mixed fibrous/colloidal substrates for fermentation and facilitating real-time removal of products of fermentation from the bioreactor so that the fermentation microorganisms are not subject to product inhibition or toxicity. Reduced water loss from the bioreactor during fermentation and in situ product recovery is an essential part of the technology in development. Consequently, zinc oxide (ZnO)/polydimethylsiloxane (PDMS)-coated superhydrophobic and super-oleophilic mesh of different sizes, 0.18 - 0.3 mm, were used to recover acetone butanol ethanol (ABE) real-time from bioreactor with and without microbial cells. The butanol and ABE concentration in the bioreactor were increased to 15 g/L and 25 g/L, respectively. Different product recovery time ranging from 20 - 120 mins were used, and the water loss in the bioreactor with 0.18 mm mesh was reduced by 2-fold when compared to the control experiment. The reduction is a 41% improvement over the value we obtained (1.42-fold reduction) in year one of the project. The water loss was further reduced in the presence of microbial cells; however, results are still preliminary and will need validation. We have also developed a preliminary baseline techno-economic analysis model for evaluating butanol production using different Clostridium species without the use of the separation system. Our analysis estimated the butanol production costs in the range $1.01-1.95 per liter (for lignocellulosic biomass) with optimization of fermentation process conditions showing promise for further cost reductions.

Publications

Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Some results from this project were part of an invited talk at an international conference listed below. USDA/NIFA support was acknowledged in the presentation. 1. TC Ezeji. 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,2020, October 12 13

Progress 08/01/19 to 07/31/20

Outputs
Target Audience:The target audiences for this reporting period included graduate and undergraduate students, research associates and Postdocs, and technicians of The Ohio State University (OSU). We did not reach wider audiences because our results were preliminary, which were contributed in part by the closure of our laboratories during the early stage of Covid-19 pandemic. However, we plan for a wider distribution as soon as we complete some ongoing experiments. Changes/Problems:There was no major change in the experiemental design or research approach in the project. What opportunities for training and professional development has the project provided?One graduate student was hired to receive training in upstream and downstream processing of biofuels and chemicals from lignocellulosic biomass, and one Postdoc is currently being trained in in technoeconomic analysis (TEA). How have the results been disseminated to communities of interest?Obtained results are preliminary to share with wider audiences at this stage and we plan for a wider distribution as soon as we complete some ongoing experiments. However, we shared some of our challenges and breakthroughs with graduate and undergraduate students, research associates and Postdocs, and technicians of The Ohio State University (OSU) during our departmental seminars. What do you plan to do during the next reporting period to accomplish the goals?The zinc oxide (ZnO)/polydimethylsiloxane (PDMS)-coated superhydrophobic and super-oleophilic mesh will be refined, new superhydrophobic and super-oleophilic plates and PDMS based membranes will be fabricated, and a preliminary model for techno-economic analysis of the vacuum-assisted gas stripping technology will be developed.

Impacts
What was accomplished under these goals? Work during the first year (2019/2020) of the project focused on Objective 1 of the proposal, which aims to fabricate and use a water-butanol separation system for real-time recovery of butanol from a bioreactor during ABE fermentation. The major accomplishment was the construction and testing of zinc oxide (ZnO)/polydimethylsiloxane (PDMS)-coated superhydrophobic and super-oleophilic stainless-steel mesh for ABE fermentation with in situ product recovery by vacuum-assisted gas stripping (VAGS). This aspect of the work is necessary as it will provide insight on the concentration of butanol in the condensate as well as water loss reduction capacity of a superhydrophobic and super-oleophilic plate in a 5-liter New Brunswick BioFlo 310 bioreactor. Using different concentrations (4.8 - 12.8 g/L) of model ABE solution, we evaluated water loss from the bioreactor and butanol concentration in the condensate (recovered butanol) during ABE fermentation with real-time in situ product recovery. Afterward, we found that the concentration of butanol and ABE in the condensate obtained with the help of the superhydrophobic and super-oleophilic stainless-steel mesh was 23- and 3.6-fold greater, respectively, than the control (uncoated stainless steel mesh), when the starting ABE concentration in the bioreactor was 4.8 g/L, with corresponding 1.42-fold (42%) reduction in water loss from the bioreactor. Notably, the VAGS operation time was 40 min. When the starting ABE concentration in the bioreactor was increased to 8.0 g/L, the concentration of butanol and ABE in the condensate obtained with the help of the superhydrophobic and super-oleophilic stainless-steel mesh was 40- and 1.75-fold, respectively, greater than the control; with corresponding 1.41-fold (41%) reduction in water loss from the bioreactor. However, when the starting ABE concentration in the bioreactor was increased to 12.8 g/L, although the superhydrophobic and super-oleophilic stainless-steel mesh produced condensates with greater butanol and ABE concentration, its superiority over the control was significantly reduced. These results are significant because the superhydrophobic and super-oleophilic stainless-steel mesh performed well at low butanol concentration in the bioreactor. Indeed, in situ butanol recovery process is typically initiated when the ABE concentration in the bioreactor is between 2.5 and <5.0 g/L. Although we plan to improve the process, we are pleased that the fabricated water-solvent separation system is effective at low solvent concentration in the bioreactor.

Publications