Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to NRP
CONVERSION OF FOOD WASTE INTO 2,3-BUTANEDIOL WITH BIOFILM FERMENTATION AND METABOLIC ENGINEERING OF NEWLY-ISOLATED BACILLUS LICHENIFORMIS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1019354
Grant No.
2019-67012-29601
Cumulative Award Amt.
$163,647.00
Proposal No.
2018-08057
Multistate No.
(N/A)
Project Start Date
May 1, 2019
Project End Date
Apr 30, 2022
Grant Year
2019
Program Code
[A7201]- AFRI Post Doctoral Fellowships
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
Food Science and Technology
Non Technical Summary
Americans lead the world in food waste. Though it may not be a statistic to be proud of there is a way to turn things around! Each year we throw away 60 million tons of food, almost half of all produce sold. While a small portion of the food waste is used as animal feed most of it ends up in landfills, contributing to global warming andgasses that are 25 times more powerful then fumes from motorized vehicles (carbon dioxide). One possible solution is to turn this food waste into something we can all benefit from. Through the use of a special waste eating bacterium, Bacillus licheniformis YNP5-TSU, we can convert waste into a useful biofuel called butanediol (BDL).Our goal for this project is to determine the best fermentation settings and genetic modifications for B. licheniformis YNP5-TSU so the maximum amount of biofuel can be produced. Many fermentation parameters such as; pH, temperature, dissolved oxygen content, and feedstock composition have significant effects on BDL output and will be calculated through use of 2-liter New Brunswick® fermenters and high performance liquid chromatography (HPLC). In order to remove other unwanted fermentation end-products, genetic modifications to B. licheniformis YNP5-TSU through the newly discovered CRISPR-Cas9 genome editing tool will also be performed.Through these techniques mentioned we will be able to provide valuable data on how this unique microorganism degrades food waste and converts it into biofuel. Completion of this project will also give insight towards the next step; implementation of B. licheniformis YNP5-TSU into large scale production and ultimately lead to a new sustainable energy resource from food waste.
Animal Health Component
30%
Research Effort Categories
Basic
70%
Applied
30%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5114010110090%
6055010101010%
Knowledge Area
511 - New and Improved Non-Food Products and Processes; 605 - Natural Resource and Environmental Economics;

Subject Of Investigation
4010 - Bacteria; 5010 - Food;

Field Of Science
1010 - Nutrition and metabolism; 1100 - Bacteriology;
Goals / Objectives
Overall Goal and Aim of ProposalOur goal is to convert food waste into value-added2,3-Butanediol (BDL), throughoptimization offermentation parameters andmetabolic engineering of the novel and newly-isolated Bacillus licheniformis YNP5-TSU.Nearly 40% of food is being wasted in the U.S., accounting for the single largest component of the U.S. municipal solid waste, resulting in $165 billion economic loss including the food itself, and associated water, energy, chemicals spent in the food supply chain (Gunders, 2012). While a small portion of the food waste is recovered through food banks or as animal feed, most of it ends up in landfills. Food waste rotting in landfills significantly contributes to methane emission, a greenhouse gas at least 25 times more powerful for global warming as carbon dioxide. With the worldwide economic development and population growth, the food waste associated economic and environmental problems will only become more challenging. Therefore, appropriate management and valorization of food waste is urgently needed.Two main objectives and an optional third have been proposed in order to reach the project goalObjective 1. Identify the key process variables that determine the performance of BDL production from glucose and food waste using wild-typeB. LicheniformisYNP5-TSU.In this objective, the key fermentation conditions and feedstock composition affecting the fermentation efficiency and yield will be identified. After completion of this objective, the generated knowledge will lead to the optimized fermentation conditions and maximized production of BDL from food waste.Task 1.1.Identify and optimize the fermentation conditions (temperature, pH and dissolved oxygen) to convert glucose to 2,3 BDLTask 1.2. Investigate several different carbon feedstocks (i.e. glucose, cellulose and food waste) and determine their impact on fermentation end-productsObjective 2. Genetically engineeringBacillus licheniformisYNP5-TSU for enhancement of BDL yield, through a knockout of mixed acid fermentation pathway genesTask 2.1Delete lactate, acetate and ethanol pathway genes using CRISPR-Cas9 genome editing system.One current strategy for enhancing butanol/BDL production is to disrupt the butyrate, acetone, lactate and acetate pathways. This is done through a knockout or knockdown of genes associated with mixed acid end products. In the similar fermentation process of butanol, byClostridium acetobutylicum,the deletion of CRE (catabolic responsive element) caused a 7.5 fold increase in butanol production (Xu et al., 2017) and deletion of the fatty acid pathway genes with the CRISPR-Cas9 system inClostridium saccharoperbutylacetonicumhas increased ABE production by >10% (Wang et al., 2017).Objective 3 (optional, dependenton completion of objective 1 and 2). Design, fabricate and evaluate a novel continuous biofilm fermentation system to boost the conversion of food waste to BDL.BDL fermentation is conventionally carried out using planktonic cells (suspended cells free-floating in bulk solution). The low planktonic cell density and the susceptibly of planktonic cells to product inhibition (i.e. BDL and acids) are the two limiting factors constraining BDL production rate and yield. To overcome these technical barriers we will develop the novel continuous biofilm fermentation system that immobilizes cells on heavy carrier materials to greatly increase theBacilluscell density. The continuous biofilm fermentation system will produce BDL much faster than the planktonic fermentation, and the application of the system will significantly reduce fermentation time, reactor size and associated cost for BDL production from food waste.Task 3.1. Develop the continuous biofilm reactor with cell immobilization for food waste fermentation into 2,3 BDL.
Project Methods
Proposed MethodologyObjective 1. Task 1.1 Fermentation parameter designExperimental design. We will investigate the effect of different key fermentation variables, including temperature, dissolved oxygen and pH, on the BDL yield and productivity. These three variables were chosen because 1) our preliminary results revealed that showed that fermentation temperature significantly affected the Bacillus growth and fermentation rates ; 2) Recent studies have reported that increasing the dissolved oxygen level to a certain level in fermentation broth lead to increased BDL yield and reduced acid yield (Qiu et al., 2016); 3) and Literature indicated that the starting pH was one of the primary factors in determining BDL fermentation yield (Li et al., 2013). We will first investigate different temperatures (50, 55, 60 and 65 ºC), dissolved oxygen levels (0, 5, 10, and 20%) and pH (6.0, 7.0, 8.0, and 9.0) to identify the best fermentation conditions for BDL production using the standard Carboxymethylcellulose (CMC) media. This media is generally used for cellulase testing but has been modified to contain 10% glucose, and 1% LB (lysogeny broth) for increased cell mass.Fermentation procedures. Fermentation will be conducted in 2-liter New Brunswick® fermenters in Dr. Huang's lab (Figure 3), where temperature, pH, and dissolved oxygen can be precisely controlled. Briefly, CMC medium (1% LB broth, 1% Carboxymethylcellulose, CMC, and 10% glucose) will be sterilized and inoculated with 1% broth culture of YNP5-TSU (growth density at OD600) for incubation up to 48 hrs. Temperature will be maintained at either: 50, 55, 60, and 65°C with a pH range of each 7.0, 8.0, and 9.0, maintained with continuous addition of .05M NaOH and stabilized dissolved oxygen level at 0, 5, 10, or 20%.Task 1.2. Investigate the effects of feedstock on the fermentation characteristics of glucose, cellulose and food waste.Experimental design. Once the optimal fermentation condition is identified for BDL production, the feedstock is also critical to determine the fermentation characteristics of B. licheniformis. In this task, we will first conduct the fermentation with different concentrations of glucose (10, 30, 50, 70, 90 and 110 g/L) to investigate the up-limit glucose concentration that leads to highest BDL titer and yield. Since the B. licheniformis has the unique cellulase activities as identified in objective 1, it has the great potential to avoid the energy-intensive pretreatment and expensive enzymatic hydrolysis to convert cellulose to glucose. Thus, we will then conduct the second set of experiment to investigate the effect of the ratio of glucose to cellulose on BDL fermentation characteristics. With the identified up-limit glucose concentration, we will replace 10, 30, 50, 70, and 90% of glucose with cellulose based on the equivalent sugar content, and evaluate the BDL titer, yield and productivity during and after fermentation. In the last set of experiment, we will use food waste as feedstock to investigate the feasibility of converting food waste to BDL via B. licheniformis fermentation. We will adjust the food waste concentration in fermentation medium based on its composition.Objective 2. Task 2.1. Delete lactate, acetate and ethanol pathway genes using CRISPR-Cas9 genome editing system.Experimental design. A majority of the genes and transcriptional regulators (Figure 5) have been identified in our B. licheniformis YNP5-TSU strain from previously collected genomic and proteomic data. CRISPR-Cas9 is the proposed method of choice to disrupt unwanted fermentation pathways and generate BDL as the main end-product.CRISPR-Cas9 procedure. First, sgRNAs (single guide RNAs) will be designed based off Illumina Hiseq3000 YNP5-TSU genomic data to target and knockdown each LDH, AoDH ES, and PTA enzymes individually. YNP5-TSU will be transformed using plasmid pJOE8999 as a shuttle vector, carrying the cas9 gene and sgRNA under the Bacillus mannose-inducible promoter (Fu et al., 2016). Plasmid purification, PCR amplification, and Sanger sequencing will be done to confirm the correct sequence has been disrupted. The knockdown of LDH, AoDH and PTA will be done individually first, then performed in a single YNP5-TSU strain to measure the compounded effects.Objective 3. (optional)Task 3.1. Develop the continuous biofilm reactor with cell immobilization for food waste fermentation to 2,3 BDL.Reactor build-up and cell immobilization. A continuous biofilm reactor, as illustrated in Figure 4, made of a glass column (60 mm (i.d.) x 500 mm (L)) packed with heavy supporting materials with an average size of 3 to 5 mm. The estimated working volume of the reactor will be 500 mL. The temperature of the reactor will be maintained at B. licheniformis optimal fermentation temperature using a heating tape. For cell immobilization, the reactor will be filled with CMC medium (1% LB broth, 1% Carboxymethylcellulose, CMC, and 10% glucose) followed by inoculation with 100 mL of actively growing B. Licheniformis. Cell growth will be allowed for 6-12 h, after which fresh glucose medium will be fed at 100 mL/h using a peristaltic pump to immobilize cells onto the packed materials. The supporting material is expected to be covered with a thin layer of B. Licheniformis after 24-36 h. We will first test the clay bricks as the heavy supporting materials because they are cheap, widely available and poly-porous materials that bacteria can be immobilized. We will also test other commonly used materials for cell immobilizations, including bonechar, active carbons, and calcium alginate.Conversion of food waste to BDL using the system. Once the bacterial cells are immobilized in the reactor, milled food waste slurry produced from subtask 2.2 will be supplied as substrate from the bottom of the biofilm reactor. Peristaltic pumps will be used to create a constant continuous upflow rate in the reactor as shown in figure 4. The upflow rate will be controlled at 100, 200, 400, 600, and 800 mL/hr to the reactor to investigate the effect of substrate loading on the continuous fermentation characteristics. Samples will be taken from the outlet of the biofilm reactor every 12 hours to monitor the performance of the continuous fermentation system to convert food waste to BDL. The key parameters to evaluate include BDL concentration, productivity, and yield. The morphology of biofilm will be imaged and analyzed using the Scanning electron microscope (SEM) to estimate the biofilm thickness and distributions surrounding the supporting materials. This experiment will continue until fermentation ceased. The outcome from this task will determine the optimal food waste feeding rate for successful conversion of food waste to BDL.

Progress 05/01/19 to 09/20/21

Outputs
Target Audience:The audience impacted from this project include(s): 1. Faculty, graduate students, undergraduate students in the Food Science department at Virginia Tech 2. Faculty, graduate students, undergraduate students in the Agricultural Science department at Tennessee State University 3. Conference members and field related scientists at the ACS Spring 2021 Conference 4. Agricultural Department graduate seminar, students and board members 5. Montana State University Biology Dpt. and search committee during in-person assistant professor job interview 6. Environmental Research and Education Foundation (EREF foundation) review panel from our proposal submission 7. Students in the Food and Science Dpt. lecture (Food and Bioprocessing class) as guest speaker 8. Metro Nashville elementary, middle and high school students involved in STEM activities 9. Attendees and conference members of the Society of Industrial Microbiology and Biotechnology (SIMB) 10. HBCU schools such as Tennessee State University where this research andUSDA funding opportunities can be shared to minority groups. 11. Students attending BIO2400, BIO2401, and Seminar Courses at Tennessee State University. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the 2-year post-doctoral program I have gained much experience and have had many opportunities for training and professional development. Training Activities Dr. Huang, his doctoral students, and masters students at Virginia Tech have helped to increase my proficiency in HPLC analysis/operation, batch/fed-batch fermentation using 2 L bioreactors, food waste compositional analysis and procedures for measuring food waste sugars and organic acids, manuscript writing and preparation, procurement/track of funds and the job application process. In the past 6 months Dr. Huang has trained me for the job application process for Assistant Professor positions. I have now undergone 5 virtual interviews and 2 on-campus interviews. Professional development Every week Dr. Huang, his lab members, and I meet to discuss our overall progress. We also have personal one-on-one meetings weekly for personalized mentorship. I have found this especially helpful for developing professional skills as it has helped me to plan, explain, and understand my own research better. It has kept me accountable to my goals and objectives.Outside of our lab, I have been in several virtual meetings with secondary mentor Dr. Zhou at TSU to discuss future plans at TSU and update her on our progress as mentioned in our research plan. I have attended weekly symposiums held in the food science department and joined many sensory labs to observe the other research being conducted at the VT food science department. Current update*** As of today 08/20/2021 I have officially joined the faculty as an Assistant Professor of Microbiology at Tennessee State University. Without the professional development and training from Dr. Huang and Dr. Zhou and the opportunity this funding has provided me, I would not be where I am today. This is an excellent program I hope to see continued for many years! How have the results been disseminated to communities of interest?Results from the research have been disseminated in the form of lectures (undergraduates and graduates) (FST 4304), and group presentations at Virginia Tech(monthly), conference oral presentation (SIMB 2019 and ACS, 2021), Montana State Univeristy research seminar during interview (informed search committee), TSU research seminar during interview, TSU agricultural graduate student association as guest lecturer, TSU instructor in BIO2400, and TSU guest lecturer for Biology seminar. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact Statement Each year we throw away 60 million tons of food, almost half of all produce sold. While a small portion of the food waste is used as animal feed most of it ends up in landfills, contributing to global warming and gasses that are 25 times more powerful then fumes from motorized vehicles (carbon dioxide). One possible solution is to turn this food waste into something we can all benefit from. Through the use of a special waste eating bacterium, Bacillus licheniformis YNP5-TSU, we can convert waste into a useful biofuel called butanediol (BDO). In the last year we were able to produce yields of 0.41 g/g 2,3-BDO (on average) with food waste in non-sterile fermentation. Several types of food waste including, cabbage, pineapple, peppers, and mixed all showed they had sufficient nutrients to support strain YNP5-TSU without addition of extra supplements. Conversion of all soluble sugars was achieved and 2,3-BDO was produced without the aid of autoclaving media before innoculation. Furthermore, novel starch enzymes from Bacillus licheniformis YNP5-TSU have been cloned and expressed in Escherichia coli. These enzymes have been shown to be as active as current commercial enzymes and are necessary for conversion of bakery waste into 2,3-BDO. If done on a larger scale can, these new procedures can and alter the bio-2,3-BDO industry and help remove waste from our planet. Objective 1: Complete Task 1.1: Completed Task 1.2: Completed 1. Major activities/experiments conducted: • Sugar loading capacity, Temperature effect on cell generation time, glucose consumptionand 2,3-BD production, effect of pH on 2,3-BD fermentation, sugar sources for 2,3-BD fermentation, and nitrogen sources for 2,3-BD fermentation. • Hot spring minerals for optimal growth and fermentation capability. Minerals including CaCO3 , MgSO4 , NaCl, KI, MnSO4 , and CuSO4 , found in hot springs where YNP5-TSU was isolated from Yellowstone National Park were scrutinized for their potential to increase 2,3-BD yield and carbon utilization rate. • Fed-batch fermentation analysis. Fed-batch fermentation was performed at 50 °C, with airflow of 2 L/min, and agitation of 150 rpm. When the glucose concentration in broth was below 20 g/L, a nutrient solution of 150 ml H2O, 60 g/L glucose, 1 g/L peptone, and 1 g/L yeast extract was added. • Food-waste fermentation. Three unitary food waste samples, i.e., cabbage waste, pepper waste, pineapple waste, and one miscellaneous food waste (a mixture of potato, pepper, strawberry, tomato, onion, cabbage, and pineapple). Fermentations were carried out at 50 °C in a shaking incubator at 150 rpms. 2. Data Collected: Data in the form of pictures and HPLC analysis were collected 3. Summary: B. licheniformis YNP5-TSU has the ability to survive in pH ranges from 8.0 to 10.0 and temperature ranges from 40 to 55 °C, which greatly reduces the chance of contamination from other microorganisms. At its optimum condition with temperature of 50 °C and pH of 8 - 9, YNP5-TSU can produce 27.4 g/L 2,3-BD from 60 g/L glucose, with a high yield of 0.46 g/g (92% of theoretical value) and productivity of 1.1 g/L/h in a batch fermentation. YNP5-TSU also can ferment different carbohydrates including fructose, sucrose, arabinose, galactose, mannose, xylose, as well as alcohols such as glycerol. In a fed-batch fermentation, 99.3 g/L of 2,3-BD was obtained within 96 hrs, with a yield of 0.41 g/g. All four types of food waste were successfully fermented by YNP5-TSU generating 0.32, 0.31, 0.48, and 0.39 g BDO/g consumed sugar for the fermentation of peppers, pineapple, cabbage, and miscellaneous wastes, which corresponds to 64%, 62%, 96%, and 78% of the theoretical yield of 0.5 g/g The results provided indicate that B. licheniformis YNP5-TSU is a very adaptable strain to withstand harsh environments while still producing 2,3-BDO near theoretical values. 4. Key outcomes or other accomplishments realized: Through objective 1 we have gained knowledge of the environmental parameters to produce maximum 2,3-BDO from sugar and food waste sources. We have taken that knowledge and put it into action for all future fermentations. Once our manuscripts have been accepted and published we believe this will lead to a conditional change on how B. licheniformis YNP5-TSU is used to generate 2,3-BDO. Objective 2: Complete Task 2.1 AlternativeComplete 1. Major activities/experiments conducted: • Suppression of contaminant food waste microbes and analysisof genetic information from B. licheniformis YNP5-TSU. Before attempting cloning of the CRISPR-Cas9 system into YNP5-TSU a much more significant investigation into the lack of starch degradation by YNP5-TSU was completed. From preliminary results YNP5-TSU showed almost no amylase activity and thus did not convert starch into 2,3-BDO. Starch is a major carbohydrate in food waste (found in the top 3 food waste types) and must be utilized to maximize 2,3-BDO production. Genome sequencing from YNP5-TSU was utilized to identify several alpha-amylase and alpha-glucosidase genes for cloning and expression. • Expressed starch enzymes. Alpha-amylase (amyS) and alpha-glucosidase (malL) were cloned into host cell E. coli BL21 via pET100 plasmid for recombinant protein expression. Each enzyme was tested for optimal temperature and pH using soluble starch and p-Nitrophenyl beta-D-glucopyranoside, respectively. Results showed AmyS had a maximal temperature of 70 °C and pH 8. Alphaglucosidase was shown to be optimally active at 50 °C and pH 9. GOPOD assay to detect glucose was used after the assays to determine glucose concentration and conversion ratio of starch/free glucose was calculated using Megazyme starch calculator. • Enzymatic activity of expressed starch degrading enzymes using bakery waste. Milled corn and Bakery waste (collected from the VT canteen) were inoculated with an enzyme cocktail (300 U/ml AmyS & 300 U/ml MalL). Alpha-amylase (AmyS) was added separately for 1 hour and 30 min to milled corn and bakery waste at 70 °C and pH 8 before the addition of alpha-glucosidase (MalL) which was then incubated at 50 °C. Sugars were calculated using the GOPOD assay and ran next to commercial starch enzymes (Megazyme) for comparison of activity. Results from this experiment showed a conversion of starch to glucose in the 30-40% range for both starch feedstocks. Commercial enzymes were similar and within the same range given a 2 hour incubation period. 2. Data Collected: Data in the form of pictures, HPLC analysis, and new modified strains were collected 3. Summary: Non-sterile food waste is a viable feedstock for 2,3-BDO production. Food waste containing waste starch can now be converted to 2,3-BDO. 4. Key outcomes or other accomplishments realized: We have accomplished a 30-40% conversion of unutilized starch from food waste. This will provide a 15-20% boost in 2,3-BDO production from all waste starch feedstocks. Objective 3. (optional) Task 3.1. Partial complete 1. Major activities/experiments conducted: •3D printed immobilized bioreactor for continuous fermentation. Continuous fermentation was investigated using a column reactor and glucose feedstock output (60 g/L) set to 0.5 ml/min. A second bioreactor was used in which O2 was supplemented at 1 L/min and cells were immobilized via 3D printed 10mm x 10mm cubes with 10% infill, creating a large surface area for adhesion of cells. 2. Data Collected: Data in the form of pictures, HPLC analysis, and 3D printed parts 3. Summary: Creating an immobilized bioreactor for continuous fermentation is possible. However, more work is needed to adjust parameters and dissolved oxygen. 4. Key outcomes or other accomplishments realized: To complete a continuous fermentation system that can convert 2,3-BDO while simultaneously hydrolyzing food waste sugars.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: OHair, J. A.; Jin, Q.; Li, H.; Yu, D.; He, Y.; Thapa, S.; Bhatti, S.; Huang, H.; Zhou, S. Biochemical and Genomic Identification of Novel Thermophilic Bacillus Licheniformis Strains YNP1-TSU, YNP2-TSU, and YNP3-TSU with Potential in 2,3-Butanediol Production from Non-Sterile Food Waste Fermentation. Food Bioprod. Process. 2021. https://doi.org/https://doi.org/10.1016/j.fbp.2021.07.003.
  • Type: Websites Status: Submitted Year Published: 2021 Citation: https://foodbioprocess.weebly.com/news.html


Progress 05/01/20 to 04/30/21

Outputs
Target Audience:The audience impacted from this project include: 1. Faculty, graduate students, undergraduate students in the Food Science department at Virginia Tech 2. Faculty, graduate students, undergraduate students in the Agricultural Science department at Tennessee State University 3. Conference members and field related scientists at the ACS Spring 2021 Conference 4. Agricultural Department graduate seminar, students and board members 5. Montana State University Biology Dpt. and search committte during in-person assistant professor job interview 6. Environmental Research and Education Foundation (EREF foundation) review panel from our proposal submission 7. Students in the Food and Science Dpt. lecture (Food and Bioprocessing class) as guest speaker 8. Department of Agricultural Seminar at TSU, included faculty and students Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training activities. During the second year of our funding I have learned to become an independent researcher, with experience in leading students, conducting experiments, manuscript submission, grant writing, and job interviews. I have gained experience in food analysis, HPLC maintenance, pre-treatment of brewer's spent grain, preparation of teaching and research seminars for job interviews as an assistant professor, and preparation of teaching statements, research statements, and in-person interview skills. Professional Development. Every week Dr. Huang, his lab members, and I meet to discuss our overall progress. We also have personal one-on-one meetings weekly for personalized mentorship. I have found this especially helpful for developing professional skills as it has helped me to plan, explain, and understand my own research better. It has kept me accountable to my goals and objectives. Outside of our lab, I have been in several virtual meetings with secondary mentor Dr. Zhou at TSU to discuss future plans at TSU and update her on our progress as mentioned in our research plan. According to the progress plan I have been working in her lab conducting the genetic editing and expression of proteins under her guidance. I have attended monthly symposiums held in the agricultural department at TSU and presented my project. I have also been a guest speaker at the graduate association of TSU. How have the results been disseminated to communities of interest?Results from the research have been disseminated in the form of lectures (undergraduates and graduates) (FST 4304), group presentations at VT (monthly), conference oral presentation (ACS, 2021), Montana State Univeristy research seminar during interview (informed search committee), and TSU agricultural graduate student association as guest lecturer. What do you plan to do during the next reporting period to accomplish the goals?During this one-year extension reporting period I plan to continue interviewing for jobs foran assistant professor in Microbiology/Biology/Agriculture and submitting the 3 manuscripts that are in the pre-submission/writing stage. As for Objective 2 which is partially complete I plan to genetically modify ourBacillus licheniformisrestriction enzyme cloning and transformation with shuttle vectorpHY300PLK (VET1395) to introduce starch degrading enzymes that have already been confirmed through expression experiments earlier this year. We have decided to focus on the modification of starchwith already expressed enzymes instead of lactate dehydrogenase or acetate dehydrogenase as originally proposed. The main reasoning being our yield is already currently within 10-12% of theoretical maximums and starch is highly abundant in food waste. I also, time willing, plan to continue investigation into 3D printed immobilized reactors which were briefly examined in Objective 3.

Impacts
What was accomplished under these goals? Impact Statement Each year we throw away 60 million tons of food, almost half of all produce sold. While a small portion of the food waste is used as animal feed most of it ends up in landfills, contributing to global warming and gasses that are 25 times more powerful then fumes from motorized vehicles (carbon dioxide). One possible solution is to turn this food waste into something we can all benefit from. Through the use of a special waste eating bacterium,Bacillus licheniformisYNP5-TSU, we can convert waste into a useful biofuel called butanediol (BDO). In the last year we were able to produce yields of 0.41 g/g 2,3-BDO (on average) with food waste in non-sterile fermentation. Several types of food waste including, cabbage, pineapple, peppers, and mixed all showed they had sufficient nutrients to support strain YNP5-TSU without addition of extra supplements. Conversion of all soluble sugars was achieved and 2,3-BDO was produced without the aid of autoclaving media before innoculation. Furthermore, novel starch enzymes from Bacillus licheniformis YNP5-TSU have been cloned and expressed in Escherichia coli. These enzymes have been shown to be as active as current commercial enzymes and are necessary for conversion of bakery waste into 2,3-BDO. If done on a larger scale can, these new procedures can and alter thebio-2,3-BDO industry and help remove waste from our planet. Objective 1:Complete Task 1: Completed during first year of funding (2019-2020) Task 2: Completed during first year of funding (2019-2020) Objective 2:Genetically engineeringBacillus licheniformisYNP5-TSU for enhancement of BDL yield, through a knockout of mixed acid fermentation pathway genes.Partially Complete Task 2.1 Delete lactate, acetate and ethanol pathway genes using CRISPR-Cas9 genome editing system. Partially Complete Major activities/experiments conducted: Preparation of non-sterile food waste. Before investigating genetic modification of B. licheniformis YNP5-TSU, food waste fermentation from Objective 1 was extended through implementation of non-sterile fermentation. All non-sterilized food wastes were incubated in 150 mL baffled flasks at 20, 37, and 50 °C for 72 hrs in a shaking incubator at 50 rpm. The pH was left unaltered at 6.5 or raised to 9.0 using 1 M NaOH. B. licheniformis YNP5-TSU was not added to the food waste. The initial concentration (colony forming units (CFU)/mL) of bacteria in the food waste slurry was determined by the surface plating method after serial dilution. Suppression of contaminant food waste microbes. Results from this experiment showed sugars in food waste slurries were completely consumed by indigenous microorganisms by 72 hrs and resulted in 4.2 g/L lactic acid, 3.3 g/L acetic acid, and 2.6 g/L ethanol when incubated at 37 °C. The ethanol observed here is most likely from wild yeast as they are typically found in food waste and have optimal growth temperatures around 32 °C (Salvadó et al., 2011). When unsterilized food waste was incubated at 20 °C, 4.5 g/L of soluble sugars was consumed at 72 hrs, producing 0.5 g/L acetic acid and 0.3 g/L lactic acid (Figure 1B). These products are produced most commonly by acetic acid producing Acetobacter pasteurianus and lactic acid producing Lactobacillus (Sampaio et al., 2014). When unsterilized food waste slurry was incubated at 50 °C or at a pH of 9.0 for 72 hrs, no sugars were consumed and no metabolites were produced, indicating that the indigenous microorganisms in food slurry were suppressed. Genetic information from B. licheniformis YNP5-TSU. Before attempting cloning of the CRISPR-Cas9 system into YNP5-TSU a much more significant investigation into the lack of starch degradation by YNP5-TSU was completed. From preliminary results YNP5-TSU showed almost no amylase activity and thus did not convert starch into 2,3-BDO. Starch is a major carbohydrate in food waste (found in the top 3 food waste types) and must be utilized to maximize 2,3-BDO production. Genome sequencing from YNP5-TSU was utilized to identify several alpha-amylase and alpha-glucosidase genes for cloning and expression. Expressed starch enzymes. Alpha-amylase (amyS) and alpha-glucosidase (malL) were cloned into host cell E. coli BL21 via pET100 plasmid for recombinant protein expression. Colonies from ampicillin (100 ug/ml)plates were selected and induced with IPTG. Crude protein expression was filtered through His-tag columns for purification of His-tagged expression proteins AmyS and MalL. Each enzyme was tested for optimal temperature and pH using soluble starch and p-Nitrophenyl beta-D-glucopyranoside, respectively. Results showed AmyS had a maximal temperature of 70 °C and pH 8. Alpha-glucosidase was shown to be optimally active at 50 °C and pH 9. GOPOD assay to detect glucose was used after the assays to determine glucose concentration and conversion ratio of starch/free glucose was calculated using Megazyme starch calculator. Enzymatic activity of expressed starch degrading enzymes using bakery waste. Milled corn and Bakery waste (collected from the VT canteen) were inoculated with an enzyme cocktail (300 U/ml AmyS & 300 U/ml MalL). Alpha-amylase (AmyS) was added separately for 1 hour and 30 min to milled corn and bakery waste at 70 °C and pH 8 before the addition of alpha-glucosidase (MalL) which was then incubated at 50 °C. Sugars were calculated using the GOPOD assay and ran next to commercial starch enzymes (Megazyme) for comparison of activity. Results from this experiment showed a conversion of starch to glucose in the 30-40% range for both starch feedstocks. Commercial enzymes were similar and within the same range given a 2 hour incubation period. 2. Data Collected:Data in the form of pictures,HPLC analysis, and new modified strains werecollected 3. Summary: Non-sterile food waste is a viable feedstock for 2,3-BDO production. Food waste containing waste starch can now be converted to 2,3-BDO. 4.Key outcomes or other accomplishments realized: Through partial completion of objective 2 we have gained new knowledge and proposed a new methodology for utilizing un-treated food waste which contains an unknown amount of contaminant microorganisms. The robust species YNP5-TSU is capable of outcompeting native bacteria for sugars while using a pH of 9.0 and growth temperature of 50° C. We also have accomplished a 30-40% conversion ofunutilized starch from food waste. This will provide a 15-20% boost in 2,3-BDO production from all waste starch feedstocks. Both of these breakthroughs?will significantly decrease cost and increase efficiency of bio-2,3-BDO. Objective 3 Design, fabricate and evaluate a novel continuous biofilm fermentation system to boost the conversion of food waste to 2,3-BDO.Partially Complete Task 3.1. Develop the continuous biofilm reactor with cell immobilization for food waste fermentation into 2,3 BDL. 3D printed immobilized bioreactor for continuous fermentation. Continuous fermentation was investigated using a column reactor and glucose feedstock output (60 g/L) set to 0.5 ml/min. Complications arose early in the attempt of continuous fermentation with low consumption of glucose. Cell growth rate was extremely low and did not reach OD 600nm above 0.4. A major issue with immobilized fermentation was the introduction of oxygen. Without adequate O2 cells did not ferment glucose into 2,3-BDO. A second bioreactor was used in which O2 was supplemented at 1 L/min and cells were immobilized via 3D printed 10mm x 10mm cubes with 10% infill, creating a large surface area for adhesion of cells. In this case O2 could reach all cells while also immobilization of biofilm secreting cells took place. However, glucose consumption was moderate and under these new conditions has not been optimized yet.

Publications

  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2021 Citation: Joshua OHair, Qing Jin, Dajun Yu, Yanhong He, Nicholas Poe, Jian Wu, Hui Li, Santosh Thapa, Hengjian Wang, Suping Zhou, Haibo Huang. Thermophilic and alkaliphilic fermentation of non-sterile food waste using Bacillus licheniformis YNP5-TSU to produce 2,3-butanediol. ACS conference proceedings. 2021.(awaiting publication)
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2021 Citation: Yanhong He, Joshua OHair, Qing Jin, Jian Wu, Haibo Huang. Simultaneous production of protein concentrate and 2,3-butanediol from Brewers spent grain. ACS conference proceedings. 2021. (awaiting publication)
  • Type: Websites Status: Published Year Published: 2021 Citation: Science Focus The Home of BBC Science Focus Magazine https://www.sciencefocus.com/news/cheers-method-for-turning-spent-grain-from-beer-production-into-biofuels-and-plant-based-foods-developed/
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: J. OHair , Q. Jin, D . Yu, J. Wu, H. Wang, S. Zhou*, H. Huang*. 2021. Non-sterile fermentation of food waste using thermophilic and alkaliphilic Bacillus Licheniformis YNP5-TSU for 2,3-butanediol production. Waste Management. ( co-first author) https://doi.org/10.1016/j.wasman.2020.11.029.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: OHair, J., Jin, Q., Yu, D., Poe, N., Li, H., Thapa, S., Zhou, S., & Huang, H. 2020. Thermophilic and Alkaliphilic Bacillus licheniformis YNP5-TSU as an Ideal Candidate for 2,3-Butanediol Production. ACS Sustainable Chemistry & Engineering. https://doi.org/10.1021/acssuschemeng.0c02759.
  • Type: Journal Articles Status: Submitted Year Published: 2021 Citation: He, Y., OHair, J., Huang, H. 2021. Simultaneous Production of Protein-Rich and 2,3-Butanediol Products from Brewers Spent Grain Through Protease Hydrolysis and Non-Sterile Thermophilic Fermentation. (submission stage)
  • Type: Journal Articles Status: Under Review Year Published: 2021 Citation: J. OHair, Q. Jin, H. Li, S. Thapa, D. Yu, Y. He, S. Bhatti, S. Zhou, H. Huang. 2021. Bioconversion of mixed unsterile food waste into value added 2,3-butanediol by Bacillus licheniformis YNP1-TSU, YNP2-TSU, and YNP3-TSU isolated from Yellowstone National Park. Food and bioproducts processing. 2021. (Under review)


Progress 05/01/19 to 04/30/20

Outputs
Target Audience:The audience impacted from this project include: 1. Faculty, graduate students, undergraduate students in the Food Science department at Virginia Tech 2. Faculty, graduate students, undergraduate students in the Agricultural Science department at Tennessee State University 3. Workshop members and field related scientists at the Symposium on Biotechnology for Fuels and Chemicals 4. Metro Nashville elementary, middle and high school students involved in STEM activities 5. Attendees and conference members of the Society of Industrial Microbiology and Biotechnology (SIMB) 6. Fellow colleagues and scientists at the annual USDA NIFA PD meeting Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training activities: Through-out the course of the first year I have gained much experience and have had many opportunities for training. Dr. Huang and his doctoral and masters graduate students at Virginia Tech have helped to increase my proficiency in HPLC analysis/operation, batch/fed-batch fermentation using 2 L bioreactors, food waste compositional analysis and procedures for measuring food waste sugars and organic acids, manuscript writing and preparation, procurement/track of funds and the ordering process for managing grant funding. Professional development: Every week Dr. Huang, his lab members, and I meet to discuss our overall progress. We also have personal one-on-one meetings weekly for personalized mentorship.I have found this especially helpful for developing professional skills as it has helped me to plan, explain, and understand my own research better. It has kept me accountable to my goals and objectives outlined in this postdoctoral fellowship. Dr. Haung hosts bi-monthly lab luncheons where we are able to enjoy a more laid back atmosphere and grow closer as a research family. I feel I have gained much insight as to how a well working lab should operate. Outside of our lab, I have been in several virtual meetings with secondary mentor Dr. Zhou at TSU to discuss future plans at TSU and update her on our progressas mentioned in our research plan. I have attended weekly symposiums held in the food science department and joined many sensory labs to observe the other research being conducted at the VT food science department. How have the results been disseminated to communities of interest?Results from the research thus far have been disseminated in the form of lectures (undergraduates and graduates) (FST 4304), group presentations (monthly), and conference oral poster presentation (SIMB). What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period I plan to complete objective 2 (all tasks) and begin objective 3. This encompasses the genetic modification portion of the proposed research. To counter any issues faced during this period, several alternative genetic modification protocols have been introduced. The initial plan is to introduce modifications through CRISPR gene editing; however, if this becomes unmanageable then TOPO cloning, synthetic in vitro gene expression and co-culture expression will be utilized. With two mentors, half a dozen labmates, and resources from multiple Universities I will be able to overcome this hurdle.

Impacts
What was accomplished under these goals? Impact Statement Each year we throw away 60 million tons of food, almost half of all produce sold. While a small portion of the food waste is used as animal feed most of it ends up in landfills, contributing to global warming and gasses that are 25 times more powerful then fumes from motorized vehicles (carbon dioxide). One possible solution is to turn this food waste into something we can all benefit from. Through the use of a special waste eating bacterium,Bacillus licheniformisYNP5-TSU, we can convert waste into a useful biofuel called butanediol (BDO). Over the past year we have establishednovel harvesting protocols to produceBDO in batch and fed-batch fermentation. The maximum theoretical limit of BDO able to be produced is 0.5 g/g from all initialcarbohydrate sources. Our result show thatB. licheniformisstrain YNP5-TSU, can produce0.46 g/g BDO yield and it can do this at temperatures of 50ºC and a starting pH of 9.0. This bacterium was also able to produce BDO from a wide range of sugars including:fructose, sucrose, arabinose, galactose, mannose, xylose, as well as alcohols such as glycerol. Results such as these, if done on a large scale can alterthe bio-BDO industry and help remove waste from our planet. Objective 1. Identify the key process variables that determine the performance of BDL production from glucose and food waste using wild-typeB. licheniformisYNP5-TSU.(complete) Objective one has been completed within the first year of funding as planned in our progress outline. Task 1.1Identify and optimize the fermentation conditions (temperature, pH and dissolved oxygen) to convert glucose to 2,3 BDL(complete) Task 1.2.Investigate several different carbon feed stocks (i.e. glucose, cellulose and food waste) and determine their impact on fermentation end-products (complete) Major activities/experiments conducted: Sugar loading capacity. Conducted in batch fermentation,B. licheniformisYNP5-TSU was inoculated in (x) g/L glucose, 10 g/L yeast extract, and 5 g/L peptone with various sugar concentrations ranging from 200 g/L glucose to 5 g/L glucose to determine consumption rates (g/L/hr)and maximum starting glucose concentrations (g/L). Temperature effect on cell generation time, glucose consumption, and 2,3-BD production.Isolate YNP5-TSU was inoculated into 95 ml of YNP5 growth media (60 g/L glucose, 10 g/L yeast extract, and 5 g/L peptone) in 150 ml baffled flasks and incubated at 45, 50, and 55 °C for 72 hrs at 150 rpms. Samples (1 ml) were collected at 0, 18, 24, 42, 48, and 72 hrs for sugar and fermentation product analysis. Growth curves were constructed using a micro-plate reader (SpectraMax® M5, Molecular Devices, San Jose, CA) at 30 °C, 45 °C, and 55 °C. Effect of pH on 2,3-BD fermentation.To characterize the pH effect on 2,3-BD fermentation, experiments were conducted in a 2-L bioreactor where pH, temperature and air flow are controlled (BioFlo 120, Eppendorf North America, Hauppauge, NY). An aliquot of 37.5 ml of P2inoculum was added into 750 ml of YNP5 growth media (glucose 60 g/L, 10 g/L yeast extract, 5 g/L peptone) and incubated at 50 °C, 150 rpm, and 2 L/min air flow rate. Initial pH was adjusted to 10.0, 9.0, 8.0, 7.0 or 6.0 with either 5M NaOH or 1M HCl and the pH change during fermentation was monitored. Sugar source for 2,3-BD fermentation.YNP5 incubated inbaffled flasks at 50 °C, initial pH 8.0 and 150 rpms with various sugar sources: glucose, fructose, sucrose, mannose, xylose, galactose, and arabinose. The fermentation was conducted for 72 hrs in a shaking incubator. Nitrogen source for 2,3-BD fermentation.Different nitrogen sources were tested to support the 2,3-BD fermentation. Malt extract, peptone, tryptone, and yeast extract were added at 20 g/L, along with 60 g/L glucose and base mineral solution (0.1 g/L KH2PO4, 0.2 g/L MgSO4, 0.1g NaNO3, 0.1 g/L KCl) in 150 ml baffled flasks. Hot spring minerals for optimal growth and fermentation capability.Minerals including CaCO3, MgSO4, NaCl, KI, MnSO4, and CuSO4, found in hot springs where YNP5-TSU was isolated from Yellowstone National Park were scrutinized for their potential to increase 2,3-BD yield and carbon utilization rate. Fed-batch fermentation analysis.Fed-batch fermentationwas performed at 50 °C, with airflow of 2 L/min, and agitation of 150 rpm. When the glucose concentration in broth was below 20 g/L, a nutrient solution of 150 ml H2O, 60 g/L glucose, 1 g/L peptone, and 1 g/L yeast extract was added. Food-waste fermentation.Three unitary food waste samples, i.e., cabbage waste, pepper waste, pineapple waste, and one miscellaneous food waste (a mixture of potato, pepper, strawberry, tomato, onion, cabbage, and pineapple).Fermentations were carried out at 50 °C in a shaking incubator at 150 rpms. 2.Data Collected:Data in the form of pictures and HPLC analysis were collected 3.Summary:B. licheniformisYNP5-TSU has the ability to survive in pH ranges from 8.0 to 10.0 and temperature ranges from 40 to 55 °C, which greatly reduces the chance of contamination from other microorganisms. At its optimum condition with temperature of 50 °C and pH of 8 - 9, YNP5-TSU can produce 27.4 g/L 2,3-BD from 60 g/L glucose, with a high yield of 0.46 g/g (92% of theoretical value) and productivity of 1.1 g/L/h in a batch fermentation. YNP5-TSU also can ferment different carbohydrates including fructose, sucrose, arabinose, galactose, mannose, xylose, as well as alcohols such as glycerol. The effects of nitrogen and mineral sources on 2,3-BD fermentation were also investigated to improve the fermentation process. In a fed-batch fermentation, 99.3 g/L of 2,3-BD was obtained within 96 hrs, with a yield of 0.41 g/g. All four types of food waste were successfully fermented by YNP5-TSU generating0.32, 0.31, 0.48, and 0.39 g BDO/g consumed sugar for the fermentation of peppers, pineapple, cabbage, and miscellaneous wastes, which corresponds to 64%, 62%, 96%, and 78% of the theoretical yield of 0.5 g/gThe results provided indicate thatB. licheniformisYNP5-TSU is a very adaptable strain to withstand harsh environments while still producing 2,3-BDO near theoretical values. 4.Key outcomes or other accomplishments realized:Through objective 1 we have gained knowledge of the environmental parameters to produce maximum 2,3-BDO from sugar and food waste sources. We have taken that knowledge and put it into action for all future fermentations. Once our manuscripts have been accepted and published we believe this will lead to a conditional change on how B. licheniformis YNP5-TSU is used to generate 2,3-BDO. Objective 2.Genetically engineeringBacillus licheniformisYNP5-TSU for enhancement of BDL yield, through a knockout of mixed acid fermentation pathway genes(not started) Task 2.1 (not started) Objective 3 (optional, dependenton completion of objective 1 and 2). Design, fabricate and evaluate a novel continuous biofilm fermentation system to boost the conversion of food waste to BDL. Task 3.1.(not started)

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: O'Hair, J.; Jin, Q.; Yu, D.; Poe, N.; Li, H.; Thapa, S.; Zhou, S.; Huang, H. Thermophilic and Alkaliphilic Bacillus Licheniformis YNP5-TSU as an Ideal Candidate for 2,3-Butanediol Production. ACS Sustainable Chemistry & Engineering. Under review. 2020.