Progress 05/01/24 to 04/30/25
Outputs
Target Audience:This past year, we have continued our communication with several target audiences per our project plan. This research has been discussed multiple times in formal classroom instruction such as BPE 300: Introduction to Industrial Bioprocessing at SUNY ESF and Micro526: Physiology of Microorganisms at UW-Madison. Faculty, graduate students and undergraduate students have presented their findings at local, regional and national conferences. Likewise, we have had discussions with stakeholders in the dairy, paper and bioplastic industries. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?For this reporting period, the project again has provided multiple opportunities for training and professional development. Notably, Ph. D. student Linjing Jia completed her degree in August of 2024, and is now working as a Senior Bioprocess Scientist at RETRN Bio, Ithica, NY. The project continues to support faculty, graduate students and a research intern at SUNY ESF and UW-Madison. During this past year, the graduate students have gained new skills in maintaining bioreactors, new molecular biology methodologies, new to them analytical techniques such as HPLC, NMR, and mass spectrometry. They have also learned how to work with new production organisms. Graduate students gained professional development by attending and presenting posters at SIMB and ASABE. The student who attended SIMB also went to a pre-conference workshop on industrial scale fermentation. Dr. Jia was selected for an oral presentation- information below.Students also presented to stakeholders at the Fall Research meeting put on by the Center for Dairy Research in Madison, WI. Jia, L., Kaur, G., Majumder, E. L. W., Juneja, A., & Kumar, D. (2024) Production of polyhydroxybutyrate from non-recyclable fiber rejects and acid whey as mixed substrate by recombinant Escherichia coli. Abstract No. 2400855, Oral Presentation at ASABE 2024 Annual International Meeting, Anaheim, California, July 28-31, 2024 How have the results been disseminated to communities of interest?For this reporting period, to communicate our findings to the scientific community, we presented them at several conferences including SIMB and ASABE. We have published papers reporting our results so far and several more papers will be submitted very shortly. Dr. Majumder gave the bioeconomy keynote address highlighting the work of this project at the American Society for Microbiology Council on Microbial Sciences (COMS) retreat for the Applied and Environmental Sciences track. To communicate with stakeholders, we have presented at industry events and also had meetings with various paper and dairy companies. Dr. Kumar had a meeting with David Magda, Sr. Director, Product Innovation & Business Development, Pixelle Specialty Solutions, in March 2025 to discuss this project and possibilities of testing fiber rejects from their paper mills. Dr. Majumder has continued to interact with OuroBio. Through the Center for Dairy Research, we have continued to receive new dairy processes residues and co-products to test including buttercream whey and whey permeates from multiple companies including Arena Cheese, Schrieber Foods and more. What do you plan to do during the next reporting period to accomplish the goals?In this last year of the award, we plan to finish our objectives. TEA and LCA will be a key areas of focus. Optimizing production from mixes of real acid whey and paper fines will be the target for objective 1. For Objective two, we will expand the range of co-polymers produced and characterize the metabolic pathways generating those. We will also work on strain optimization. We plan to publish several papers and present our findings to target audiences.
Impacts
What was accomplished under these goals?
The major issue that our project targets is use of pure sugar feedstocks in the production of biobased and biodegradable plastics. Pure sugar feedstocks are the largest portion of the cost of bioplastics, which are too expensive to compete with petroleum-based plastics, and also compete with food production. Our project works to utilize and valorize current negative cost residues and waste streams from the dairy and paper industries as feedstock for bioplastic production, the groups that will most immediately benefit from our work. The outcomes of our project will help dairy processors and paper manufacturers turn a negative cost residue into a revenue-generating co-product. This will also help bioplastic companies to use these streams and reduce cost of bioplastics. For Objective 1, during last reporting period, Acid whey was collected from a commercial dairy facility (Chobani), and AW was stored in a freezer until use. Waste fiber rejects were collected from a local paper mill in Syracuse, New York. Fibers were dried (<10 moisture) and stored in a refrigerator at 4 °C until needed for experimentation. The chemical composition of both waste streams was analyzed. Then, waste fibers were pretreated using an optimized two-step hydrothermal pretreatment (hot water treatment followed by disk refining) and hydrolyzed using commercial cellulase and hemicellulose enzymes yielding a good concentration of fermentable sugars. During this reporting period, PHB fermentation experiments were conducted on these optimized hydrolysates and acid whey. This included at least 5 sets of experiments: Pure AW, pure hydrolysate, 3 mix of AW and hydrolysate (1:1 mix, 1:2 mix, 2:1 mix, o mass basis). All fermentation experiments were performed at 20 g/L sugar concentrations (sugars: glucose, xylose, lactose, galactose). Lactic acid and acetic acid are additional carbon sources, but experiments were performed keeping only the sugar concentrations fixed among all experiments. Flasks are harvested at various time points: 6,12, 18, 24, 30, 36, 48, 72 h to determine pH, OD, sugars and acid concentrations, cell dry weight and PHB inclusion. Additional variables were tested. The waste streams were also characterized prior to testing. The results showed that the mix of AW and paper fines hydrolysates produced better yields and titers of PHB than pure sugars or either waste stream alone. These are promising results that confirmed on of our hypotheses. We are now working to increase the yields on this self-neutralized waste mixture. For Objective 2, we have continued to work on improving our engineered E. coli bioproduction chassis by inserting the genes at different positions in the chromosome after seeing low expression from some positions. We are also working with some native producers and microbial communities to test for an improved range of co-polymers, which we are seeing. We are seeing longer chain lengths of 10-12 C units incorporated, which is encouraging. We obtained new sources of dairy fats in addition to WPPC including Whey Cream Buttermilk. The fats in buttermilk co-product appear to be more easily incorporated than those in WPPC. For Objective 3, work will ramp up during this reporting period as we have now acquired more of the necessary data to begin populating the TEA and LCA models. Key outcomes are in the higher yields of bioplastic from the waste mixing approach compared to pure feedstocks. We are also observing new types of polymers from mixing various dairy and paper wastes.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Jia, L., Kaur, G., Juneja, A. et al. Polyhydroxybutyrate production from non-recyclable fiber rejects and acid whey as mixed substrate by recombinant Escherichia coli. Biotechnol Sustain Mater 1, 12 (2024). https://doi.org/10.1186/s44316-024-00013-y
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Progress 05/01/23 to 04/30/24
Outputs
Target Audience:This past year, we have communicated with several target audiences per our project plan. This research has been discussed multiple times in formal classroom instruction such as BPE 300: Introduction to Industrial Bioprocessing at SUNY ESF and Micro526: Physiology of Microorganisms at UW-Madison. Faculty, graduate students and undergraduate students have presented their findings at local, regional and national conferences, this includes engineering and microbiology events as well as dairy industry events. Likewise, we have had discussions with stakeholders in the dairy, paper and bioplastic industries. The review paper in the products section was tangential to our work on this grant as it emphasizes lignin, which is why NIFA was not acknowledged, but we included it in this progress report because it highlights our overall philosophy in utilizing agricultural wastes as sustainable and low-cost feedstocks for bioproduction of valuable molecules and polymers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project during this year has provided multiple opportunities for training and professional development.Training included graduate students being trained in many new skills including new to us uses of Gas Chromatography, better recombineering technology for strain development, culturing microbial communities for PHA production, bioreactor experiments and waste mixing. Rachel Rovinsky and PI Erica Majumder also attended a 4-day workshop on decolonizing microbiology. Additional professional development was met by students attending conferences and presenting posters such as ASABE and SIMB. The first PhD student graduating with work on this project will defend her thesis in early July 2024. How have the results been disseminated to communities of interest?To present our current findings to the scientific community, we presented at several relevant conferences during this reporting period. That included the USDA project director's meeting in Kansas City during summer 2023. Dr. Majumder presented at SBFC. A graduate and undergraduate student each presented at SIMB. Dr. Kumar and two graduate students presented at ASABE. We participated in a review paper on the bioconversion of lignin. We have also been in contact with different suppliers in both New York state and Wisconsin. In New York, our local dairy company Byrne Dairy made some production line changes, so we had to contact Chobani and explain our project to get acid whey. Dr. Kumar also met with Westrock, a paper mill from where we get fiber rejects. He also has a meeting with a start-up (Capro-X) working on dairy waste later in April at the end of this reporting period. In Wisconsin, Dr. Majumder has continued to work with the Center for Dairy Research to acquire WPPC and acid whey from various producers in the state. She also presented at the CDR's annual research forum and the World Cheese Expo about this project. More and more producers are getting excited about the dairy industry being a part of the bioeconomy and valorizing their waste streams. Dr. Majumder has also been meeting with bioplastic startup company OuroBio. What do you plan to do during the next reporting period to accomplish the goals?We anticipate the next reporting period will be very fruitful in terms of publishable data and subsequent publications. The first graduate student trained on the project is defending her thesis in July. All the method development and preliminary experiments are now primed to begin full experiments. Improvements to the microbial chassis will continue. Microbial communities are being tested for co-polymer production with WPPC. Scaling and fermentation conditions will be tested on the Acid Whey-waste fines hydrolysate mixture. TEA will be started.
Impacts
What was accomplished under these goals?
The major issue that our project targets is the use of pure sugar feedstocks in the production of biobased and biodegradable plastics. Pure sugar feedstocks are the largest portion of the cost of bioplastics, which are too expensive to compete with petroleum-based plastics, and also compete with food production. Our project works to utilize and valorize current negative cost residues and waste streams from the dairy and paper industries as feedstock for bioplastic production, the groups that will most immediately benefit from our work. The outcomes of our project will help dairy processors and paper manufacturers turn a negative cost residue into a revenue-generating co-product. This will also help bioplastic companies to use these streams and reduce cost of bioplastics, ultimately helping all Americans to decrease the amount of petroleum-based plastics consumed by replacing them with biobased and biodegradable plastics. For Objective 1, Acid whey was collected from a commercial dairy facility (Chobani), and AW was stored in a freezer until use. Waste fiber rejects were collected from a local paper mill in Syracuse, New York. Fibers were dried (<10 moisture) and stored in a refrigerator at 4 °C until needed for experimentation. The chemical composition of both waste streams was analyzed. Then, Waste fibers were pretreated using an optimized two-step hydrothermal pretreatment (hot water treatment followed by disk refining) and hydrolyzed using commercial cellulase and hemicellulose enzymes yielding a good concentration of fermentable sugars.PHB fermentation experiments are in progress. This includes at least 5 sets of experiments: Pure AW, pure hydrolysate, 3 mix of AW and hydrolysate (1:1 mix, 1:2 mix, 2:1 mix, o mass basis). All fermentation experiments are being performed at 20 g/L sugar concentrations (sugars: glucose, xylose, lactose, galactose). Lactic acid and acetic acid are additional carbon sources, but experiments were performed keeping only the sugar concentrations fixed among all experiments. Flasks are harvested at various time points: 6,12, 18, 24, 30, 36, 48, 72 h to determine pH, OD, sugars and acid concentrations, cell dry weight and PHB inclusion. Preliminary results indicate improved fermentation with the AW-waste fines mixture. For Objective 2, we obtained WPPC from a local company. We determined the fatty acid composition of the WPPC with Gas Chromatography and the microbial community present with 16S rRNA gene community sequencing on a Miseq. We performed initial experiments testing for co-polymer production on a synthetic mixture of AW and waste fines with WPPC supplemented in the growth medium. Preliminary results indicate the presence of co-polymers of PHAs for some of the native producer bacteria and communities tested. We have also continued to improve our engineered E. coli strain for producing bioplastics. After some failures with gene insertion using our previous genome engineering method, we established a new to the Majumder lab method for gene insertion into the chromosome and are testing if the newly generated strains are capable of PHB and PHA production. For Objective 3, we plan to begin work during this reporting period as we have now acquired some of the necessary data to begin populating the TEA and LCA models. Key outcomes are in the validation of the waste mixing approach to neutralize the solution for better hydrolysis and fermentation. Likewise, use of WPPC was confirmed with initial evidence for incorporation into co-polymers, and microbial chassis strain engineering methods have been established.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2024
Citation:
Shilva Shrestha; Shubhasish Goswami; Deepanwita Banerjee; Valentina Garcia; Elizabeth Zhou; Charles N. Olmsted; Erica Majumder; Deepak Kumar; Deepika Awasthi; Aindrila Mukhopadhyay; Steven W. Singer; John M. Gladden; Blake A. Simmons; Hemant Choudhary. Perspective on Lignin Conversion Strategies that Enable Next Generation Biorefineries. Accepted. ChemSusChem.
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Progress 05/01/22 to 04/30/23
Outputs
Target Audience:In the first few months of this project, we have begun to communicate with the various target audiences reached by this project. We have attended regional meetings with representatives from stakeholder industries including dairy, paper and plastics. We have reached out to several of these companies to provide samples of the residues and waste streams. We have presented posters atmicrobiology and bioengineering conferences. We have started training graduate students directly on this project and have presented in undergraduate and graduate seminars. Changes/Problems:There has not been a major change or problem with the work proposed. We did want to make one note on the timeline. The award was backdated to starting 5/1/22 even though we did not receive the award until autumn. So, this progress report only represents a few months of work rather than a full year. What opportunities for training and professional development has the project provided?To date, one new PhD graduate student has been recruited for the microbiology aspect. Rachel Rovinsky started in January of 2023 and is learning about strain engineering. Current graduate students at SUNYESF have been getting training on these waste streams and we plan to recruit 2 more students this fall at ESF for the process engineering and TEA and LCA aspects of the project. Joint group meetings have been established for professional development in interdisciplinary communication. How have the results been disseminated to communities of interest?In the first few months of the project, we have communicated the early findings to several of the audiences outlined in the proposal. Poster presentations were given at several national scientific conferences including American Society for Microbiology in June 2022, ASABE summer 2002 and SIMB SBFC spring 2023. Research presentations have also been given to local stakeholders such as the Center for Dairy Research Fall research forum in 2022 and NY Department of Environmental Conservation meetings.Results from our findings have been described in the courses we teach but also in guest lectures to graduate seminars other departments like Food Science and Materials Engineering. What do you plan to do during the next reporting period to accomplish the goals?For Objective 1, we will begin mixing the waste streams. We are also sourcing raw AW and WF from companies in WI and NY. For Objective 2, we will begin testing our newly engineered strains on the mixed waste and start testing which co-polymers are produced with inclusion of WPPC in with AW and WF. We are making a standard synthetic AW WF mixture to be able to compare across labs. For Objective 3, we are beginning to collect the data needed to start the TEA and LCA calculations. Manuscripts and presentations are in progress. We will also develop and activity for our courses that connects this research and teaching.
Impacts
What was accomplished under these goals?
For Objective 1, we have improved the hydrolysis conditions and sugar release from the paper waste fines. Several pretreatment methods were tested, and a method was found that uses lower energy inputs. A paper manuscript is in progress.Mixing of acid whey and waste fines hydrolysates will start soon. For Objective 2, we have done proof of concept experiments that show conversion of WPPC phospholipids into PHA co-polymers. We have finally established a genome insertion method for our base strain and are currently integrating target genes of interest for better carbon conversion and production of unique PHA co-polymers. We contributed to a review paper in this topic area that is under review at Chemical Reviews (ACS) journal. Work on Objective 3 will begin in the next phase once baseline process parameters are established.
Publications
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