COTREATMENT FOR LOW-COST FERMENTATION OF CELLULOSIC BIOMASS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1009868
• Grant No.: 2016-10008-25319
• Project No.: NH.W-2015-10012
• Proposal No.: 2015-10012
• Program Code: BRDI
• Project Start Date: Aug 15, 2016
• Project End Date: Aug 14, 2020
• Grant Year: 2016

Project Director
Lynd, L.

Recipient Organization
DARTMOUTH COLLEGE
8000 CUMMINGS HALL
HANOVER N H,NH 03755

Performing Department
Chemical/Biochemical

Non Technical Summary
We propose a potentially transformative new approach for overcoming the lignocellulose recalcitrance barrier. Instead of thermochemical pretreatment prior to biological processing, we propose physical disruption during biological processing - termed "cotreatment". Although milling is known to be too energy-intensive to be practical for pretreatment followed by fungal cellulase-mediated hydrolysis, several compounding factors suggest substantially lower energy requirements for cotreatment-enhanced microbial fermentation. First-of-a-kind data supporting this concept demonstrate a) that fermentation proceeds readily in the presence of physical disruption at an intensity sufficient to substantially increase lignocellulose solubilization, and b) high extents of solubilization comparable to conventional pretreatment. Economic analysis is presented indicating potential for far lower cost than conventional lignocellulose conversion, particularly at small scale. Cotreatment-assisted lignocellulose fermentation will be investigated using both ethanol-producing thermophilic bacteria and mixed thermophilic methanogenic consortia, and will be carried out by a team from Dartmouth and Pennsylvania State University familiar with both of these systems as well as fluid mechanics. Task 1 involves lab-scale comparison of mechanical disruption strategies, Task 2 involves pilot-scale testing with a selected milling strategy, and Task 3 involves tests at increasing solid concentration. Task 4 addresses the mechanics of cotreatment. Process evaluation and industry outreach are addressed in Task 5. Completion of these tasks is expected to allow definitive assessment of the cotreatment concept while progressing from TRL3 to TRL6. Enchi Corporation will lead industry outreach and be involved in cost estimation. The DOE Bioenergy Science Center will contribute engineered thermophilic strains and characterization of fermentation residues.

Animal Health Component

0%

Research Effort Categories

Basic

35%

Applied

65%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
511	7410	2020	100%

Knowledge Area
511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
7410 - General technology;

Field Of Science
2020 - Engineering;

Keywords

cellulosic biomass

bacterial processing

biofuel

cotreatment

low-cost

Goals / Objectives
The overall goal of the project is to test the central hypothesis that mechanical disruption can be a practical, and indeed highly cost effective, approach to overcoming the recalcitrance of cellulosic biomass if carried out in a cotreatment rather than pretreatment mode with conversion mediated by anaerobic bacterial cultures rather than fungal cellulase. Pursuant objectives are listed below.1. Characterize and compare at laboratory scale and low (20 g/L) feedstock concentration the impact of mechanical disruption via various modes and intensities on a) the extent of solubilization enhancement as a result of mechanical disruption, and b) microbial activity. 2. Maximize enhancement of lignocellulose solubilization by cotreatment at the 65 L scale using a selected cotreatment mode and a moderate (~ 75 g/L) feedstock loading. 3. Maximize cotreatment-enhanced lignocellulose solubilization yields at high lignocellulose concentrations, (> 150 g/L). 4. Systematically characterize cotreatment-assisted lignocellulose solubilization from a fluid mechanical(s) perspective, including: a) characterizing properties of reacting slurries without cotreatment, b) characterizing the impact of cotreatment on slurry properties and fermentation rates and yields, and c) delineating productive energy used for particle disruption and viscous energy dissipation. 5. Evaluating the efficacy of the cotreatment approach in terms of economics, life cycle analysis, and industrial acceptance. 6. Pursue the objectives above with both defined thermophilic ethanol-producing cultures and mixed thermophilic methanogenic consortia, thereby increasing the breadth of application and affording opportunities for fundamental insights.

Project Methods
Feedstocks. Corn stover and switchgrass will be provided by Penn State University for use at both Penn State and Dartmouth. Stover will be harvested in October after manual corn ear removal, and chopped to 2 cm minus with a plot forage harvester modified for corn stover. Corn ears will be stripped of grain, chopped, and blended with the stalks and leaves in proportion to simulate a commercial stover harvest. Switchgrass (Kanlow variety) will be harvested in October after senescence. These lignocellulosic feedstocks will be air-dried to less than 18% moisture and stored dry until needed.Microorganisms. Dartmouth is actively developing engineered strains of the cellulolytic thermophile Clostridium thermocellum with sponsorship from the Bioenergy Science Center (BESC) and in collaboration with other BESC institutions. Dartmouth further has access to engineered strains of the hemicellulose-fermenting Thermoanaerobacter saccharolyticm that produce ethanol at commercially-viable titers and yields as a result of a substantial DOE-funded project (Herring et al., 2012), and is engineering other thermophilic, hemicellulose-utilization strains - again with BESC support. As detailed in the supporting letter from BESC Director Paul Gilna, the project will have access to strains developed by BESC. We will choose the best strain available at the time that various experimental campaigns are initiated.Following Kinet et al. (2015), the Richard lab at PSU will develop a thermophilic methanogenic consortia from a mixture of two source inocula - a thermophilic composting facility and a mesophilic anaerobic digester. The compost inocula will come from Penn State's institutional composting facility, which composts 7 tons per day of food waste, switchgrass, leaves and bedded dairy and poultry manure. Samples will be collected from pile regions at temperatures between 55C and 70C. The mesophilic inoculum will be sourced from a commercial dairy farm that digests bedded dairy manure in a high solids plug flow digester, enriched on a ground corn stover feed through five one week temperature cycles stepping up, from 35C to 55C in 5C increments, and blended with freshly collected thermophilic compost inoculum. This thermophilic methanogenic culture will be maintained in a seed reactor at 55C.Growth medium. The low-carbon (LC) medium developed in the P.I.'s lab will be used for both defined thermophilic cultures and mixed methanogenic consortia with modifications as necessary for growth at high feedstock concentrations.Fermentation and cotreatment systems. For laboratory-scale work with both defined cultures and mixed anaerobic consortia, custom reactors will be used fabricated from stainless-steel process pipe based on the design used for experiments involving cotreatment with maintained biocatalyst activity. Ball milling and knife milling will be implemented in the reactor by adding stainless steel ball bearings or modifying the impeller design and speed as appropriate. Wet disc milling at both small and large scale will be carried out with a MD-3000 mill, with identical mills purchased at Dartmouth and Penn State.For pilot-scale work on defined cultures, we will use a 65 L custom fermentor in the Lynd lab that is equipped for agitation and SIP sterilization. A less elaborate system is sufficient for work with mixed methanogenic consortia, for which aseptic operation is not necessary. The Richard lab will modify a 75 L plastic drum with agitation and an external pipe circuit driven by a 50 mm diameter high-solids slurry pump. Reactors will be fed dried, chopped corn stover or switchgrass at the loading rates described below. Methanogenic reactors will be monitored daily for pH, and should the pH drop below pH 6.6 will be neutralized with 2M NaOH. Depressurization studies will be carried out first for defined cultures, using a modified Parr reactor, and if results are encouraging for mixed cultures by a visiting student from the Richard lab using the same reactor.Measurements. Liquid-phase fermentation products will be analyzed by HPLC, and gas-phase products by GC. Capability for dynamic off-gas analysis is in both the Lynd and Richard labs and will be used to infer rates. Fermented solids will be routinely analyzed gravmetrically and via quantitative saccharification in order to calculate fractional carbohydrate conversion. The Richard lab has capability to measure particle size distributions for wet or dry material with mesh sieves down to 20 microns for dried samples, laser diffraction from 0.05 to 900 microns for wet and 0.5 to 900 microns for dry samples, and dynamic light scattering from 0.3nm - 10.0 microns as well as instruments to measure surface area, internal porosity, and other factors that correlate to rheological properties. In addition, the project will have access to the extensive analytical capabilities of BESC, as indicated in the letter of support from Director Paul Gilna. Viscosity will be characterized using a TA Instruments DHR-3 Discovery Hybrid Rheometer equipped with a Peltier environmental chamber for temperature control. This instrument controls shear rate mode using either plate/plate (used by Viamajala et al., 2009) or vane-in-cup (used by Dunaway et al., 2010) geometries. Electrical current flow to agitators and mill motors will be measured and used to calculate energy inputs as done by the P.I.'s group previously (Fan et al., 2003).Evaluation and industry outreach. Aspen Plus will be used to build process simulations of cotreatment-enhanced production of ethanol or methane from cellulosic biomass based on performance parameters obtained from experimental work. From the resulting material and energy balances, we will develop capital cost and operating cost estimates drawing on literature sources, discussions with industry experts, and our own prior work. We will then evaluate profitability using discounted cash flow rate of return analysis. Process simulations will be used to perform life cycle analysis, focusing on parameters expected to be impacted by substituting cotreatment for conventional pretreatment such as greenhouse gas emissions, and net energy ratio (i.e. bioenergy produced/fossil energy invested). Bill Brady of Enchi. Corp. will lead industry outreach as described in his supporting letter.Project success metricsRelative to targeted performance goals Carbohydrate solubilization: > 85% Milling energy: < 5% feedstock LHV Initial solids concentration: > 150 g/L Reaction time (defined cultures): < 5 days Reaction time (methanogenic): < 8 days Methane yield: > 0.25 L/g initial CODComment: ethanol titer and yield for defined culture work will be noted. Engineered strains with high titer and yield developed by BESC will be available to the project but development of such strains is not a part of this project.Competitiveness relative to best-in-class technology Return on investment (payback time) Energy output:input Life cycle greenhouse gas emissionsComment: reports from NREL and the private sector will be used as a basis of comparison.Understanding relative to the literature Factors impacting cotreatment effectiveness Microbial mechanical disruption tolerance Cotreatment mechanicsComment: except for general understanding of cell disruption mechanics, there has been no directly relevant prior work.Dissemination Archival papers will be the primary dissemination mechanism, with all five project tasksrepresented in submitted manuscripts. Quantitative impact factors are well established.Impacts of small-scale operation on rural economic development and sustainability Economic Rate of replication Integration with agricultural material flowsComment: distinctive opportunities and features engendered by viability at small scale will be explored.

Progress 08/15/16 to 08/14/20

Outputs
Target Audience:The primary target audience for this study includes university and government researchers, science discovery science and biomass processing companies interested in both fundamental science and commercial application of biomass conversion technology, public and private funding organizations including venture capital and government agencies, and others interested in rural communities and in bio-based economic development. An additional audience is the research community interested in the rheology and fluid dynamics of biomass slurries and how physical properties change with reaction. These various stakeholders are being engaged through presentations at conferences and other venues as detailed in this report. Changes/Problems:Brenden Epps left Dartmouth College mid-way through the project. After publishing a definitive paper on rheology led by Dr. Epps, we shifted attention to other objectives following his departure. What opportunities for training and professional development has the project provided?Dartmouth Drs. Brenden Epps and Xiongjun Shao gained experience with project management as Co-PIs. 4th year engineering graduate student Mr. Michael Balch (engineering, supported by a fellowship from the Thayer School) and first year engineering graduate student Sanchari Ghose (fluid mechanics, supported by NIFA) devoted 100% of their research effort to the project. Undergraduate Dartmouth Senior Megan Chamberlain did project-related laboratory research full-time during an off-term, and is expected to be a coauthor on a publication to be submitted during the second year of the project. In August, 2016, Epps and Ghosh traveled to NREL to meet with several scientists, including rheology expert Jonathan Stickel. In September, 2016, Epps traveled to U. Illinois to deliver a seminar and meet with Prof. Randy Ewoldt, an esteemed rheologist, to conduct an initial battery of experiments. In May, 2017, Ghosh attended a 3-day short course on rheology organized by TA Instruments, which was very effective in furthering her knowledge and expertise in the field of rheology. In addition, Epps developed a three-week module on rheology for his graduate-level course ENGS 150 Intermediate Fluid Mechanics; Ghosh (and other project student Balch) were students in the course. In June and November 2017, Shao traveled to NREL twice and conducted corn stover fermentation at various scales from 10L to 3,500L. First year graduate student Galen Moynihan has joined the Lynd lab (100% supported by NIFA). William Eduardo Herrera Agudelo and Sindelia Freitas Azzoni, two researchers from the University of Campinas, Brazil have visited the Lynd lab at Dartmouth to gain experience in culturing anaerobic thermophilic bacteria and cotreatment. In December of 2017 professor Lee Lynd together with Dr. Evert Holwerda, Michael Balch, Sanchari Ghosh and Galen Moynihan visited the Process Development Center at the University of Maine, Orono to do milling tests with once fermented corn stover generated at NREL. The two-day visit included doing milling experiments and interacting with the process engineering staff gaining insight from their experience with milling technologies and generating nano-cellulose materials. Professor Lynd gave a presentation for an audience of 75 people including undergraduate, graduate students and faculty at UMO. Professor Lee Lynd visited with Union Process in Akron Ohio in September of 2017 to discuss milling strategies (Union Process commercially develops attrition milling technology). Dartmouth graduate students Sanchari Ghosh and Galen Moynihan also visited Union Process to do milling tests with once fermented corn stover generated in the Lynd lab (described earlier in this report). Pennsylvania State University In addition to her regular coursework, Penn State doctoral student Anahita Bharadwaj participated in a microbiome seminar series at Penn State during Fall 2017 and Spring 2018. Ms Bharadwaj and Penn State laboratory technologist Ms. Kay DiMarco traveled to Dartmouth twice to learn cotreatment, fermentation, and biomass characterization methods in the Lynd lab, and Ms. Bharadwaj also traveled to the Oak Ridge National Lab to learn metagenomics methods. Ms. Bharadwaj also traveled the 39th Symposium on Biotechnology for Fuels and Chemicals to both learn from experts and develop her own science communication skills. She won the conference poster competition with her cotreatment poster listed in this report. The anaerobic mixed culture pilot reactor's also served as the subject of a senior design project on pH sensing and control systems. Four female undergraduates formed the team that successfully designed that solution. Anahita took a leadership role in the lab and student management along with cotreatment project research, mentoring and training 8 undergraduate students and 2 graduate students in reactor operations, experimental and laboratory techniques, and data analysis. As a PhD student Dr. Bharadwaj presented at eight conferences and symposiums and won several best presentation awards. Currently, manuscripts are being prepared for submission into various peer-reviewed journals. Dr. Bharadwaj graduated in August 2020. MS student Isamar Amador-Diaz visited NREL for a week to work along the lab researcher's (Mary Biddy, Nick Grundl and Tao Ling) on TEA modeling using Aspen Plus. She also visited ORNL to learn about their efforts on LCA and biomass logistics modeling. Isamar participated in 5 conferences during Fall 2017-Spring 2018, presenting cotreatment related posters in 4 of them. She had the opportunity to attend a TEA/LCA Workshop at one of the conferences. During Fall 2017-Spring 2018, Isamar also mentored a group of 5 senior undergraduate capstone students. The senior students successfully finished their capstone, offering a high solids content pumping solution for cotreatment at a 80L scale. An additional undergraduate capstone team is expected to implement the external loops system for the 80L reactor this year. How have the results been disseminated to communities of interest?In addition to the papers and conference presentations mentioned in the following section, and in anticipation of several in-preparation papers, we were active in presenting the cotreatment concept and project results at many meetings in the US and abroad. Due to restricted space to report the complete list in the final report, both conference oral presentations as well as poster presentations have now been included in 'Products' section of the Final Report. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Goal 1 a): Three substrates have been tested at low feedstock concentrations with continuous ball milling: switchgrass (October and December harvest dates), corn stover, and poplar. For all substrates, significant increase in solubilization was documented in the presence of cotreatment as compared to non-cotreated controls. These results have been published (Balch et al 2020). Dartmouth purchased two different size conical disc mills, both able to mill during fermentation via an external loop. Experiments on switchgrass and corn stover resulted in a significant increase in solubilization in the presence of cotreatment. At Penn State three different proposed cotreatment mechanisms; cutting forces (laboratory blender), hammering forces (lab-scale ball mill) and shearing forces (bench-top conical disc mill) were evaluated in preliminary trials for cotreatment efficacy. Other parameters such as milling time, number of passes and microbial adaptation were also tested. The extent of solubilization enhancement was studied for mixed culture systems at mesophilic conditions producing biogas as the final product. Two different mechanical disruption techniques, the ball mill and the colloid mill, were further studied in replicated trials and improvements in sugar solubilization and biogas production quantified. Results indicate an increase in biomass sugar solubilization and biogas production (10 - 15% with the ball mill and about 5% with the colloid mill as compared to the status-quo control with just one round of milling). Importantly, the colloid mill has been shown to be a more energy efficient cotreatment strategy compared to the ball mill as described in the Goal 5 section below. Goal 1b): Dartmouth has concluded testing compatibility of pure cultures with milling during fermentation. To date, seven organisms have been examined to determine the effect of cotreatment. A published manuscript (Balch et al., 2020) provides comparative data for fermentation by four different microorganisms in the presence and absence of milling. Combined with our published comparison of C. thermocellum and yeast (Balch et al., 2018), we have comparative data for seven different microbes in pure culture. At Penn State two bench-scale anaerobic digesters have been established with mixed microbiome inocula and senescent switchgrass as the feed. The mixed culture microorganisms involved in the conversion of switchgrass to biogas under anaerobic conditions were characterized using 16s rRNA amplicon sequencing. A novel live/dead assay was developed in order to assess the growth and compatibility of mixed culture microorganisms under cotreatment milling.. Goal 2: Cotreatment studies were also conducted using corn stover samples acquired from the Great Lakes Bioenergy Research Center (less recalcitrant compared to NREL material) with 80 g/L solids loading. Total carbohydrate solubilization of 60% was achieved without cotreatment. Solubilization was increased to 86% with two sequential milling and fermentation steps, one with 5 minutes and one with 8 minutes milling. The results of this set of experiments indicated that cotreatment on higher solids loadings is promising and milling time can be significantly decreased. At the Pennsylvania State University two prototype pilot reactors were constructed in a trickle-bed configuration, with a high-solids bed with liquid recirculation. These reactors were routinely operated in fed-batch mode with loading rates of 75 g/L to 125 g/L, making material available for cotreatment using different pilot-scale milling technologies. Penn State also acquired two 80 L CSTR commercial bioreactors for the 65 L testing. One of these bioreactors was adapted to run cotreatment experiments in a continuous mode with milling in an external loop by a pilot scale conical disc mill. The reactor was operated for four months without cotreatment to optimize media and achieve steady-state baseline conditions, and was then operated for 40 days with daily milling a moderate milling gap size. Two cotreatment intensities were tested prior to having to cease operations due to COVID-19. Microbiome data to characterize changes in the microbiome are scheduled for sequencing after the COVID-19 backlog eases. Goal 3: In work at Dartmouth we tested cotreatment milling at 10% solids during year 1 as reported earlier. Implementing milling in an external loop was the most efficient cotreatment configuration, although pumping a high solids fermentation slurry around that external loop was the most challenging bottleneck. Fortunately, the material flow characteristics of lignocellulosic biomass slurries improve significantly during fermentation (see goal 4). An undergraduate research team at Penn State did a series of trials with unfermented switchgrass to evaluate different pumping options. Initial trials with a progressive cavity pump (auger type) pump were promising so this was purchased. Internal mixing is also problematic at high solids loading and agitation mechanisms were also evaluated at Penn State. A pitched plate impeller was capable of adequate mixing above 100 g/L. However, pumping remained a limitation for a cotreatment recirculation loop at this scale, so cotreatment tests in the 80L reactors were operated at <75 g/L and are reported in goal 2. The high solids material was milled without recirculation as summarized in goal 4. Goal 4: As anticipated in the proposal, most of the activity pursuant to this goal was carried out at Dartmouth. The rheological properties of corn stover slurries were tested. An archival paper on this topic has been published (Ghosh et al., 2018). In the most thorough documentation to date of the rheological properties of unpretreated cellulosic biomass during biological reaction, we showed that the viscosity of corn stover slurries at process-relevant solids loadings decreases by 2000-fold due to fermentation by C. thermocellum Penn State also measured energy requirements for milling with a data logger connected to the large conical disc mill. This equipment was used to test cotreatment energy requirements at different solid contents, particle sizes and fermentation times as reported in Drew May's M.S. thesis (May, 2020). Goal 5: Economic and life cycle analysis was carried out by researchers from Dartmouth in collaboration with colleagues from the NREL, ANL, and UC-Riverside. Results were published in Lynd et al., Current Opin. Biotechnol. 45:202-211. A technoeconomic model of cotreatment-aided anaerobic digestion was developed in ASPEN+ and published in an M.S. thesis (Amador-Diaz 2019). This model indicated the opportunities for cotreatment improving process economics would be strongly affected by economies of scale and by solids concentration. Goal 6: Penn State led implementation efforts of cotreatment for mixed culture microbial consortia, while Dartmouth led implementation effort for pure culture activities. The team communicated regularly, including biweekly teleconferences and face-to-face meetings several times a year to leverage experiences and coordinate experiments. One of the fundamental research questions related to the interaction of cotreatment with fermentation is the potential impact of milling on microorganisms (as reported under 1a). Penn State initiated a set of experiments to evaluate the effects of both high solids and milling on microbial populations. The novel live/dead assay developed for Goal 1a was applied to these microbiomes, allowing for the study of cotreatment impacts on a wide range of microbiome populations. Metagenomics analysis of samples with and without milling indicated some adaptation of bacterial populations and several promising organisms, as reported in Bharadwaj (2020).

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2017 Citation: Balch, M.L., E.K. Holwerda, M.F. Davis, R.W. Sykes, R.M. Happs, R. Kumar, C.E. Wyman, L.R. Lynd. 2017. Lignocellulose fermentation and residual solids characterization for senescent switchgrass fermentation by Clostridium thermocellum in the presence and absence of continuous in-situ ball-milling. Energy Env. Sci. 10:1252-1261. DOI:10.1039/c6ee03748h.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Ghosh, S, Holwerda EK, Worthen RS, Lynd LR, Epps BP. 2018. Rheological properties of corn stover slurries during fermentation by Clostridium thermocellum.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Fermentation with continuous ball milling: Effectiveness at enhancing solubilization for several cellulosic feedstocks and comparative tolerance of several microorganisms.2020. Balch M. L., Meghan B. Chamberlain, Robert S. Worthen, Evert K. Holwerda, Lee R. Lynd
• Type: Journal Articles Status: Other Year Published: 2021 Citation: Technoeconomic analysis of consolidated bioprocessing with cotreatment. Ghosh, S., E. K. Holwerda, T. Pschorn, and L. R. Lynd.
• Type: Journal Articles Status: Other Year Published: 2021 Citation: Development and operation of a fermentation system capable of metered, aseptic delivery of lignocellulose slurries at high solids loadings. Kubis, M., G. Moynihan, E. K. Holwerda, and L. R. Lynd.
• Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Amador-Diaz, I. 2018. Anaerobic digestion of lignocellulosic biomass via cotreatment: a technoeconomic analysis. M.S. thesis. The Pennsylvania State University.
• Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Balch, M. 2019 Cotreatment of lignocellulose to enhance solubilization in lieu of thermochemical pretreatment. Ph.D. thesis. Dartmouth College.
• Type: Theses/Dissertations Status: Published Year Published: 2020 Citation: Bharadwaj, A. 2020. Microbial adaptation and cotreatment-enhanced biomass solubilization in lignocellulosic anaerobic digestion. Ph.D. thesis. The Pennsylvania State University.
• Type: Theses/Dissertations Status: Published Year Published: 2020 Citation: May, A.J. 2020. Cotreatment enhanced anaerobic digestion of lignocellulosic biomass. M.S. thesis. The Pennsylvania State University
• Type: Journal Articles Status: Other Year Published: 2021 Citation: Bharadwaj A., Holwerda E.H., Lynd L.R and Richard T.L.. Accelerating lignocellulosic anaerobic digestion by cotreatment. To be submitted in Bioresource Technology.
• Type: Journal Articles Status: Other Year Published: 2021 Citation: Bharadwaj A., Wamea P., Holwerda E.H., Thomas R.S., Lynd L.R and Richard T.L. Colloid milling as an energy efficient cotreatment strategy for lignocellulosic anaerobic digestion. To be submitted in Biotechnology for Biofuels.
• Type: Journal Articles Status: Other Year Published: 2021 Citation: Bharadwaj A., Shreve, M. J., Regan J.M., Lynd L.R and Richard T.L. Assessing viability, adaptation and robustness of anaerobic microbiome during cotreatment. To be submitted in Journal of Industrial Microbiology & Biotechnology
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Lynd, Lee. Low Cost Cellulosic Biofuels: New Questions and New Answers. Green Chemistry Course. Center for Bioethanol Technology, Campinas, Brazil. September 28, 2016.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Lynd, Lee. Low cost cellulosic biofuels: New Questions and New Answers. Andlinger Center for Energy and the Environment. Princeton, October 24, 2016.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Lynd, Lee. African Human Development: Land, Agriculture, and Bioenergy. Stellenbosch Water Institute and Center for Complex Systems in Transition. University of Stellenbosch, South Africa. November 7, 2016.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Lynd, Lee. Low Cost Cellulosic Biofuels: New Questions and New Answers. 2G Ethanol Workshop. Center for Bioethanol Technology, Campinas, Brazil. December 1, 2016.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Lynd, Lee. Low-Carbon Fuels. Workshop on Ensuring Energy Security through a Low-Carbon Energy Economy. National Renewable Energy Laboratory. December 8, 2016.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Lynd, LR. Low-Cost Cellulosic Biofuels: New Questions in Pursuit of New Answers. Department of Chemical Engineering, Stanford University. January 20, 2017.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Holwerda, EK. Microbial cellulose utilization and multiple levers for overcoming recalcitrance of cellulosic biomass. LIFE Master Study Delft & Leiden University, Hanover, New Hampshire. July 17 2017.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Lynd, LR. Thermophilic consolidated bioprocessing with cotreatment: A potentially disruptive paradigm for biological production of cellulosic biofuels. Society of Industrial Microbiology Meeting, Denver, Colorado. August 3, 2017.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Lynd, LR. Thermophilic consolidated bioprocessing with cotreatment: A potentially disruptive paradigm for biological production of cellulosic biofuels. National Renewable Energy Laboratory, August 4, 2017.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Lynd, LR. Cellulosic biofuels: Need, hope, assessment, diagnosis, and prescription. Advanced hardwood biofuels meeting. August 10, 2017.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Epps, BP. Low-cost cellulosic biofuels: cotreatment mechanics and rheology. University of Illinois Urbana-Champaign, Department of Mechanical Science and Engineering, Fluid Mechanics Seminar, September 16, 2016.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Ghosh, S; Epps, BP and Lynd, LR. Rheology of corn stover slurries during fermentation to ethanol. Conference presentation, 70th Annual Meeting of the American Physical Society Division of Fluid Dynamics, Denver, CO, November 19, 2017.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Richard T.L. 2017. Next Generation Anaerobic Digestion. Presented at the Mid-Atlantic Biomass Conference and Expo, Sept. 12-14, 2017, State College, PA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Lynd, L.R. Cellulosic biofuels: Making the next decade more important than the last. 7th International Congress on Biofuels and Bioenergy. Toronto. October 2, 2017.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Lynd, L.R. Thermophilic consolidated bioprocessing with cotreatment: A potentially disruptive paradigm for second generation ethanol production. BBEST 2017. Campos do Jordao, Brazil. October 22, 2017.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Lynd, L.R. Thermophilic consolidated bioprocessing with cotreatment: A potentially disruptive paradigm for second generation ethanol production. Penn State University. December 5, 2017.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Lynd, L.R. Thermophilic consolidated bioprocessing with cotreatment: A potentially disruptive paradigm for second generation ethanol production. University of Maine, Orono. December 7, 2017.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Lynd, L.R. Low cost cellulosic biofuels: New questions in pursuit of new answers. University of Massachusetts, Department of Chemical Engineering 2018.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Lynd, L.R. Low cost cellulosic biofuels: New questions in pursuit of new answers.Biomass Refinery: From Biomass Crops to Chemicals and Fuels. Hokkaido University. February 5, 2018.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Lynd, L.R. Low cost cellulosic biofuels: New questions in pursuit of new answers.Tufts University, Department of Chemical Engineering. April 17, 2018.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Lynd, L.R. Low cost cellulosic biofuels: New questions in pursuit of new answers. KTH Royal Institute of Technology. Stockholm, April 23, 2018.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Lynd, L.R. 2G 2.0. Keynote presentation at the Symposium on Biotechnology for Fuels and Chemicals, April 28 - May 2, 2018, Clearwater, FL.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Lynd, L.R. Microbial cellulose utilization: From applications to fundamentals and back again. Keynote presentation at the Canadian Society of Microbiology. University of Manitoba. June 19, 2018.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Bharadwaj, A., Holwerda, E., Lynd, L.R., and Richard, T. L. (2019). Accelerating lignocellulose anaerobic digestion using cotreatment. College of Engineering Symposium, Pennsylvania State University. University Park. PA. (2nd place  Best oral presentation in the Environmental & Sustainable Infrastructure category)
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Bharadwaj, A., Holwerda, E., Lynd, L.R., and Richard, T. L. (2019). A study on the effect of cotreatment on anaerobic digestion process. Environmental Chemistry and Microbiology Student Symposium, March 15-16, Pennsylvania State University. University Park. PA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Richard, T.L., A. Bharadwaj and I. Amador-Diaz. (2019) Biomimetic Rumination: Cotreatments Energy Return on Energy Investment. AIChE annual meeting. Nov. 11, 2019. Orlando, FL.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Sanchari G., M. Balch, G. Moynihan, E.K. Holwerda, B. Epps, and L.R. Lynd. Cotreatment of Cellulosic Biomass: Effectiveness on Various Feedstocks, Impact on Different Microbes, and Various Milling Modalities. AIChE annual meeting. Nov. 12-th, 2019. Orlando, Florida.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Holwerda, EK. Evaluation of Multiple Levers for Overcoming the Recalcitrance of Cellulosic Biomass. Genomic Sciences Program Annual Principal Investigators meeting, Arlington, Virginia. February 5-8
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Bharadwaj, A. Effect of cotreatment (mechanical disruption) on microbial consortia involved in anaerobic digestion of lignocellulosic biomass. 39th Symposium for Fuels and Chemicals. Society of Industrial Microbiology. San Francisco, CA. May 1-4, 2017 (Best Poster).
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Balch et al., Cotreatment to increase solubilizationof lignocellulose and its impact on microorganisms. BESC retreat. Chattanooga, TN. July 12th, 2017.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Ghosh, S; Epps, BP and Lynd, LR. Rheological properties of corn stover slurries during fermentation to ethanol. Technical poster, Society or Rheology 89th Annual Meeting, Denver, CO, October 8, 2017.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Amador-Diaz et al. 2018. Cotreatment enhanced mixed culture fermentation of switchgrass. Presented at Symposium on Biotechnology for Fuels and Chemicals, April 28 - May 2, 2018, Clearwater, FL.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Bharadwaj, A., Whitham J., Brown S., Holwerda E.H., Lynd L.R., and Richard, T.L. 2018. Anaerobic Digestion of Lignocellulose Using Mixed Microbial Populations. Symposium for Biotechnology in Fuels and Chemicals, Apr 29  May 2, 2018, Clearwater FL.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Amador-Diaz et al. 2018. Cotreatment enhanced mixed culture fermentation of switchgrass. Presented at Energy Days May 30 - 31, 2018, State College, PA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Ghosh, S; Balch, ML; Moynihan, GD; Holwerda, EK; Epps, BP and Lynd, LR. Cotreatment Update: Effectiveness on Various Feedstocks, Impact on Different Microbes, and Changes in Biomass Physical Properties During Fermentation. Technical poster, Center of Bioenergy Innovation Annual Meeting, Asheville, NC, June 6th, 2018
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Ghosh, S; Worthen, RS; Holwerda EK; Lynd LR and Epps BP. Rheological properties of corn stover slurries during fermentation by Clostridium thermocellum. Conference presentation, Society of Rheology 90th Annual Meeting, Houston, TX, October 17, 2018
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Amador-Diaz et al. 2018. Cotreatment enhanced mixed culture fermentation of switchgrass. Presented at institute of Biological Engineering annual meeting, April 5 - April 7, 2018, Norfolk, VA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Bharadwaj, A., May, J. A., Holwerda, E., Lynd, L.R., and Richard, T. L. (2019). Cotreatment-enhanced anaerobic digestion of lignocellulosic biomass. The Science of Drawdown: Research to Action, Sep 16  18, 2019, University Park. PA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Bharadwaj, A., Holwerda, E., Lynd, L.R., and Richard, T. L. (2019). Accelerating lignocellulose anaerobic digestion using cotreatment. Symposium for Biotechnology in Fuels and Chemicals, Apr 27  May 1, 2019, Seattle WA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Bharadwaj, A., Holwerda E.K., Lynd, L.R., and Richard, T. L. (2019). Accelerating lignocellulose anaerobic digestion using cotreatment. Graduate exhibition. Mar 22  24, Pennsylvania State University. University Park PA. (2nd place  Best Poster, Best Data Visualization)
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Bharadwaj, A., May, J. A., Holwerda, E., Lynd, L.R., and Richard, T. L. (2019). Cotreatment enhanced anaerobic digestion of lignocellulosic biomass. The Science of Drawdown: Research to Action, Sep 16  18, 2019, University Park. PA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Ghosh, S., Holwerda E.K., T Pschorn, Lynd L.R. (2020): Disc Milling of Fermented Corn Stover to Increase its Accessibility to Fermentation by Clostridium thermocellum. Virtual poster, 16-th November AIChE conference.

Progress 08/15/18 to 08/14/19

Outputs
Target Audience:The primary target audience for this study includes university and government researchers, science discoveryscience and biomass processing companies interested in both fundamental science and commercialapplication of biomass conversion technology, public and private funding organizations including venturecapital and government agencies, and others interested in rural communities and in bio-based economicdevelopment. An additional audience is the research community interested in the rheology and fluid dynamicsof biomass slurries and how physical properties change with reaction. These various stakeholders are beingengaged through presentations at conferences and other venues as detailed in this report. Changes/Problems:Brenden Epps has left Dartmouth College as well as the project, Isamar Amador-Diaz has graduated and left the Richard lab, Mikayla Balch has graduated and left the Lynd lab. What opportunities for training and professional development has the project provided?PhD student Anahita Bharadwaj presented various conferences and symposiums and has won several best presentation awards. She has also trained several undergraduate students in various fermentation techniques and good laboratory practices. Currently, manuscripts are being prepared for submission into various peer-reviewed journals. How have the results been disseminated to communities of interest?Dartmouth College: In addition to the papers mentioned in the following section, and in anticipation of several in-preparation papers, we were active in presenting the cotreatment concept and project results at many meetings in the US and abroad, to be detailed in the final report. Amador-Diaz, I. 2018. Anaerobic digestion of lignocellulosic biomass via cotreatment: a technoeconomic analysis. M.S. thesis. The Pennsylvania State University. Balch, M. 2019 Cotreatment of lignocellulose to enhance solubilization in lieu of thermochemical pretreatment. Ph.D. thesis. Dartmouth College. What do you plan to do during the next reporting period to accomplish the goals?Dartmouth college: During the no-cost extension period, we plan to finish experimental work as described above, submit manuscripts to the archival literature, and submit a comprehensive final report to NIFA. Pennsylvania State University: Experiments to assess the resilience and robustness of mixed cultures under cotreatment conditions are currently under way and will be reported soon. Experiments using the 60 L thermophilic anaerobic digester with intermittent colloid milling are also currently underway. This will provide a deeper understanding of both process efficiency and microbiome adaptation under continuous milling.

Impacts
What was accomplished under these goals? Pursuant to goal 1: a) The extent of solubilization enhancement as a result of mechanical disruption. Dartmouth College: This is a key focus of continued activity, to be detailed in the final report. Pennsylvania State University: The extent of solubilization enhancement has been studied for mixed culture systems at mesophilic conditions producing biogas as the final product. Two different mechanical disruption techniques, the ball mill and the colloid mill, have been studied and improvements in sugar solubilization and biogas production quantified. This trial was configured with single stage cotreatment in a ferment-mill-ferment process. Secondary measurements such as particle size change and volatile solids degradation have been quantified. Furthermore, the energy consumed by the mills was quantified to determine the energy efficiency and economic feasibility of cotreatment. Results indicate an increase in biomass sugar solubilization and biogas production (10 - 15% with the ball mill and about 5% with the colloid mill as compared to the status-quo control with just one round of milling). Importantly, the colloid mill has been shown to be a more energy efficient cotreatment strategy compared to the ball mill as described in the Goal 5 section below. b) Testing compatibility micro-organisms with cotreatment. Dartmouth College: A submitted manuscript (Balch et al., in review) provides comparative data for fermentation by five different microorganisms in the presence and absence of milling. Combined with our published comparison of C. thermocellum and yeast (Balch et al., 2018), we have comparative data for 7 different microbes in pure culture. More detail will be provided in the final report. Pennsylvania State University: The mixed culture microorganisms involved in the conversion of switchgrass to biogas under anaerobic conditions have been characterized using 16s rRNA amplicon sequencing. A novel live/dead assay has been developed in order to assess the growth and compatibility of mixed culture microorganisms under cotreatment milling. This technique will be used to determine the resilience and robustness of the mixed culture system when subjected to cotreatment. Pursuant to goal 2: Work at Dartmouth College with defined cultures has proceeded at a 15L scale; work at Pennsylvania State University is underway at an 60 L scale. Results will be presented in the final report. Pursuant to goal 3: Dartmouth College: Work toward this goal is proceeding, including development of an unprecendented system able to aseptically feed solids to a defined culture at high solids. Because this job proved bigger than anticipated, we are using funding from the DOE Center for Bioenergy Innovation grant as well as the NIFA grant. Acknowledgement of both sources will be acknowledged. Pennsylvania State University: A 60L large scale thermophilic anaerobic digestion reactor has been set up, complete with pH and temperature control as well as biogas volume and composition measurement capabilities. This reactor uses senescent switchgrass as the sole carbon source. A pilot-scale colloid mill has been attached to this reactor in order to study intermittent cotreatment milling strategy for enhanced biomass solubilization. Initial measurements of this pilot-scale colloid mill with fresh switchgrass were used to estimate scale-up energy requirements as reported in Goal 5. Trials are currently under way to study intermittent cotreatment for anaerobic digestion at this larger scale, and will include both milling energy requirements and resultant changes in energy outputs as measured by biogas methane production. Pursuant to goal 4: An archival paper on this topic has been published (Ghose et al., 2018). In the most thorough documentation to date of the rheological properties of unpretreated cellulosic biomass during biological reaction, we showed that the viscosity of corn stover slurries at process-relevant solids loadings decreases by 2000-fold due to fermentation by C. thermocellum. Pursuant to goal 5: Dartmouth College: This is ongoing for both defined cultures (Dartmouth College) and methanogenic microbiomes (Pennsylvania State University). TEA is informed by the results of efforts aimed at goal 5. Pennsylvania State University: A technoeconomic model of cotreatment-aided anaerobic digestion was developed in ASPEN+ and published in an M.S. thesis (Amador-Diaz 2019). This model indicated the opportunities for cotreatment improving process economics would be strongly affected by economies of scale and by solids concentration. The Penn State bench-scale cotreatment experiments summarized under goal 1 also included quantification of energy requirements of the mill as well as the energy content of the additional energy produced in the methane fraction of the biogas. For this bench-scale trial the Energy Return on Energy Investment (EROI) was significantly better for the colloid mill than the ball mill, at 1.59 and 0.79 respectively, doubling the amount of energy per unit of milling energy invested. To estimate the EROI at closer to commercial scale, a larger colloid mill by the same manufacturer (IKA) was used to determine EROI for higher flowrates (60 L/minute) and higher solids concentrations (6%). Matching flowrates and energy requirements from our pilot scale measurements at different flowrates and solids contents with manufacturer provided scale-up data for higher flowrates resulted in estimates of EROI as high as 200:1 for a 10% solids mixtures at a 4000 L/minute flowrate, as indicated in the figure below. These estimates are expected to be conservative as the cotreatment regime has not been optimized, and the 60L/min energy measurements were made with fresh rather than digested switchgrass. As a point of comparison, ruminant digestion has been estimated to have an EROI of 50, equivalent to using 2% of the feedstock energy for processing. Pursuant to goal 6: Dartmouth College: This is embodied in our approach to goals 1 through 5, as described above. Pennsylvania State University: Mixed culture microbiomes capable of degrading senescent switchgrass (as the sole carbon source) into biogas have been operated at both mesophilic and thermophilic conditions at Penn State. The novel live/dead assay developed for Goal 1a is being applied to these microbiomes, allowing for the study of cotreatment impacts on a wide range of microbiome populations. Both methane-producing and organic acid producing microbial populations are targeted with this study in order to broadly understand the tolerance to cotreatment and associated resilience of microorganisms involved in the conversion of lignocellulosic biomass into various value-added fuels and chemicals.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Bharadwaj A., Holwerda E.H., Lynd L.R and Richard T.L. (exp. 2019). Accelerating lignocellulosic anaerobic digestion by cotreatment. To be submitted in Bioresource Technology.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2020 Citation: � Bharadwaj A., Wamea P., Holwerda E.H., Thomas R.S., Lynd L.R and Richard T.L. (exp. 2020). Colloid milling as an energy efficient cotreatment strategy for lignocellulosic anaerobic digestion. To be submitted in Biotechnology for Biofuels. � Bharadwaj A., Shreve, M. J., Regan J.M., Lynd L.R and Richard T.L (exp. 2020). Assessing viability, adaptation and robustness of anaerobic microbiome during cotreatment. To be submitted in Journal of Industrial Microbiology & Biotechnology
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Bharadwaj, A., Holwerda, E., Lynd, L.R., and Richard, T. L. (2019). Accelerating lignocellulose anaerobic digestion using cotreatment. College of Engineering Symposium, Pennsylvania State University. University Park. PA. (2nd place  Best oral presentation in the Environmental & Sustainable Infrastructure category) � Bharadwaj, A., Holwerda, E., Lynd, L.R., and Richard, T. L. (2019). A study on the effect of cotreatment on anaerobic digestion process. Environmental Chemistry and Microbiology Student Symposium, March 15-16, Pennsylvania State University. University Park. PA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Bharadwaj, A., May, J. A., Holwerda, E., Lynd, L.R., and Richard, T. L. (2019). Cotreatment-enhanced anaerobic digestion of lignocellulosic biomass. The Science of Drawdown: Research to Action, Sep 16  18, 2019, University Park. PA. � Bharadwaj, A., Holwerda, E., Lynd, L.R., and Richard, T. L. (2019). Accelerating lignocellulose anaerobic digestion using cotreatment. Symposium for Biotechnology in Fuels and Chemicals, Apr 27  May 1, 2019, Seattle WA. � Bharadwaj, A., Holwerda, E., Lynd, L.R., and Richard, T. L. (2019). Accelerating lignocellulose anaerobic digestion using cotreatment. Graduate exhibition. Mar 22  24, Pennsylvania State University. University Park PA. (2nd place  Best Poster, Best Data Visualization)

Progress 08/15/17 to 08/14/18

Outputs
Target Audience:The primary target audience for this study includes university and government researchers, science discovery science and biomass processing companies interested in both fundamental science and commercial application of biomass conversion technology, public and private funding organizations including venture capital and government agencies, and others interested in rural communities and in bio-based economic development. An additional audience is the research community interested in the rheology and fluid dynamics of biomass slurries and how physical properties change with reaction. These various stakeholders are being engaged through presentations at conferences and other venues as detailed in this report. Changes/Problems:Galen Moynihan joined the Lynd lab as a first year graduate student. Dr. Xiongjun Shao has left the project as indicated in last year's report. What opportunities for training and professional development has the project provided?Dartmouth First year graduate student Galen Moynihan has joined the Lynd lab (100% supported by NIFA). William Eduardo Herrera Agudelo and Sindelia Freitas Azzoni, two researchers from Campinas, Brazil have visited the Lynd lab at Dartmouth to gain experience in culturing anaerobic thermophilic bacteria and cotreatment. In December of 2017 professor Lee Lynd together with Dr. Evert Holwerda, Michael Balch, Sanchari Ghosh and Galen Moynihan visited the Process Development Center at the University of Maine, Orono to do milling tests with once fermented corn stover generated at NREL. The two-day visit included doing milling experiments and interacting with the process engineering staff gaining insight from their experience with milling technologies and generating nano-cellulose materials. Professor Lynd gave a presentation for an audience of 75 people including undergraduate, graduate students and faculty at UMO. Professor Lee Lynd visited with Union Process in Akron Ohio in September of 2017 to discuss milling strategies (Union Process commercially develops attrition milling technology). Dartmouth graduate students Sanchari Ghosh and Galen Moynihan also visited Union Process to do milling tests with once fermented corn stover generated in the Lynd lab (described earlier in this report). Pennsylvania State University PhD student Anahita Bharadwaj presented posters at 4 conferences during Fall 2017-Spring 2018. Anahita has taken a leadership role in the lab and student management along with cotreatment project research. She has mentored and trained 8 undergraduate students and 2 graduate students in reactor operations, experimental and laboratory techniques, and data analysis. MS student Isamar Amador-Diaz visited NREL for a week to work along the lab researcher's (Mary Biddy, Nick Grundl and Tao Ling) on TEA modeling using Aspen Plus. She also visited ORNL to learn about their efforts on LCA and biomass logistics modeling. Isamar participated in 5 conferences during Fall 2017-Spring 2018, presenting cotreatment related posters in 4 of them. She had the opportunity to attend a TEA/LCA Workshop at one of the conferences. During Fall 2017-Spring 2018, Isamar also mentored a group of 5 senior undergraduate capstone students. The senior students successfully finished their capstone, offering a high solids content pumping solution for cotreatment at a 80L scale. An additional undergraduate capstone team is expected to implement the external loops system for the 80L reactor this year. How have the results been disseminated to communities of interest?Acknowledged NIFA in presentation: Richard T.L. 2017. Next Generation Anaerobic Digestion. Presented at the Mid-Atlantic Biomass Conference and Expo, Sept. 12-14, 2017, State College, PA. Lynd, L.R. Cellulosic biofuels: Making the next decade more important than the last. 7th International Congress on Biofuels and Bioenergy. Toronto. October 2, 2017. Lynd, L.R. Thermophilic consolidated bioprocessing with cotreatment: A potentially disruptive paradigm for second generation ethanol production. BBEST 2017. Campos do Jordao, Brazil. October 22, 2017. Lynd, L.R. Cellulosic biofuels: Broad benefit realization and technology. Similar (although not identical) talks with same title given to: • Penn State University. December 5, 2017. • University of Maine, Orono. December 7, 2017. Lynd, L.R. Low cost cellulosic biofuels: New questions in pursuit of new answers. Similar (although not identical) talks with the same title given to: • University of Massachusetts, Department of Chemical Engineering. • Biomass Refinery: From Biomass Crops to Chemicals and Fuels. Hokkaido University. February 5, 2018. • Tufts University, Department of Chemical Engineering. April 17, 2018. • KTH Royal Institute of Technology. Stockholm, April 23, 2018. Lynd, L.R. 2G 2.0. Keynote presentation at the Symposium on Biotechnology for Fuels and Chemicals, April 28 - May 2, 2018, Clearwater, FL. Lynd, L.R. Microbial cellulose utilization: From applications to fundamentals and back again. Keynote presentation at the Canadian Society of Microbiology. University of Manitoba. June 19, 2018. Acknowledged NIFA in poster: Ghosh, S; Worthen, RS; Holwerda EK; Lynd LR and Epps BP. Rheological properties of corn stover slurries during fermentation by Clostridium thermocellum. Conference presentation, Society of Rheology 90th Annual Meeting, Houston, TX, October 17, 2018. Amador-Diaz et al. 2018. Cotreatment enhanced mixed culture fermentation of switchgrass. Presented at institute of Biological Engineering annual meeting, April 5 - April 7, 2018, Norfolk, VA. Amador-Diaz et al. 2018. Cotreatment enhanced mixed culture fermentation of switchgrass. Presented at Symposium on Biotechnology for Fuels and Chemicals, April 28 - May 2, 2018, Clearwater, FL. Amador-Diaz et al. 2018. Cotreatment enhanced mixed culture fermentation of switchgrass. Presented at Energy Days May 30 - 31, 2018, State College, PA. Ghosh, S; Balch, ML; Moynihan, GD; Holwerda, EK; Epps, BP and Lynd, LR. Cotreatment Update: Effectiveness on Various Feedstocks, Impact on Different Microbes, and Changes in Biomass Physical Properties During Fermentation. Technical poster, Center of Bioenergy Innovation Annual Meeting, Asheville, NC, June 6th, 2018. What do you plan to do during the next reporting period to accomplish the goals?Goal 1: A paper exploring the impact of cotreatment on feedstocks (goal 1a) and organism growth (goal 1b) is in the final stages and will be submitted for publication before 2019. At Dartmouth graduate student Michael Balch will test intermittent milling by modifying the existing cotreatment reactors (described in Balch et al., 2017) to test the effect of reduced milling duration. We plan to explore the impact of total milling duration on solubilization to obtain more insight in milling energy requirements and efficiency. Graduate student Sanchari Ghosh will use the small conical disc mill to mill once-fermented biomass slurries at various solids loadings in order to test solubilization enhancement by varying milling parameters like milling time, milling gap, and milling speed. The objective of conducting these tests is to narrow down what milling parameters will result in low energy utilization of the milling process while still being effective. At Penn State laboratory experiments with small scale milling technologies will be completed and the results used to optimize the tradeoffs between milling time, energy requirements, and enhanced solubilization with mixed anaerobic cultures. Impacts on biomass solubilization, product formation, and microbial populations will be characterized. Goal 2: Large scale fermentation and milling Dartmouth is planning on developing a 30-50 L scale bioreactor set-up that can operate in semi continuous mode at high solids loadings. This set up includes an aseptic sample delivery system and automated operation of feed regimes. The set-up will be able to operate in batch mode with the ability to do cotreatment on the fermented solids. We are planning purchasing a mill from Union Process so we have the possibility to use two milling technologies (attrition mill from UP or a conical disc mill already purchased). Penn State will complete the modifications of the 80L fermenter to accommodate an external milling loop, and then run trials with switchgrass feedstock and a mixed anaerobic microbiome. Goal 3: At Dartmouth once the set-up described above will be operational we will test the ability of the system to operate aseptically at increasing solids loadings. At Penn State pilot trials in the 80L reactor described above will begin at 75 g/L, and the solids loading will be increased gradually to >150 g/L to the extent pumping and milling technologies allow. Goal 4: Energy requirements will be evaluated at for both stand-alone and integrated (external loop) milling strategies for a range of solids contents and fermentation extents. A publication on those energy requirements will be prepared. Goal 5: The anaerobic digestion TEA will be completed and used to evaluate economics and scale-up issues and limitations for use of cotreatment at farm, municipal, and industrial scales. Life Cycle Analysis will also be used to assess environmental impacts and opportunities. A publication will be prepared to share the TEA-LCA analysis. Goal 6: We have begun discussions with both start-up and established bioenergy firms interested in cotreatment, and will be sharing results over the next year to continue to build interest in commercial implementation. There is strong interest from Enchi Corporation in submitting an SBIR proposal based on the success of this BRDI project.

Impacts
What was accomplished under these goals? Pursuant to goal 1: a) The extent of solubilization enhancement as a result of mechanical disruption At Dartmouth testing feedstocks has been completed; experiments on switchgrass and corn stover resulted in a significant increase in solubilization in the presence of cotreatment. Dartmouth purchased two different size conical disc mills, both able to mill during fermentation via an external loop. The smaller size bench-top mill was tested using once fermented corn stover generated in the Lynd lab. A 30-minute ex-situ milling session increased solubilization from 65.5 % to 83.9% of the total carbohydrate content of the initial material. As the parameter space for mills and this mill in particular is fairly large (milling time/milling speed/solids loading/gap width/milling head configuration) more in-depth experiments will be done in an ex-situ ferment-mill-ferment setting as this allows for a higher throughput of tested variables. The small conical mill allows for milling energy to be measured during milling and this will be one of the reported metrics going forward. At Penn State three different proposed cotreatment mechanisms; cutting forces (laboratory blender), hammering forces (lab-scale ball mill) and shearing forces (bench-top conical disc mill) are being tested for cotreatment efficacy. Other parameters such as milling time, number of passes and microbial adaptation will also be tested. b) Testing compatibility micro-organisms with cotreatment Dartmouth has concluded testing compatibility of pure cultures with milling during fermentation. In addition to Clostridium thermocellum, Zymomonas mobilis, Saccharomyces cerevisiae, Bacillus subtilis and Escherichia coli we included the gram-positive thermophile Thermoanaerobacterium saccharolyticum. Growth of T. saccharolyticum was found not to be inhibited by milling consistent with the results for Escherichia coli and Clostridium thermocellum. Tests with Bacillus subtilis were finalized and found that fermentative activity was halted by milling, similar to what was found for Zymomonas mobilis and Saccharomyces cerevisiae. At Penn State two bench-scale anaerobic digesters have been established with mixed microbiome inocula and senescent switchgrass as the feed. These digesters serve as the source material for small-scale cotreatment trials. PhD student Anahita Bharadwaj is using the material from these reactors for cotreatment trials with different milling technologies. Pursuant to goal 2: The larger size conical disc mil at Dartmouth was used for milling during fermentation at 15 Liter scale. The conical mill also functions as a pump and this test demonstrated the ability to mill via an external loop without the need of a pump at the solids loadings up to 60 g/L. The test also demonstrated that milling in this configuration had no adverse effects on a Clostridium thermocellum culture. Once fermented corn stover generated at NREL was used for external-lab ex-situ milling tests. In a continuation of last year we worked with the company Union Process (UP), which commercially develops attrition milling technology. Together with UP we performed milling tests using different milling media showing a clear reduction in particle size during ex-situ milling. The milled material was returned to Dartmouth where the increase in solubilization after milling was determined; from 38.5% up to 95.0% for a 40-minute milling session. Two graduate students from Dartmouth visited with UP in August of 2018. The one-day visit included milling once-fermented corn stover slurries at different time durations. All tests resulted in reduced average particle sizes. The UP attrition mills are able to handle high solids loadings required for this project. In the next few months Dartmouth plans to purchase a mill from UP to do in lab tests with, as well as determining the energy requirements. Members from the Dartmouth team also tested a ceramic disc mill at the Process Development Center at the University of Maine, Orono. The milled once-fermented corn stover increased in solubilization from 38.5% to 69.3% for a 75-minute milling session. Penn State acquired two 80 L bioreactors for the 65 L testing planning for the coming year. These bioreactors are being adapted to run cotreatment experiments in a continuous mode with milling in an external loop. Pursuant to goal 3: Implementing milling in an external loop is expected to be the most efficient cotreatment configuration. However, pumping a high solids fermentation slurry around that external loop is likely to be the most challenging bottleneck. While the material flow characteristics of lignocellulosic biomass slurries improve significantly during cotreatment (see goal 4), solids loading rates of >150 g/L will be challenging. For unfermented and unmilled biomass, solids above 75 g/L are difficult to pump, so this year an undergraduate research team at Penn State did a series of trials with unfermented switchgrass to evaluate different pumping options. Peristaltic and centrifugal pumps did not work well at high solids, but initial trials with a progressive cavity pump (auger type) pump were promising so a progressive cavity pump was purchased. Internal mixing is also problematic at high solids loading, so alternative agitation mechanisms including a ribbon blender are being evaluated at Penn State. Pursuant to goal 4: At Dartmouth the rheological properties of corn stover slurries were tested. Simple shear experiments showed that corn stover slurries (before and after fermentation) are well-described by a power law model and have a power-law exponent of 0.10, which is indicative of shear-thinning behavior. Upon fermenting (with Clostridium thermocellum) a 16 wt.% solids (160 g/L) corn stover slurry, the plastic viscosity decreases dramatically by a factor of 2000, with the first 8-fold reduction occurring in the first 10% conversion. Large amplitude oscillatory shear experiments revealed only minor changes in the slurry's rheological footprint, with the notable change being a reduction in the slurry's critical strain amplitude needed for the onset of nonlinearity. All slurries were found to be elastoviscoplastic, showing an elastic-to-viscous crossover at roughly 100% strain amplitude. Overall, the dramatic reduction in viscosity combined with the relatively-unchanged rheological fingerprint indicates that cotreatment may be energetically favorable; that is, milling and mixing energies are expected to substantially decrease beyond just 10% conversion. Viscosity measurements confirm the hypothesis that the physical properties of corn stover slurries change significantly during fermentation by C. thermocellum, and indicate that the energy expended on overcoming slurry viscosity will be far less for partially-fermented corn stover than for unfermented corn stover. All of these results have been compiled into a paper that has been submitted to Biotechnology for Biofuels and is currently under review. Penn State has also been measuring energy requirements for milling with a data logger connected to the large conical disc mill. This equipment will is being used to test cotreatment energy requirements at different solid contents, particle sizes and fermentation times. Pursuant to goal 5: At Penn State, MS student Isamar Amador-Diaz is developing a techno-economic analysis of mixed culture anaerobic digestion with cotreatment as part of her thesis. This Aspen plus model will inform research questions regarding the cost-efficiency of cotreatment for mixed culture systems. Pursuant to goal 6: Penn State is leading implementation efforts of cotreatment for mixed culture microbial consortia, while Dartmouth is leading implementation effort for pure culture activities. The team is communicating regularly, including biweekly teleconferences and face-to-face meetings several times a year to leverage experiences and coordinate experiments.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Ghosh, S, Holwerda EK, Worthen RS, Lynd LR, Epps BP. Rheological properties of corn stover slurries during fermentation by Clostridium thermocellum.

Progress 08/15/16 to 08/14/17

Outputs
Target Audience:The primary target audience for this study includes university and government researchers, science discovery science and biomass processing companies interested in both fundamental science and commercial application of biomass conversion technology, public and private funding organizations including venture capital and government agencies, and others interested in rural communities and in bio-based economic development. An additional audience is the research community interested in the rheology and fluid dynamics of biomass slurries and how physical properties change with reaction. These various stakeholders are being engaged through presentations at conferences and other venues as detailed in this report. Changes/Problems:Co-PI Xiongjun Shao will leave the project by accepting a faculty position in China. Dr. Evert Holwerda will take over his role in managing collaboration with the Natonal Renewable Energy Laboratory and milling equipment providers. What opportunities for training and professional development has the project provided?Dartmouth Drs. Brenden Epps and Xiongjun Shao gained experience with project management as Co-PIs. 4th year engineering graduate student Mr. Michael Balch (engineering, supported by a fellowship from the Thayer School) and first year engineering graduate student Sanchari Ghose (fluid mechanics, supported by NIFA) devoted 100% of their research effort to the project. Undergraduate Dartmouth Senior Megan Chamberlain did project-related laboratory research full-time during an off-term, and is expected to be a coauthor on a publication to be submitted during the second year of the project. In August, 2016, Epps and Ghosh traveled to NREL to meet with several scientists, including rheology expert Jonathan Stickel. In September, 2016, Epps traveled to U. Illinois to deliver a seminar and meet with Prof. Randy Ewoldt, an esteemed rheologist, to conduct an initial battery of experiments. In May, 2017, Ghosh attended a 3-day short course on rheology organized by TA Instruments, which was very effective in furthering her knowledge and expertise in the field of rheology. In addition, Epps developed a three-week module on rheology for his graduate-level course ENGS 150 Intermediate Fluid Mechanics; Ghosh (and other project student Balch) were students in the course. In June and November 2017, Shao traveled to NREL twice and conducted corn stover fermentation at various scales from 10L to 3,500L. Pennsylvania State University In addition to her regular coursework, Penn State doctoral student Anahita Bharadwaj participated in a microbiome seminar series at Penn State during Fall 2017 and Spring 2018. Ms Bharadwaj and Penn State laboratory technologist Ms. Kay DiMarco traveled to Dartmouth twice to learn cotreatment, fermentation, and biomass characterization methods in the Lynd lab, and Ms. Bharadwaj also traveled to the Oak Ridge National Lab to learn metagenomics methods. Ms. Bharadwaj also traveled the 39th Symposium on Biotechnology for Fuels and Chemicals to both learn from experts and develop her own science communication skills. She won the conference poster competition with her cotreatment poster listed in this report. The anaerobic mixed culture pilot reactor's also served as the subject of a senior design project on pH sensing and control systems. Four female undergraduates formed the team that successfully designed that solution. How have the results been disseminated to communities of interest?In addition to the paper mentioned in "Products" section, and in anticipation of several in-preparation papers, we were active in presenting the cotreatment concept and project results: Lynd, Lee. Low Cost Cellulosic Biofuels: New Questions and New Answers. Green Chemistry Course. Center for Bioethanol Technology, Campinas, Brazil. September 28, 2016. Lynd, Lee. Low cost cellulosic biofuels: New Questions and New Answers. Andlinger Center for Energy and the Environment. Princeton, October 24, 2016. Lynd, Lee. African Human Development: Land, Agriculture, and Bioenergy. Stellenbosch Water Institute and Center for Complex Systems in Transition. University of Stellenbosch, South Africa. November 7, 2016. Lynd, Lee. Low Cost Cellulosic Biofuels: New Questions and New Answers. 2G Ethanol Workshop. Center for Bioethanol Technology, Campinas, Brazil. December 1, 2016. Lynd, Lee. Low-Carbon Fuels. Workshop on Ensuring Energy Security through a Low-Carbon Energy Economy. National Renewable Energy Laboratory. December 8, 2016. Lynd, LR. Low-Cost Cellulosic Biofuels: New Questions in Pursuit of New Answers. Department of Chemical Engineering, Stanford University. January 20, 2017. Holwerda, EK. Evaluation of Multiple Levers for Overcoming the Recalcitrance of Cellulosic Biomass. Genomic Sciences Program Annual Principal Investigators meeting, Arlington, Virginia. February 5-8 Bharadwaj, A. Effect of cotreatment (mechanical disruption) on microbial consortia involved in anaerobic digestion of lignocellulosic biomass. 39th Symposium for Fuels and Chemicals. Society of Industrial Microbiology. San Francisco, CA. May 1-4, 2017. Balch et al., Cotreatment to increase solubilizationof lignocellulose and its impact on microorganisms. BESC retreat.Chattanooga, TN. July 12th,2017. Holwerda, EK. Microbial cellulose utilization and multiple levers for overcoming recalcitrance of cellulosic biomass. LIFE Master Study Delft & Leiden University, Hanover, New Hampshire. July 17 2017. Lynd, LR. Thermophilic consolidated bioprocessing with cotreatment: A potentially disruptive paradigm for biological production of cellulosic biofuels. Society of Industrial Microbiology Meeting, Denver, Colorado. August 3, 2017. Lynd, LR. Thermophilic consolidated bioprocessing with cotreatment: A potentially disruptive paradigm for biological production of cellulosic biofuels. National Renewable Energy Laboratory, August 4, 2017. Lynd, LR. Cellulosic biofuels: Need, hope, assessment, diagnosis, and prescription. Advanced hardwood biofuels meeting. August 10, 2017. Epps, BP. Low-cost cellulosic biofuels: cotreatment mechanics and rheology. University of Illinois Urbana-Champaign, Department of Mechanical Science and Engineering, Fluid Mechanics Seminar, September 16, 2016. Ghosh, S; Epps, BP and Lynd, LR. Rheological properties of corn stover slurries during fermentation to ethanol. Technical poster, Society or Rheology 89th Annual Meeting, Denver, CO, October 8, 2017. Ghosh, S; Epps, BP and Lynd, LR. Rheology of corn stover slurries during fermentation to ethanol. Conference presentation, 70th Annual Meeting of the American Physical Society Division of Fluid Dynamics, Denver, CO, November 19, 2017. What do you plan to do during the next reporting period to accomplish the goals?Goal 1: Two papers are in preparation and will be completed during the next period. The first, addressing goal 1a, looks at the effect of cotreatment on multiple feedstocks (switchgrass, corn stover, and poplar). The second, pursuant to goal 1b, and will report on the effect of cotreatment on the growth of various organisms (E. coli, Z. mobilis, B. subtilis). Goal 2: We will continue to screen the once-fermented material prepared at NREL. We will also continue to work with Union Process on continuous attrition milling to evaluate energy consumption at large scale milling. We have also purchased a conical mill at Dartmouth. We are planning to continue testing milling at less severe conditions with milling-time as the main parameter; we have purchased a conical mill and will use this in our lab for in-situ milling but under intermittent conditions. Via side loop from a bioreactor the mill will be fed, this allows for testing milling time as a parameter as well as intensity of milling. We have adapted an existing 15 L bioreactor with 10 L working volume to be able to work in-line with milling. Goal 3: At Penn State we will be adapting the pilot reactor recirculation system with a high solids pump and cotreatment milling technology to allow intermittent milling at pilot scale. That pilot reactor has been operating in fed-batch mode, and with a cotreatment loop we anticipate switching to operation in a continuous mode which we hypothesize will allow us to exceed solids loading rates of >150g/L. Goal 4: Current viscometric results show that cotreatment is a favorable process due to its decreased viscosity post fermentation. We plan to present these findings in relevant conferences and journals later this year. Our current focus is in planning these publications and developing and executing a comprehensive test plan that covers the dimensions of feedstock (corn stover and switchgrass), biocatalyst (C. therm, fungal cellulace, mixed anaerobic microbiomes), and mechanical tests (as described above). The objective of these experiments will be to characterize the change in material properties as a function of fractional conversion. Final selection of tests and related procedures is underway. These efforts will culminate in two major publications and should fulfill Objective 4a. To our knowledge these will be the most comprehensive description of physical property changes in lignocellulose slurries to date. Subsequent experiments will address Objectives 4b and c. Goal 5: We will continue to reach out to industry to investigate commercial interest and application possibilities. We are considering submitting an SBIR proposal with Enchi Corporation that would include cotreatment. Goal 6: At Penn State a set of studies have been designed to investigate the interactions of inoculum, pH, solids loading rate and cotreatment on the anaerobic digestion microbiome. The aim is to understand whether mechanical disruption has a differential impact on different microbial species.

Impacts
What was accomplished under these goals? Pursuant to goal 1: a. Three substrates have been tested at low feedstock concentrations with continuous ball milling: switchgrass (October and December harvest dates), corn stover, and poplar. For all substrates, significant increase in solubilization was documented in the presence of cotreatment as compared to non-cotreated controls. Fermentations were carried out in the reactor and conditions described in Balch et al 2017. b. To date, five organisms have been examined to determine the effect of cotreatment. We measure substrate consumption in the presence and absence of cotreatment. Clostridum thermocellum is able to continue fermentation in the presence of cotreatment. Escherichia coli is as well. Conversely, Saccharomyces cerevisiae and Zymomonas mobilis both halted fermentation activity in the presence of cotreatment. Tests with Bacillus subtilis are ongoing, but indicate a halting of fermentation as well. Fermentations were carried out in the reactor described in Balch et al 2017. E. coli and B. subtilis were grown on an LB broth and Z. mobilis was grown on the DSM recommended media. All organisms were grown for 24 hours unless halted by cotreatment, which were allowed to continue for an additional 2 days with reinoculation at 48h to ensure fermentation was truly halted. Pursuant to goal 2: Cotreatment studies were carried out using industrial milling equipment. Fermentation of corn stover at a scale of 3,500L with 60 g/L solids was performed at NREL. The fermentation solubilized 38% carbohydrate. The remaining solids were collected and dewatered to about 20% solids. Part of solids were milled at NREL with a disc refiner and a Szego mill. Most of the solids were shipped to Dartmouth to be distributed to various equipment providers for milling tests. Milling tests were performed at 10% solids. The milled material was shipped back to Dartmouth for another round of fermentation to evaluate the effect of milling. Substantially higher solubilization was observed upon refermentation of once-fermented solids following milling in disc refining, Szego milling, and attrition milling. Single pass disc milling (with plate B) increased solubilization from 51% to 66%, single pass Szego milling increased solubilization from 51% to 65%, and two-pass Szego milling increased solubilization to 72%. Attrition milling for 5, 15, 30 minutes incased solubilization from 51% to 74%, 78%, and 83%. We are now working with Union Process in their continuous attrition mills to evaluate power consumption at industrial scale. Cotreatment studies were conducted using corn stover samples acquired from the Great Lakes Bioenergy Research Center (less recalcitrant compared to NREL material) with 80 g/L solids loading. Total carbohydrate solubilization of 60% was achieved without cotreatment. To increase solubilization and test lower severity-milling conditions, intermittent milling in an ex-situ ball mill was applied to 80 g/L corn stover fermentations. Two milling instances were chosen, one session of 5 minutes milling time and one session of 8 minutes. All experiments started with a fermentation and harvesting step to acquire residual solid substrate, then either followed by milling or a second fermentation and harvesting step (this repeated). Solubilization was increased to 86% with two sequential milling and fermentation steps, one with 5 minutes and one with 8 minutes milling. The results of this set of experiments indicated that cotreatment on higher solids loadings is promising and milling time can be significantly decreased. Work was initiated on a pilot reactor system for anaerobic mixed culture fermentation. Two prototype pilot reactors were constructed in a trickle-bed configuration, with a high-solids bed with liquid recirculation. A jacket heater and biogas collection and monitoring systems were installed and tested. A group of four biological engineering undergraduates developed and tested a pH control system. This reactor now routinely operates in fed-batch mode with loading rates of 75 g/L to 125 g/L, and is ready for cotreatment modifications once a milling technology has been selected. Pursuant to goal 3: In the work pursuant to goal 2, we have tested cotreatment milling at 10% solids. The main effort toward this goal will occur after year 1. Pursuant to goal 4: a. Mechanical properties (viscosity, storage and loss moduli, and deformation work) of low-solids-concentration corn stover slurries (2-10% solids by weight) were measured for a two-by-two test matrix of reaction time (unreacted and once-fermented) and milling time (not milled vs ball milled for 5 minutes). Significant effort was invested in developing robust testing procedures to obtain repeatable mechanical measurements. Results showed a dramatic 10-100-fold decrease in the viscosity of once-fermented slurries when compared to unreacted slurries of the same solids concentration, as well as the characterization of these slurries as heavily shear-thinning and showing a power law dependence. Another set of experiments using a test matrix of the milling time (not milled vs milled for 5 minutes) and the particle size (1 mm vs 0.42 mm) is currently being conducted to investigate the degree of dependence of viscosity on particle size and particle stiffness, which is affected by milling time. b and c. The main effort toward these goals will occur after year 1. Pursuant to goal 5: Economic and life cycle analysis was carried out by researchers from Dartmouth in collaboration with colleagues from the National Renewable Energy Laboratory, Argonne National Laboratory, and the University of California at Riverside. An advanced scenario consisting of ethanol production from corn stover by thermophilic fermentation with cotreatment in lieu of thermochemical pretreatment. Compared to current technology, the advanced scenario is projected to offer 8-fold shorter payback period, economic feasibility at 10-fold smaller scale, and greater greenhouse gas emission benefit per ton biomass. Results were published in Lynd et al., Current Opin. Biotechnol. 45:202-211. Pursuant to goal 6: One of the fundamental research questions related to the interaction of cotreatment with fermentation is the potential impact of milling on microorganisms. Preliminary evidence suggests yeast are highly sensitive to milling and may be destroyed or severely set-back by cotreatment, whereas some other organisms (e.g. C. thermocellum) are tolerant. Penn State has initiated a set of experiments to evaluate the effects of both high solids and milling on microbial populations. The initial experiments investigated milling with a mortal and pestal and a blender, and ball milling will be tested next. Preliminary metagenomics analysis of samples with and without milling have not indicated a major impact on major bacterial populations, but we are particularly interested in archaea and will next be introducing a eukaryote (yeast) control. Mixed culture studies have been with switchgrass, introducing an additional feedstock to the project.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Balch, M.L., E.K. Holwerda, M.F. Davis, R.W. Sykes, R.M. Happs, R. Kumar, C.E. Wyman, L.R. Lynd. 2017. Lignocellulose fermentation and residual solids characterization for senescent switchgrass fermentation by Clostridium thermocellum in the presence and absence of continuous in-situ ball-milling. Energy Env. Sci. 10:1252-1261. DOI: 10.1039/c6ee03748h.