COMBINED PHYSICOCHEMICAL AND BIOLOGICAL PROCESSES FOR CONVERTING LIGNIN TO BIOFUEL

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: MCINTIRE-STENNIS
• Reporting Frequency: Annual
• Accession No.: 0222611
• Project Start Date: Jul 1, 2010
• Project End Date: Jun 30, 2014
• Program Code: [(N/A)]- (N/A)

Recipient Organization
RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY
3 RUTGERS PLZA
NEW BRUNSWICK,NJ 08901-8559

Performing Department
Environmental Sciences

Non Technical Summary
This project addresses the forestry research need (6) utilization of wood and other forest products. Lignin is the second largest carbon source on earth after cellulose. It is considered waste product and typically burned after cellulose extraction. Considering the high carbon value of lignin, conversion to methane fuel (and other products) would be a much better use of this natural renewable source material. The greatest challenge of biologically converting lignin to methane fuel is the extremely slow rate of microbial degradation of lignin. In general, unaltered lignin conversion to biofuels cannot occur without some manner of pretreatment. Prior studies showed that, when decomposed to much simpler organic molecules of <1,000 Dalton MW, lignin can be converted by microbes to methane under anaerobic conditions. The decomposition of lignin requires caustic physical and chemical conditions such as strong base and high temperature up to 300 degree Celsius. Pretreatment methods that are milder and less energy intensive are highly sought. We propose to treat lignin with acid/base under supercritical CO2 condition which is above 31.1 degree Celsius and 72.9 atmospheric pressures. We will search for optimal supercritical CO2 conditions, will characterize the molecular properties of the decomposed lignin segments, and will measure the methane conversion efficiency for the decomposed lignin. We plan to patent the technologies developed and publish the scientific findings in peer-reviewed journals. Expected Impact: The proposed study meets the current research need of innovative technologies for producing renewable energy from biomass. As the second largest carbon source on earth, lignin is typically left as waste after cellulose conversion to products. The complex aromatic structure of lignin potentially can be harvested for use as chemical feedstock. In addition, it is highly desired to converting lignin into certain forms of biofuel such as methane that can be easily transported and readily utilized. We will develop a combined physical and chemical technique for rapidly decomposing tightly-knit, three dimensional lignin structures to smaller organic molecules that can later be converted by microbes to methane. Compared to the widely used hot water treatment (200-300 degree Celsius), the technique to be developed in this study is milder, faster, and less energy intensive. If it is successfully developed, our technology could potentially overcome the limits of current technologies to break up complex structures of lignin, advancing both technical and scientific frontiers for converting lignin to proper biofuels.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
123	0699	2020	100%

Knowledge Area
123 - Management and Sustainability of Forest Resources;

Subject Of Investigation
0699 - Trees, forests, and forest products, general;

Field Of Science
2020 - Engineering;

Keywords

lignin and lignin degradation

methane and biofuel

supercritical co2

physicochemical pretreatment

molecular size of organic compounds

Goals / Objectives
Objectives: We propose to employ a chemical treatment process under supercritical CO2 conditions (at or above 31.1 degree Celsius and 72.9 atmospheric pressure) for transforming lignin into smaller organic molecules, which will be converted anaerobically to methane. It is known that diffusion coefficients of chemicals in supercritical CO2 are 2-3 orders of magnitude greater than under ambient conditions. Another major advantage of using CO2 fluid for such treatment is that lignin macromolecules can swell in supercritical CO2 fluid and the organic matrix volume can be increase by as much as 50%. The voids created by swelling allow reactive chemicals diffuse freely, thus should substantially enhance the rate of lignin oxidation. Because of much lower temperature and pressure conditions employed, the proposed supercritical CO2 treatment is much less energy intensive compared to supercritical water or hot water treatments. The specific objectives of the study are: 1) to investigate the optimal supercritical CO2 conditions for decomposing macromolecular lignin to smaller organic segments of appropriate molecular sizes that can be efficiently and rapidly converted to methane; 2) to characterize molecular properties of the lignin segments degraded under different supercritical CO2 conditions; 3) to quantify the efficiency of the biological conversion of the degraded lignin to methane using a microcosm technique. The ultimate goal of this study is to develop a combined physicochemical and biological process for effectively converting lignin to methane. Outputs: We will develop a combined physical and chemical technique for rapidly decomposing tightly-knit, three dimensional lignin structures to smaller organic molecules that can later be converted by microbes to methane. Compared to the widely used hot water treatment (200-300 degree Celsius), the technique to be developed in this study is milder, faster, and less energy intensive. If it is successfully developed, our technology could potentially overcome the limits of current technologies to break up complex structures of lignin, advancing both technical and scientific frontiers for converting lignin to proper biofuels.

Project Methods
The proposed research project will be completed within 3 years. The project will consist of two phases, each having three integrated research tasks. In Phase I, we will use purified lignin as the starting material for pretreatment under various supercritical CO2 and chemical conditions and for the microbial conversion test. Purified lignin will be purchased from Aldrich-Sigma Chemical Company. It has relatively uniform structure and elemental composition, which is key to investigating optimal conditions for decomposition. In Phase II, we will use woodchips as starting materials to further test the supercritical CO2-based process as an advanced technique for pretreating lignin materials. Since woodchips are mixture of cellulose, hemicelluloses and lignin, interference in degradation by other labile components will be closely monitored. In Task 1 we will investigate degradation of lignin under different supercritical CO2 conditions (by varying temperature and pressure) with or without addition of acid or base. A 2-vessel simultaneous oven-based extraction system (Model 7071) purchased from Applied Separations (Allentown, PA) will be used as the supercritical CO2 reactor. In Task 2, we will characterize the molecular properties of the small organic molecules derived from lignin degradation under both supercritical CO2 and normal treatment conditions. Conversion rates (mass of the produced small organic molecules versus total lignin mass) will be quantified using a total dissolved organic carbon analyzer. It is assumed smaller organic molecules degraded under both supercritical CO2 and normal conditions are highly water soluble. The percentage of organic carbon in dissolved form on the total organic carbon basis is thus indicative of the conversion efficiency under specific treatment conditions. In Task 3, we will conduct biological tests for converting degradation products to methane under anaerobic conditions. We will use a Biochemical Methane Potential (BMP) assay to determine the extent of lignin conversion to methane as mediated by an anaerobic microbial consortium. The assay will compare the rate and extent of methane formation between lignin treated under different supercritical CO2 conditions with lignin that has not been treated and with an active control containing acetate/propionate. The total gas and methane production in each microcosm will be monitored using a gas chromatography (GC) regularly over a 2-3 week period of time. After completion of the studies outlined above for purified lignin materials, we will select woodchips as the starting materials and will repeat the three research tasks in Phase II. The selection and characterization of woodchips will be performed in the second half of the second year of the project. The procedures used in Phase I will be exactly followed. Special attention will be paid to the effect of cellulose and hemicelluloses commonly present in woodchip on the conversion rate during supercritical CO2 treatment and the efficiency of biological conversion of lignin to methane.

Progress 07/01/10 to 06/30/14

Outputs
Target Audience: Undergraduate students and graduate students at Rutgers University and several Chinese universities. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project provides hands-on experience to the undergraduate and graduate students who have worked on the experiments. The studentshave been trained on thetechniques for preparing and characterizing nanomaterials. How have the results been disseminated to communities of interest? In 2014, the PI attended a workshop on biomass and biofuel held in Guangzhou, China. The results of this study (although not so promising) were discussed in the oral presentation. It attracted attention from another research group who has studied pulverized lignin as fiber supplement. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The main goal of the physicochemical treatment of lignin materials is to enhance the biodegradability of lignin by breaking down the material into much smaller particles.Our study showed that lignin materials can be pulverized with a wet ball mill technique into particleswith hydrodynamic diameters of100-200 nanometers. However, the powdered lignin materials did not show improved biodegradability under anaerobic conditions. It appears that the pulverized sub-mircon sized lignin is still very difficult to be converted biologically to methane or alcohols. Lignin with much small sizes (on the order of nanometers or molecular weightsmaller than 1000 Dalton) may be required for rapid degradation by anaerobes. In 2013 and 2014, we have focused on breaking up of lignin to nanoparticles. In the previous year (2012), we employed a wet ball mill technique to break the lignin materials of 5 to 10 micrometers (um) to about 200 nanometers (nm) within 1-2 hrs. In the past two years (2013-2014), we have explored a spectrum of milling conditions with lignin pretreated under very low temperatures (-80 Celsius) for breaking up the organic materials to the sizes of sub-microns. Lignin materials were stored in ultra-low freezer to simulate a freezing condition that may increase the rigidity of the organic matrix. The pretreated lignin materials were ground in a solution phase over extended time period (accumulatively to 24 hrs in a intermittent mode) with the wet ball milling system. The resulting aqueous suspensions were characterized using a light scattering method for characterizing the dynamic sizes of the suspended particles. Our results showed that the sizes of the lignin were of 100 nm range, which is much smaller than the sizes of the lignin materials ground under normal conditions (observed in 2012). The smaller sizes of lignin particles are much desired since lignin macromolecules are tightly knit and are highly resistant to microbial degradation, a key step for converting lignin-based biomass (such as wood) to biofuel consisting of low molecular weight organic compounds such as methane or alcohols.However, the subsequent microcosm study for investigating the biodegradability of the ground lignin materials of 100 nm did not show improved rates of degradation.

Publications

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Undergraduate students and graduate students at Rutgers University and several Chinese universities, who took the courses or attended the seminars given by the PI. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? In 2014, we will complete three tasks related to the project. Firstly, we will continue characterizing the ground lignin materials of 100 nm sizes using spectroscopic (FTIR and 13C-NMR) methods. We believe the milling process is largely physical, hence the major functionality of lignin should remain unchanged. However, the spectroscopic properties may provide certain evidence that the cleavage of lignin may start at weak bonding of the organic macromolecules. Secondly, we will conduct microcosm study for investigating the biodegradability of the ground lignin materials of 100 nm. We will use a Biochemical Methane Potential (BMP) assay to determine the extent of lignin conversion to methane as mediated by an anaerobic microbial consortium. The assay will compare the rate and extent of methane formation between lignin ground to 100 nm sizes with lignin that has not been treated and with an active control containing acetate/propionate. The total gas and methane production in each microcosm will be monitored using a gas chromatography (GC) regularly over a 2-3 week period of time. Thirdly, we will compile the information obtained from this project, and will try to draft a manuscript for publication.

Impacts
What was accomplished under these goals? The main goal of the physicochemical treatment of lignin materials is to enhance the biodegradability of lignin by breaking down the material into much smaller particles. In 2013, we have focused on breaking up of lignin to nanoparticles. In the previous year (2012), we employed a wet ball mill technique to break the lignin materials of 5 to 10 micrometers (um) to about 200 nanometers (nm) within 1-2 hrs. In the past year (2013), we have explored a spectrum of milling conditions with lignin pretreated under very low temperatures (-80 Celsius) for breaking up the organic materials to the sizes of sub-microns. Lignin materials were stored in ultra-low freezer to simulate a freezing condition that may increase the rigidity of the organic matrix. The pretreated lignin materials were ground in a solution phase over extended time period (accumulatively to 24 hrs in a intermittent mode) with the wet ball milling system. The resulting aqueous suspensions were characterized using a light scattering method for characterizing the dynamic sizes of the suspended particles. Our results showed that the sizes of the lignin were of 100 nm range, which is much smaller than the sizes of the lignin materials ground under normal conditions (observed in 2012). We are currently conducting microcosm study for investigating the biodegradability of the ground lignin materials of 100 nm.

Publications

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: The results of this study were presented in multiple seminars at universities and institutions of China during PI's visits in 2012. Some data were included in course lectures that were delivered to undergraduate and graduate students. One research paper was drafted and submitted to a journal for peer review. PARTICIPANTS: PI: Dr. Weilin Huang; Collaborators (2): Dr. Lily Young, Department of Environmental Sciences, Rutgers University; Dr. Ping'an Peng, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Wushan, Guangzhou, China; Graduate Students (1): Qiang Huang. TARGET AUDIENCES: Undergraduate students and graduate students at Rutgers University and several Chinese universities; Research scientists in the area of biofuel and bioenergy. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
In the past 12 months, we focused on two separate studies that were designed for converting lignin materials to methane. The first study was a laboratory experiment which was set out to break lignin particles to nanoparticles. The lignin materials used in this study were purchased from Aldrich-Sigma Chemical Company. They have particle sizes of 5-10 micrometers with molecular weight on the order of 1 million Dalton. Using a wet ball mill system, lignin was ground at room temperature to 180-200 nanometers within 1-2 hours. Characterization of particle sizes showed that lignin particle sizes were rapidly decreasing to sub-micron diameters within the first 20 minutes of milling. After 60 minutes of milling, lignin was gradually broken down to 200 nanometers. Further milling had no measureable change of particle sizes, suggesting that lignin of 180-200 nanometers may be highly elastic and flexible. We plan to use the ground lignin materials for testing their biodegradability and efficiency of converting to methane. In a second study, we reviewed key pathways and important enzymes that are responsible for biodegradation of lignin related aromatic compounds under anaerobic conditions. It is known that anaerobic monoaromatic degradation can be initiated in a number of different ways, but the signature metabolite for these pathways is benzoyl-CoA. Chemicals with different upstream degradation pathways can therefore be compared by targeting the downstream benzoyl-CoA pathway. Different steps of the benzoyl-CoA pathway could be used as a biomarker, however the ring opening hydrolase, encoded by the bamA gene, is ideal because it is detected under a number of respiratory strategies, including denitrifying, iron reducing, sulfate reducing, and fermentative conditions. In this study, we systematically reviewed bamA gene with respect to usage as a biomarker in enrichment cultures and environmental DNA extracts. When we compared the bamA gene diversity of several sites from the literature, we found that there was little similarity between them. The bamA gene has a wide distribution in the environment, although thus far there does not appear to be a correlation between location or initial substrate and the gene sequence. Given the number of potential upstream inputs from natural and manmade monoaromatic compounds, the benzoyl-CoA pathway and the bamA gene play an important role in the global carbon cycle that has thus far been overlooked. In our next phase of study, we will pay particulate attention to the bamA gene in the conversion of nano-size lignin to methane under laboratory conditions.

Publications

• No publications reported this period

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: We proposed to employ a chemical treatment process under supercritical CO2 conditions (at or above 31.1oC and 72.9 atmospheric pressure) for transforming lignin into smaller organic molecules, which would be converted anaerobically to methane. We have treated lignin materials using a supercritical CO2 reactor at different temperature and pressure conditions. For comparison, we also prepared lignin materials using NaOH and H2O2 at different conditions. A total of 22 treated lignin samples were generated and used for quantifying methane production rates using the Biochemical Methane Potential (BMP) assay. The results were shared in an undergraduate class, and will be presented in conference. PARTICIPANTS: Dr. Weilin Huang (Principal Investigator), Dr. Lily Young (co-PI), Dr. Nengwu Zhu (visiting scientist), Mr. Wanhui Zhang (visiting graduate student) TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: In next year of study, we plan to explore the treatment procedures in two different ways. Firstly, we will use a modifier (methanol) to increase the polarity of the supercritical CO2 fluid. With the modifier, the CO2-MeOH fluid should better penetrate the interior of lignin molecules, hence enhancing the decomposition reactions. This treatment is currently undergoing in our laboratory. Secondly, we will employ a high-speed ball milling technique that can break organic particles to nanosize materials. For this second treatment, we will use both pure lignin and woodchips. The high-speed ball mill is available at Food Science Department (contact: Dr. Qingrong Huang). We are currently discussing the procedures and will likely start the test in April.

Impacts
The results of the BMP assays showed the total methane production for all lignin samples treated differently and active control reactors. Among all the systems tested, the active control of acetate/propionate had the highest methane production. The sterile control reactors had no methane production as expected. The treated lignin samples had methane production falling between the two types of control systems. This is consistent with our expectations that the acetate/propionate in the active control reactors are fully bioavailable for producing methane under the testing conditions whereas sterile control reactors should not produce measureable methane since methanogenic microorganisms were killed in the system. For all treated lignin samples, the general trend was that all treated lignin samples had methane production higher than the background reactor, indicating that the carbon of the treated lignin was partially bioavailable and could be converted to CH4 under the testing conditions. It appeared that the lignin materials treated only with the supercritical CO2 fluid had the same methane productions as the original lignin without any treatment, suggesting that the supercritical CO2 treatment may not be effective for enhancing methane production. However, the lignin materials double treated by the supercritical CO2 followed by H2O2 at either room temperature or 80oC had high methane production than the original lignin and the lignin materials treated only with the supercritical CO2 fluid, suggesting H2O2 treatment was very effectively enhancing methane production. Currently, we are searching for the interpretation of the data, and further modification of treatment procedures will be made to enhance production of methane from treated lignin materials.

Publications

• No publications reported this period

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Over the past six months, we have developed a standard operating procedure for conducting chemical modification of lignin using the newly purchased supercritical CO2 extractor. Three different reaction chambers were configured for loading lignin and other organic matter samples ranging from 50 mg to 100 g under various temperature and pressure conditions. The smaller sizes will be used for searching the optimal temperature and pressure conditions for modifying lignin and other organic matter in the supercritical fluid. The larger sizes will be used to generate larger batches of modified organic matter samples for subsequent biogas production by anaerobes. Meanwhile, we have started chemical treatment process under ambient pressure condition. Lignin and hemicellulose samples were treated with acids and bases at different temperatures (25, 50, 60 and 70oC). The treated samples will be analyzed for their total dissolvable organic carbon contents. The data, along with the data to be obtained from supercritical fluid treated samples, will be used for assessing the treatability of lignin and other organic matter under supercritical conditions. In the following six months, we will start the treatment of lignin and hemicellulose under supercritical CO2 conditions. The samples will also be used for testing of biogas production under anaerobic conditions. PARTICIPANTS: Dr. Nengwu Zhu, an associate professor in environmental chemistry from South China University of Technology, is working on this project at Rutgers University as a visiting scholar. His 12-month visit from June 2010 is funded by the Chinese Scholar Council. He spends 50% of his time on this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Although the research project is still in its early stage, the experimental study has showed that the supercritical CO2 extractor newly purchased by our laboratory can be adjusted to treat lignin and other organic matter. Our goal is to develop the supercritical CO2-based technology for modifying and partially decomposing lignin and other organic matter such that they can be rapidly converted biologically to methane fuel.

Publications

• No publications reported this period