Source: AUBURN UNIVERSITY submitted to
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Dec 1, 2009
Project End Date
Nov 30, 2013
Grant Year
Project Director
Wang, J.
Recipient Organization
Performing Department
Non Technical Summary
In view of rising concerns over energy sustainability and global warming, the need for biofuels is expected to increase sharply in the coming years. At present, the U.S. biofuels market is dominated by corn-derived ethanol. However, substantial future growth of fuel ethanol will depend upon the development of cellulosic ethanol processes due to the following reasons: (1) further growth of corn ethanol is restrained by the availability of agricultural land, water resources, and the food vs. fuel tradeoff which is already causing concern; (2) it is estimated that cellulosic ethanol reduces both energy input and greenhouse gas emission by over 85% compared to corn ethanol; and (3) cellulosic biomass is the most abundant and inexpensive renewable feedstock for ethanol production. Due to its high abundance in cellulosic biomass, xylose utilization is critical for commercial cellulosic ethanol processes. To make cellulosic ethanol the long-term renewable energy source, besides the need of cost-effective enzymes for cellulose/hemicellulose hydrolyzation, there is a need of optimal microbes that can ferment xylose as well as glucose into ethanol with a high yield and productivity. To address the later, numerous microorganisms have been constructed in the last two decades to simultaneously ferment mixture of glucose and xylose. In parallel to the on-going improvement of the recombinant strategies, we believe that a viable alternative for glucose/xylose co-fermentation is the coculture strategy, as in nature all environmental bioconversions are catalyzed by mixed microbial cultures. In addition, the coculture systems offer greater flexibility and economic advantages compared to monoculture. Specifically, cocultures provide more versatile metabolic machinery due to a larger pool of collective genes, and tend to be more robust to different disturbance such as variations in feedstock composition and operation condition. At the same time, there are certain challenges associated with coculture systems and limited research has been done to understand coculture systems. In this project, we designed and customized a novel bioreactor to facilitate the investigation of coculture systems. Through experimental design, we propose to optimize the fermentation of glucose/xylose mixture using the coculture of S. cerevisiae and P. stipitis, and to develop a mathematical model to describe the dynamic interactions between the two strains.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Goals / Objectives
The overall goal of the proposed study is to identify the optimal conditions for fermenting mixture of glucose and xylose using coculture of S. cerevisiae and Pichia stipitis, as well as to develop an unstructured model to describe the dynamics of the coculture system. The supporting objectives are: 1) to setup and test the bioreactor; 2) to identify the optimal conditions for ethanol production using the coculture; 3) to develop an unstructured dynamic model for the co-culture system; and 4) to test the hypothesis that adaptation improves strains' ethanol tolerance.
Project Methods
There are three major challenges associated with ethanol production using the proposed coculture system: the incompatible optimal DO levels of S. cerevisiae and P. stipitis for ethanol production; diauxic growth of P. stipitis on the mixed substrate of glucose and xylose; and the weak ethanol tolerance of P. stipitis. To address these challenges, first we have designed and customized a novel bioreactor that enables us to independently control the DO levels as well as to track the cell population changes for both strains; in addition, we propose to operate in the pseudo-continuous mode (continuous fermentation with cell retention). In this way, by adjusting the dilution rate, we can maintain the bulk glucose concentration at a very low level to ensure that P. stipitis consumes xylose only. More importantly, the proposed pseudo-continuous fermentation provides an ideal condition for cell adaptation, and enable us to test the hypothesis that adaptation improves strains' resistance to inhibitors such as ethanol. In this project, we perform mixed-level fractional factorial experimental design to study the coculture system systematically and quantitatively. In addition, unstructured dynamic model will be developed for the coculture system, which will provide valuable information to help elucidate the metabolic interactions between the two different strains.

Progress 12/01/11 to 11/30/12

OUTPUTS: The following major tasks were conducted in the third year. 1. Modification of the two-chamber bioreactor for co-culture fermentation We spent significant amount of time solving the equipment leakage and cell retention module blockage problems after switching the reactor material to polycarbonate in order to stand high temperature during autoclave. Subtask 1: bioreactor leakage. The biggest difficulty with the new material is to find suitable glue to put different parts together without leakage. After extensive experiments with different glues and annealing/curing processes, we found that by applying silicone gel on top of Weld-on, a type of cement, to seal the connection, we solved the leakage problem. The developed reactor has been tested using 8 runs of autoclave sterilization and initial co-culture run which lasted over a week. In addition, we adopted the pseudo-continuous fermentation from the beginning of the experiment which enables the simultaneous control of both cell growth and ethanol production, and complete utilization of glucose and xylose. Subtask 2: cell-retention module redesign. To address the cell retention module blockage caused by bubbles generated closed to membrane surface, we conducted several different designs and modifications and a vertically positioned cell retention module was found to be the best. The selection of appropriate membrane for the cell retention module was conducted by testing and comparing outflow rate performances over extended period. Supor0.8 performs the best among tested membranes due to its superior material type and high water flow rate property. The outflow rate measurements showed the stability of the flow rate. 2. Optimization of growth condition for both strains. In order to determine the experimental condition that optimizes cell growth and ethanol production at the same time, we performed exploratory experiments to determine the growth phase condition for the co-culture experiment. We found that in order to control cell grow rate, for S. cerevisiae, we have to control sugar consumption rate, while for S. stipitis, we can control OTR. In our initial co-culture experiment, we confirmed our findings. 3. Accurate measurement and control of oxygen transfer rate. Existing literatures on the effect of OTR to xylose fermentation of S. stipitis are inconsistent mainly because of the lack of accurate control of OTR. To obtain accurate measurement and control of OTR, we are systematically investigating the effect of different factors on OTR, including cell density, agitation speed, and substrate concentration. We modified existing dynamic method to measure the OTR by considering the mass transfer between the reactor head space and the broth as well as the mass transfer between the bubbles and the broth. Experimental work is still ongoing. In order to achieve an accurate control of the oxygen transfer rate (OTR) in the co-culture system, we also developed a gas mixing apparatus to maintain the fixed overall gas flow rate while changing the ratio of air and nitrogen, which has been verified experimentally. PARTICIPANTS: Participants: Jin Wang, PhD, PI, Auburn University, led the research. Led the design of the two-chamber bioreactor; design of experiments; taught the students on data analysis and presentation preparation; paper writing and revision. Q. Peter He, PhD, co-PI, Tuskegee University, co-led the research. Led the design, modification of the two-chamber bioreactor; constructed the pH, temperature control system; design of experiments; paper writing and revision. Meng Liang, PhD student at Auburn University. Carried out some of the designed experiments, constructed the two-chamber bioreactor, carried out the data analysis. Min Hea Kim, PhD student at Auburn Univesity. Carried out some of the designed experiments, helped with the construction of the two-chamber bioreactor, helped with data analysis. Alyson Charles, undergraduate student at Auburn University. Helped with preparation of experiments. Collaborators: Brian Schwieker, technician at Department of Chemical Engineering, Auburn University. Helped with the construction of the two-chamber bioreactor TARGET AUDIENCES: Researchers in the field of biofuel production through biological pathways. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

The following major findings were obtained for the tasked performed during the third year. 1. Pseudo-continuous operation enables the simultaneous control of cell growth and ethanol production of both strains. It is worth noting that neither continuous operation nor batch operation would allow us to do so. 2. The mass transfer between the reactor head space and broth is significant and cannot be ignored in order to obtain accurate measurement of OTR. 3. Weld-on plus silicone gel can provide desired strength and sealing of polycarbonate reactor.


  • Liang M., Kim M.H., He Q.P. & Wang J. (2012), Impact of pseudo-continuous fermentation on the ethanol tolerance of Scheffersomyces stipitis, Journal of Bioscience and Bioengineering, under revision;
  • Liang M., He Q.P., Jeffries T.W. & Wang J. (2012), Elucidating xylose metabolism of Scheffersomyces stipitis by integrating principal component analysis with flux balance analysis, 2013 American Control Conference, under review;
  • Liang M., Kim M.H., He Q.P., Jeffries T & Wang, J., metabolic network modeling of redox balancing and ethanol production in Scheffersomyces stipitis, AIChE annual meeting, 2012, Pittsburgh, PA.;
  • Kim, M.H., Liang, M., He, Q.P. & Wang, J., Efficient bioconversion of glucose/xylose mixtures for ethanol production using a novel co-culture system, AIChE annual meeting, 2012, Pittsburgh, PA;
  • Kim, M.H., Liang, M., He, Q.P. & Wang, J., Pseudo-Continuous Fermentation - an effective way to study the dynamics of co-culture systems. 34th SBFC, New Orleans, 2012;
  • Liang M., He Q.P., and Wang J., Reconstruction of the central carbon metabolism of Pichia stipitis, 34th SBFC, New Orleans, 2012;
  • Liang M., Kim, M.H., He Q.P., and Wang J., Elucidation of redox balance in xylose fermentation with Pichia stipitis, 34th SBFC, New Orleans, 2012

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

OUTPUTS: 1.Development of the two-chamber bioreactor for co-culture fermentation In the preliminary work we have built a prototype of the two-chamber bioreactor which enabled us to test out the mass transfer properties of different membranes. In the past year, we continued to test and modify/improve the bioreactor to address the following practical aspects of bioreactor operation when cells are present: sterilization, agitation, air distribution, temperature control, sampling etc. The major activities are summarized below. Sterilization: the bioreactor is originally made from transparent PVC material, which is durable and very easy to work with, but cannot stand for the standard autoclave condition. We developed a two-step sterilization process which can provide satisfactory sterilization, and prevent PVC material from getting opaque and deformed. However, the sterilization results are not consistent for every run. After investigation we decided to switch to polycarbonate. The new difficulty is gluing different parts of the bioreactor together. After extensive research and trials, we solve the problem with epoxy material. We modified the design of the bioreactor to facilitate the assembling/gluing process. After testing various curing conditions, we developed the appropriate annealing and curing processes and successfully built a two-chamber co-culture bioreactor with polycarbonate material. It has successfully passed the test for leakage and autoclave. Agitation and air distribution: We first used a string bar driven by an external magnetic field to agitate the system, and modified two commercial magnetic stirring systems in order to provide a strong external magnetic field. However, compared to the propellers driven by external motors, the magnetic bar stays at the bottom of the reactor which provides limited agitation and very limited air redistribution.Aafter extensive search, we identified a commercial system which uses propeller driven by external magnetic field, and we modified the commercial system to fit our purpose. The new agitation system has been tested to provide satisfactory agitation and air redistribution. Temperature control system: Because of the special design of the bioreactor, there is no commercial temperature control system available that can be uses directly. To control the temperature, we built a temperature control system in house by using the heating tapes and a commercial PID controller. We have tested that the developed control system can maintain the reactor temperature within 1.5 C of the target. 2.Proof-of-concept run of the co-culture experiment We first conducted separate batch experiments to determine the delay time associate with two different strains, and calculated the appropriate inoculum concentration for the co-culture experiment, which would give comparable cell density of both strains at the end of the batch fermentation phase. We then successfully conducted a proof-of-concept run of the co-culture experiment. The co-culture experiment consists three phases: batch aerobic growth phase, batch anaerobic fermentation phase, and pseudo-continuous fermentation phase. PARTICIPANTS: 1. Jin Wang, PI, led the research, worked with the co-PI and the graduate students to modify the design of the bioreactor, design the batch and pseudo-continuous fermentation experiments and analyze the data. 2. Q. Qinghua He, co-PI, led the research, worked with the PI and the graduate students to modify the design of the bioreactor, design the batch and pseudo-continuous fermentation experiments and analyze the data. 3. Meng Liang, graduate student, involved with bioreactor design and manufacture and carrying out some of the experiments. 4. Min Hea Kim, graduate student, worked on the bioreactor design and manufacture, experiment design and carrying out most of the experiments. 5. Stewart Philips, undergraduate student, involved with bioreactor manufacture and carrying out some of the experiments. 6. Alison Charles, undergraduate student, involved with carrying out some of the experiments. TARGET AUDIENCES: researchers in the field of fermentation, especially those in biochemical conversion of the lignocellulosic biomass to ethanol. PROJECT MODIFICATIONS: The progress is slower than what was planned due to two reasons: 1. the graduate student who has been working on this project was diagnosed with cancer and had to be away for 8 months for treatment. 2. the unexpected difficulty related to develop the new two-chamber bioreactor. As a result, a one-year no cost extension was requested and granted.

The proof-of-concept run of the co-culture experiment confirmed our hypothesis which is the foundation of the final project goal, i.e., with pseudo-continuous fermentation, we can achieve complete consumption of both glucose and xylose simultaneously. Our results appear to be the first continuous co-culture experiment to achieve complete consumption of xylose under continuous operation; all existing results on continuous co-culture approaches only achieve partial consumption of xylose.


  • Kim, M.H., Liang, M., He, Q.P. & Wang, J. (2011), Pseudo-Continuous Fermentation Using a Novel Bioreactor to Facilitate the Study of a Co-Culture System for Ethanol Production. AIChE annual meeting, Minneapolis, MN.
  • Liang M., He Q.P., and Wang J. (2011), Improving Ethanol Tolerance of Pichia stipitis via Continuous Fermentation with Cell Retention, Journal of Industrial Microbiology & Biotechnology, under review.

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


The following major findings were obtained for the tasked performed in the first year. 1. Pseudo-continuous fermentation of single culture Pichia stipitis During the prolonged pseudo-continuous fermentation experiments, we confirmed that our hypothesis is correct, i.e., with pseudo-continuous operation, we can eliminate the possible wash-out that is common in continuous fermentation with P. stipitis, which makes it possible to test out different operation conditions; we also confirmed that the in-house developed cell retention module can provide satisfactory performance - several improvements have been made to prevent its breakage and to make sure that P. stipitis cells do not accumulate on the cell retention part so that the filtration resistance can be maintained at about the same level for the prolonged experiments; finally, we qualitatively tested the effects of different oxygen transfer rates, and collected useful information for future co-culture experiments. 2. Validation and investigation on the improved ethanol tolerance of Pichia stipitis gained through pseudo-continuous fermentation Exp1: conduct ethanol tolerance test. We found that adapted cells show significantly improved ethanol tolerance, especially at high ethanol concentration: when exposed to medium with 60g/L ethanol, the viability of adapted cells shows 8 folds and 7 folds increase compared to unadapted cells, for glucose-based and xylose-based culture media, respectively. Exp2: measure ethanol induced leakage of 260-nm-light-absorbing compounds. We found that compared to the unadapted cells, the adapted ones showed significantly reduced leakage of UV-absorbing compounds. Specifically, when exposed to 25g/L of ethanol for 6 hrs, the leakage (OD260) for the unadapted cells and the adapted cells are 0.87 and 0.21 for glucose-based substrate, and 0.86 and 0.20 for xylose-based substrate. Exp3: measure passive proton influx and active proton extrusion. The adapted cells shown significant lower passive proton influx (1.7 and 1.8 mmol/min/mg for glucose-based and xylose-based substrates for 120g/L of ethanol), compared to unadapted cells (15 and 19mmol/min/mg for glucose-based and xylose-based substrates for 120g/L of ethanol). In addition, the adapted cells showed increased proton efflux rate compared to unadpated cells. Both results on passive proton influx and active proton extrusion show that adapted cells exhibit better resistance to ethanol, probably due to improved ATPase activity as well as reduced plasma member permeability to proton. Exp4: determine the ethanol limitation to growth. For unadpated cells, the limiting ethanol concentrations are 57.29g/L and 50.24 g/L for glucose-based and xylose-based substrates, respective. For the adapted cells, the limiting ethanol concentrations were improved to 73.40g/L and 66.38g/L respectively. These numbers correspond to 28% and 32% of increase for the adapted cells.


  • 1. Liang M., He Q.P. and Wang J. (2009), Improving Ethanol Tolerance of Pichia stipitis Via Continuous Fermentation with Cell Retention, AIChE Annual Meeting, Nashville, TN
  • 2. Liang M., He Q.P. & Wang J. (2010), Pseudo-continuous operation: an effective way to improve ethanol tolerance of Pichia stipitis in hexose/pentose fermentation, The Society for Industrial Microbiology Annual Meeting, San Francisco, CA.