Open Access
Tanjore, Deepti
Graduate Program:
Agricultural and Biological Engineering
Doctor of Philosophy
Document Type:
Date of Defense:
June 16, 2009
Committee Members:
  • Thomas Lehman Richard, Dissertation Advisor
  • Thomas Lehman Richard, Committee Chair
  • Dr Patrick Cirino, Committee Member
  • Ali Demirci, Committee Member
  • John Michael Regan, Committee Member
  • Ming Tien, Committee Member
  • ensilage
  • storage
  • pretreatment
  • biomass
  • corn stover
  • fermentation
  • ethanol
  • biofuel
  • lignocellulosic biomass
  • white rot fungi
Ethanol production from renewable sources is a research area of immense interest due to increasing fuel demand and depleting fossil oil and gas resources. Environmental issues like global warming and pollution create concerns about continuing consumption of coal and other conventional fossil fuels. Lignocellulosic biomass, such as agricultural residue, can be fermented to ethanol which can act as a good alternative fuel source. Corn stover is the largest currently available lignocellulosic biomass residue in the US, and thus makes an important substrate to evaluate. The present research aims to employ and enhance biological routes of corn stover conversion to ethanol. An attempt has been made to combine three upstream unit operations: storage, pretreatment, and hydrolysis, in a simple and robust solid substrate fermentation. Ensilage was chosen to be the preferred storage method due to low dry matter losses. White rot fungi which preferentially degrade lignin over cellulose were grown on stover to pretreat the biomass. Cellulase and xylanase producing Trichoderma species were employed in an attempt to convert crystalline cellulose and hemicellulose to glucose and xylose molecules, respectively. Initially, the effect of drying, freezing and refrigeration of corn stover prior to ensilage was studied. This first experiment was essential to see if lab studies being conducted on frozen stover can be extrapolated to real world scenarios where stover is ensiled fresh or dry. The treatments were conducted for 2 and 7 days. No affect of treatments was observed when tested for 2 day time period, while 7 days produced significantly different biomass for each treatment (p < 0.05). The lowest pH levels were observed in control samples (3.73 ± 0.03) after 21 days of ensilage. All other treatments showed similar pH levels averaging 4.26 ± 0.15. Water soluble carbohydrate content was observed to be highest in control samples, i.e., freshly ensiled biomass (7.73 ± 0.26 % g/ g initial DM) and lowest in refrigerated biomass (3.86 ± 0.49 %g/ g initial DM). Addition of enzyme decreased pH values and increased sugar production significantly (p < 0.05). Fiber content of biomass was mostly not affected by the treatments. Dry and frozen corn stover produced comparable results to the control after ensilage; however refrigeration changed biomass properties considerably. Based on these results, we selected freezing as our method of storing substrate between harvest and experimentation, with the expectation that real world results would be as good as or better than our experimental trials. As demonstrated in this initial sample pre-storage study, several parameters are useful to understand the effects of treatments and ensilage on the biomass. These parameters include pH, dry matter loss, water soluble carbohydrates, fiber content (cellulose, hemicellulose, and lignin), monosaccharides (mainly glucose, xylose, mannose, and arabinose), and organic acids (lactic, acetic, propionic, isobutyric, and butyric acids). The methods developed to study these variables are highly dependent on sample handling techniques and extraction procedures. In a second methods-development investigation, the effects of particle size, post ensilage biomass drying temperatures, and modified phenol-sulfuric acid methods were evaluated to identify the best approach for post ensilage parameter measurement methods. A smaller particle size was tested to see if it would decrease the error associated with average sugar measurements. More sugars were produced in biomass with a smaller particle size, but the variance associated with the sugar averages was similar to the larger particle size for the same number of replicates. Since particle size reduction did not improve the quality of data and may create problems with translating results to the real world, larger particle sizes are recommended. Drying at a lower temperature 60°C, compared to the traditional 105ºC, did not lead to significantly higher dry matter loss by possible microbial activity (p > 0.05). This could be due to low pH values of the biomass after ensilage. Also, biomass dried at 60ºC showed higher sugar concentrations, avoiding possible Maillard reactions at higher temperatures like 105°C (p < 0.05), so the lower temperature drying appears preferable. Washing plant material without drying produced higher sugar concentrations than biomass dried at 105°C. For sugar concentration measurements, a modified phenol-sulfuric acid method with lower reaction temperatures has been previously recommended to avoid the formation of phenolsulfonic acid. The acid reduces color intensity and thereby underestimates the sugar concentration. When tested with corn stover biomass in the present study, the modified phenol-sulfuric acid exhibited much higher sugar concentrations than the traditional method (p < 0.0005), improving the sensitivity of the test. From this study, a set of post-ensilage paramenter measurement methods have been developed for use in subsequent studies. After developing pre- and post- ensilage methods, an experiment was designed to combine anaerobic ensilage treatment with an aerobic lignin degradation process in a two step procedure. The following four factors were considered as issues to be addressed in developing an efficient pretreatment-storage process. (a) Economically, for real world scenarios, it is ideal for the fermentation to be conducted in unsterile conditions. However, fungi performance might get a boost if the biomass was sterilized and the inoculated fungi do not have to compete with the indigenous microbial community. Therefore, sterilization was evaluated as an experimental treatment. (b) White rot fungi usually require 4 days to produce lignin degrading enzymes. Two time periods, 7 and 14 days, were tested to study lignin degradation levels along with dry matter losses. (c) Two storage phases, aerobic and anaerobic, were tested in both possible orders. The sequence of aerobic and anaerobic phases was important because of the following two scenarios. Starting with an aerobic phase may lead to substantial dry matter loss and also exposes cellulose and hemicellulose polysaccharides to degradation by the indigenous microbial community during ensilage which is undesirable. Biomass with an aerobic phase after ensilage already has low pH levels which can impede microbial metabolism (see section 3.2). However, exposure to oxygen can lead to the oxidation of organic acids produced during ensilage. This situation could raise the pH levels, which is undesirable. Thus testing both the orders is required to identify the potential sequence. (d) A robust competitive fungal population is required to perform in the preferred unsterile conditions. Three white rot fungi: Phanerochaete chrysosporium, Ceriporiopsis subvermispora, and Pluerotus ostreatus were tested. Sterilization through autoclaving decreased initial pH values by 1 pH unit, suggesting a mild pretreatment effect. However, the final pH values were significantly higher, presumably due to elimination of indigenous lactic acid producing bacteria during sterilization (p < 0.01). Surprisingly, sterilized biomass showed significantly higher dry matter loss (p < 0.0001). This could be due to freely available sugars and lack of competition in the biomass encouraging high metabolic rates for the surviving and inoculated organisms. Contrary to expectations, introducing an aerobic phase after ensilage did not increase pH values (p > 0.5) indicating no loss of organic acids due to oxidation. As expected, 14 days of an aerobic phase caused higher dry matter loss of biomass compared to 7 days, but also produced higher water soluble carbohydrates (p < 0.0001). P. chrysosporium showed best performance among the white rot fungi and produced the highest water soluble carbohydrate levels in unsterile conditions (5.38 ± 0.36 g/g initial dry matter). P. ostreatus exhibited reasonable lignin degradation with dry matter loss comparable to the un-inoculated control treatment. In addition to biological pretreatment, hydrolysis can also be considered during storage. To explore this option, Trichoderma reesei Rut C30 was tested in a sequenced aerobic and anaerobic system. T. reesei Rut C30 produces cellulases and xylanases, which are expected to convert polysaccharides of cellulose and hemicellulose, respectively, to simpler sugars. However, inoculation with the organism failed to produce any additional sugars. In fact, the xylose content of biomass pretreated with the T. reesei Rut C30 was lower than in the control samples. Sterilization, once again, caused higher dry matter loss (24.21 ± 2.52 %g/ g initial DM) compared to unsterile biomass (13.83 ± 1.24 %g/ initial g initial DM). Introducing an aerobic phase after ensilage increased the pH values (5.67 ± 0.23) significantly (p < 0.0005) compared to sequencing the aerobic phase before ensilage (4.79 ± 0.006), indicating loss of organic acids. The optimal hydrolysis temperature is 50°C, while maximum enzyme production for T. reesei RutC30 occurs at 30ºC. Accordingly, a higher temperature, 50°C, was tested for 7 days after 7 days of incubation at 30ºC. As expected, higher temperatures almost doubled the dry matter loss, but did not produce significantly higher water soluble carbohydrates (p > 0.1). Increasing the aerobic phase from 7 to 14 days doubled the dry matter loss and produced significantly higher carbohydrate contents (p < 0.01). The final experiment attempted to combine the three unit operations, storage, pretreatment and hydrolysis into a single stage, two phase process. P. ostreatus was chosen as white rot fungi for this test due to the low dry matter losses and reasonable lignin degradation levels produced by the organism. As previously noted, T. reesei Rut C30 failed to perform in an experiment where in was the only inoculated organism. However, this could be due to the lack of lignin degrading organisms in that system. For this study, two co-cultures: co-culture A (P. ostreatus with T. reesei Rut C30) and co-culture B (P. ostreatus with T. reesei wild type organism) were tested to identify the more robust hydrolytic fungi combinations. Autoclaving was replaced with irradiation as sterilization technique to avoid the previously observed mild pretreatment affect in chapters 5 and 6. As anticipated, the initial pH values were unaffected by irradiation. Sterilization through irradiation resulted in the lowest dry matter loss in control samples, but the highest dry matter losses when inoculated with the co-culture (0.87 ± 0.34, and 1.94 ± 0.29 g/ g initial dry matter). Increasing the number days increased the dry matter loss (p < 0.0001), while having a negative effect on sugar production. Both the co-cultures and the order of treatments had no affect on the fiber content of biomass. Again, the sugar production with inoculated cultures was lower than the controls, indicating the inefficacy of the T. reesei species in this system. A 3 X 3 factorial experiment was designed to study the effect of temperature, moisture and number of days of an aerobic phase. The three variables were tested at three levels. Temperature was tested at 30ºC, 50ºC, 70ºC, moisture was tested at 40%, 55%, 70%, and aerobic phase time period was tested at 7, 14, 21 days. The ideal results, i.e., lowest pH values, lowest dry matter loss and highest sugar production occurred at 30°C, 40% moisture, and 7 days of aerobic phase. Extending the aerobic phase to 14 days produced higher sugar concentration, but by 21 days, the sugar levels started to fall. A statistical model was developed to describe dry matter loss along with lignin degradation (ADL) based on the results from the factorial experiment. The set of experiments designed to develop a simple, low cost solid state fermentation system incorporating storage, pretreatment and hydrolysis answered several questions related to issues including the benefits of sterilization, order of treatment, length of aerobic phase, and temperature and moisture levels. However, the tested organism for hydrolysis, T reesei, was not found to be effective in this system. The designed system of sequenced aerobic and anaerobic phases did have several advantages, and can be developed with other naturally occurring or robust genetically modified organisms that can act competitively to produce the desired result.