Progress 10/01/01 to 09/30/06
Outputs
The objective was to produce high value specialty chemicals from condensed corn solubles (CCS) and other dry mill byproducts. Our approach was to 1) acclimate cultures to grow on these byproducts, 2) optimize the medium formulation, 3) create improved strains, 4) develop effective product recovery methods, and 5) conduct scaled-up fermentations. Our initial focus was to produce succinic acid using Actinobacillus succinogenes ATCC 55618. Succinic acid is an important chemical intermediate with an annual market of over $4 billion. We gradually acclimated A. succinogenes to CCS, with similar cell mass counts (109 CFU/ml) to the lab medium. However the lag phase was 6-12 h longer, and the microbe produced little succinic acid on CCS. Therefore this effort was discontinued, and the focus was switched to polyhydroxyalkaonate (PHA). PHA is a biodegradable polymer that can mimic compounds ranging from hard rubber to elastic type polymers. In shake flask trials, Pseudomonas
resinovorans ATCC 14235, P. putida KT217 and P. putida KT2442 were acclimated to grow on CCS. We then determined that 240 g/L CCS was the best formulation, and that glucose, glycerol, octanoate, and corn oil could be utilized as carbon sources. When soapstock was added initially, it inhibited growth, but it could be added in a fed-batch mode later in fermentation. P. putida KT217 yielded the highest cell mass (1.3-2.6 g/L) and PHA productivity (5.1-8.1 mg/L/h) in shake flask trials, thus it was used thereafter. We determined the optimal C:N:P ratio for cell growth and PHA production, using aerated 1 liter Erlenmeyer flasks. The best combination was 400 g/L CCS, with 1.7 g/L ammonium hydroxide and no additional phosphate. This resulted in cell dry weight increasing to over 20g/L. Trials in a 5 L bioreactor allowed continuous pH control at 7, which maintained cell viability and allowed nitrogen limitation to occur, triggering PHA accumulation. Using this basal medium, we conducted
carbon feeding trials to trigger PHA production. Feeding sterile glycerol water (at 1.5-3.5%) resulted in 25-30% PHA and 84 mg PHA/L/h after 96 h. Adding sunflower soapstock caused excessive foaming, causing us to reduce aeration, which slowed PHA production. In these trials 15 g/L sterile soapstock was added at each feeding. Cell dry weight was only 50% of the normal level, while PHA content was 15-20% of the CDW, with a productivity of 30 mg PHA/L/h. Several antifoams were evaluated, and some reduced viable counts by over 50%. The antifoam Biospumex (Cognis company) was effective, with minimal cell damage. The traditional method to recover PHA is soxhlet extraction with chloroform, followed by evaporation and methanol precipitation. We investigated supercritical fluid extraction with CO2 and ethanol. After evaluating a matrix of temperatures, pressures, and mole fractions of CO2 and ethanol, we determined optimal operating conditions of 9,000 psi, 60 C, and 15% ethanol resulted in a
98% pure PHA (based on GC/FID and GC/MS). It also decreased extraction time by 10 fold, reduced solvent usage by 15 fold, and eliminated chemical precipitation compared to the soxhlet method.
Impacts
The rapid growth of the corn ethanol industry has placed considerable pressure on the price/value of DDGS and condensed corn solubles. Alternative uses for these co-products is needed to maintain profitability. Production of high value biomaterials, such as polyhydroxyalkanoates, from these co-products could bolster the economics of corn ethanol production.
Publications
- Javers, J., W. Gibbons, D. Raynie, and F. Halaweish. 2007. Extraction of Medium Chain Length Polyhydroxyalkanoates from Pseudomonas resinovorans by Ethanol Modified Supercritical Fluid Extraction. J. Polymer Chemistry. (submitted).
- Gibbons, W.R. 2006. Production of polyhydroxyalkanoates from industrial byproducts. Celebrating Research, Scholarship and Creative Activities at SDSU. January, Brookings.
- Javers, J. 2007. Production of PHA from agricultural byproducts. Ph.D. Dissertation, South Dakota State University, Brookings, SD.
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Progress 01/01/05 to 12/31/05
Outputs
Previously we determined that Pseudomonas putida KT217 achieved the highest cell mass and PHA productivity on a condensed corn solubles medium. We also determined the organism could utilize soapstock and glycerol water, and identified an effective antifoam agent. In 2005 we sought to determine the optimal C:N:P ratio for cell growth and PHA production. Initial trials in aerated shake flasks (800 ml) compared CCS levels of 100-600 g/L without additional nutrients. The 200 and 400 g/L CCS levels yielded significantly higher cell dry weight, while the 100 and 400 g/L CCS levels had the highest growth rate. Thus, the 400g/L level was chosen as optimal. Next we investigated ammonia supplementation and found that addition of 1.7 g/L ammonium hydroxide increased final CDW significantly, to over 20g/L. The nitrogen was utilized in these trials along with the majority of the CCS dissolved solids. Phosphate increased at the end of fermentation due to cell death. We noted that
pH rose from 7 to over 8 and then dropped during fermentation, limiting PHA to less than 10% of CDW, so we next investigated pH control. Initially pH was controlled by addition of sulfuric acid and ammonium hydroxide. This reduced cell death, however ammonia climbed to over 3.5 g/L at the end of fermentation. This high level of nitrogen would retard PHA production, thus we next used sulfuric acid and sodium hydroxide for pH control. This maintained cell viability and nitrogen limitation at the end of fermentation, providing better conditions for PHA accumulation. Our next focus was to feed the culture low-cost carbon sources for PHA accumulation. Trials used 400 g/L CCS with 2.7 g/L ammonia, and pH control with sulfuric acid and sodium hydroxide. The first carbon source utilized was glycerol water from biodiesel production. Sterile glycerol water was fed to the culture to maintain glycerol levels between 1.5-3.5%. Nitrogen was limiting shortly after glycerol feeding was implemented.
After 50 h the cells contained approximately 10% PHA, resulting in a PHA productivity of 60 mg PHA/L/h. After 96 h the PHA content reached 25-30% of CDW, and productivity rose to 84 mg PHA/L/h. This was low compared productivities of 2-3 g/L/h reported in the literature. The second carbon source evaluated was sunflower soapstock. This substrate presented several problems, including excessive foaming. We were forced to reduce aeration, which slowed PHA production because oxygen is required for B-oxidation of soapstock fatty acids. Accurate and quick measurement of carbon sources in soapstock was also not possible due to lengthy sample preparation requirements, its low solubility, and the potential for multiple carbon sources in this crude byproduct. In these trials 15 g/L sterile soapstock was added at each feeding. CDW rose until the first soapstock addition, then it decreased and stabilized at 16 g/L. This is only about 50% of the total CDW obtained from 400 g/L medium in previous
trials. PHA content was 15-20% of the CDW, with a productivity of 30 mg PHA/L/h.
Impacts
The rapid growth of the corn ethanol industry has placed considerable pressure on the price/value of DDGS and condensed corn solubles. Alternative uses for these co-products is needed to maintain profitability. Production of high value biomaterials, such as polyhydroxyalkanoates, from these co-products could bolster the economics of corn ethanol production.
Publications
- Javers, J., W.R. Gibbons, and D. Raynie. 2005. Production of polyhydroxyalkanoates from industrial waste streams. SD Academy of Science, 90th Annual Meeting, April, Sioux Falls, SD.
- Gibbons, W.R. 2005. DDGS research and the Sun Grant Initiative. Midwest Consortium annual meeting "Adding value to DG from dry grind processes." November, Indianapolis, IN.
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Progress 01/01/04 to 12/31/04
Outputs
We previously determined that Pseudomonas putida KT217 achieved a higher cell mass (1.3-2.6 g/L) and PHA productivity (5.1-8.1 mg/L/h) on a condensed corn solubles (CCS) medium compared to two other strains (P. putida KT2442 and P resinovorans ATCC 14235). In 2004 we conducted trials in a 5 liter bioreactor in which agitation, aeration, and pH control could be optimized. Initially we used a 300 g/L CCS medium supplemented with soapstock. Cell yields were low (2-3 g/L) and we hypothesized that nitrogen was limiting. Subsequent trials in the 300g/L CCS medium, supplemented with nitrogen (ammonium hydroxide), showed significant improvements in nutrient utilization (glycerol and organic acids) and cell growth (15g/L). We next sought to determine the extent of soybean soapstock utilization by adding up to 15 g/L at 43 h (following complete utilization of glycerol). This caused the PHA content (as % CDW) to increase from 1% to over 18% between 43-50 h of incubation. A
copolymer consisting mainly of 3-hydroxydecanoic acid was produced with 3-hydroxyhexanoic, 3-hydroxyoctanoic, and 3-hydroxydodecanoic acid as minor components. The PHA productivity in these trials rose to 54 mg/L/h, compared to 5.1-8.1 mg/L/h in the CCS medium without nitrogen additions and aeration. Foaming became a problem in the bioreactor and several antifoams were tested for effectiveness. Some antifoams reduced viable counts by over 50%, however the antifoam Biospumex (Cognis company) was effective with minimal cell damage. A byproduct of biodiesel production (glycerol water) was also evaluated as a supplementary feedstock. Preliminary results showed no toxicity and rapid utilization, meaning that glycerol water may be a good carbon source to add in a fed-batch mode to achieve high cell density prior to PHA accumulation. We next began investigating the effects of C:N:P ratio to optimize cell growth. Carbon sources were monitored via HPLC, while nitrogen and phosphorus were
monitored using Hach test kits. Initial trials in shake flask cultures gave indefinite results (avg 1.8-2.1 g/L CDW) because oxygen became limiting early in the exponential phase (before nutrient limitation occurred). Trials are now being conduted in aerated 1L shake flasks. A matrix of CCS concentrations (C), ammonium hydroxide (N), and phosphate (P) are being evaluated to determine optimal levels.
Impacts
Condensed corn solubles (CCS) is an underutilized byproduct of dry mill ethanol production. Production of high value chemicals, such as polyhydroxyalkanoates could provide new products for these dry mill operations. This would enhance their profitability and sustainability.
Publications
- Javers, J., W.R. Gibbons, and D. Raynie. 2004. Production of polyhydroxyalkanoates from industrial waste streams. Abstract 21. Corn Utilization and Technology Conference. June, Indianapolis, IN.
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Progress 01/01/03 to 12/31/03
Outputs
During 2003, shake flask growth curves of Pseudomonas resinovorans ATCC 14235, P. putida KT217, and P. putida KT2442 were conducted to compare the condensed corn solubles medium (2 levels of CCS) vs the defined medium, blended with various carbon sources (at 2 levels). Carbon sources included glucose, glycerol, octanoate, corn oil, and soapstock. All strains were able to utilize glucose, glycerol, and octanoate for growth and PHA production. When grown on the CCS medium, the 240 g/L level of CCS outperformed the 80 g/L level, and glucose was utilized before glycerol present in CCS. P. putida KT 217 yielded the highest cell mass (1.3-2.6 g/L) and PHA productivity (5.1-8.1 mg/L/h), while P. putida KT2442 was the least efficient (at 1.1-2.4 g/L cell mass and 1.2-5.0 mg/L/h PHA). P. resinovorans was intermediate, however it was also able to produce PHA on corn oil, when added post-exponential phase. These data compare favorably with those from P. putida KT217 and KT2442
grown on a defined laboratory medium, where researchers have previously reported cell yields of 0.8 g/L and 0.7 g/L, containing 41% and 37% PHA, respectively. When the pseudomonas cultures were provided with soapstock at the start of fermentation, growth was inhibited. However, when soapstock was added after exponential phase it did not adversely affect growth, and PHA granules were visible under phase contrast microscopy. Analytical procedures are being developed to better determine concentrations of the insoluble substrates contained in soapstock. Based on this information trials are underway in 3 liter benchtop reactors so that aeration/agitation and pH control can be optimized. Future trials will also focus on nutrient supplementation (i.e. PO4 and NH4), feeding strategies, and cell recycling methods to obtain high cell density for PHA accumulation. Dual stage carbon feeding (e.g., glucose/soapstock) will be evaluated to boost PHA production. Preliminary results for ethanol
modified supercritical fluid extraction (SFE) of PHA from cell pellets were reported last year. This year we validated the method statistically against conventional Soxhlet extraction. We determined that ideal conditions were 60C and 9000 psi, that SFE extraction produced a cleaner PHA extract, and that PHA precipitated upon contact with atmospheric conditions. The modified SFE method also decreased extraction time (10X), decreased solvent usage (15X), and eliminated the need for chemical precipitants. Extracts from both SFE and Soxhlet methods were over 98% pure as determined by GC/FID and GC/MS.
Impacts
Condensed corn solubles (CCS) is an underutilized byproduct of dry mill ethanol production. Production of high value chemicals, such as polyhydroxyalkanoates could provide new products for these dry mill operations. This would enhance their profitability and sustainability.
Publications
- Raynie, D.E., J. Javers, and W.R. Gibbons. 2003. Selective separation of biopolymers from microbial fermentation. Proceedings of S.D. Academy of Science. 82: 371.
- Smith, R. and W.R. Gibbons. 2003. Use of condensed corn solubles for production of succinic acid by Actinobacillus succinogenes. Proceedings of S.D. Academy of Science. 82: 235.
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Progress 01/01/02 to 12/31/02
Outputs
Our objective is to produce high value, specialty chemicals from condensed corn solubles (CCS), a byproduct of dry-mill ethanol production. Initially we will focus on polyhydroxyalkanoate (PHA) and succinic acid. PHA is a biodegradable polymer with properties (elasticity, thermal resistance) such that it can mimic compounds ranging from hard rubbers to elastic type polymers. Succinic acid is an important chemical intermediate with an annual market of over $4 billion. In year one, our objective was to identify microbes with the highest productivity and yield, then acclimate them to a CCS-based medium. For PHA production we selected Pseudomonas resinovorans ATCC 14235, P. putida KT217, and P. putida KT2442, all of which grew immediately on the CCS medium. Next we will optimize the basal medium (CCS level) and evaluate different supplemental carbon sources (eg. corn oil). Another major effort in year one has been to develop a more efficient and less costly method to
recover PHA from the cell (for both analytical and commercial purposes). Traditionally this is done by soxhlet extraction with chloroform, followed by evaporation, and then methanol precipitation. Supercritical fluid extraction (SFE) with carbon dioxide (9,000 psi, 60 C) and 15% ethanol modifier showed excellent results, producing a high purity PHA (mass spec analysis yet to be performed). Preliminary trials with accelerated solvent extraction (ASE) have also been conducted with mixed results. A matrix of temperatures, pressures, mole fractions will be performed in both SFE and ASE to optimize extraction. For succinic acid production we selected Actinobacillus succinogenes ATCC 55618. Over several month it was gradually acclimated to grow on a CCS medium. Growth curves comparing the lab medium with the CCS medium, showed a slightly longer lag phase (6-12 h longer), but similar maximum cell counts (10 E9 CFU/ml). Trials in year two will determine the best CCS concentration, was well as
the optimal sugar level.
Impacts
Condensed corn solubles (CCS) in an under-utilized byproduct of dry mill ethanol production. Production of high value chemicals, such as polyhydroxyalkanoates and succinic acid, could provide new "profit-center" products from these dry mill operations.
Publications
- No publications reported this period
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