Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849
Performing Department
Biosystems Engineering
Non Technical Summary
In order to reduce the dependence on foreign supply of petroleum, the United States is in need of alternative energy sources as well as material resources. Lignocellulosic biomass (LCB) is a potential and competitive source for bioenergy and biomaterial production. There are two main reasons: biomass is one of the few energy and biomaterial sources that can actually be utilized to produce several types of energy (motor fuel including butanol and ethanol, electricity, heat) and materials (acetone, polyhydroxybutyrate (PHB)); and cellulosic biomass is renewable and commonly found.Researchers and scientists within and outside of Auburn University are working on how to convert algae into bioenergy . Currently, there is very little research on production of ethanol and PHB from algae and none of them is systematic. Therefore, we propose a study on comprehensively utilizing algae: using algae residuals (by-products) after bioactive elements are extracted, as feed stocks to produce bioenergy and biomaterials. Studies on pretreatment of raw algae, enzymatic hydrolysis, and fermentation for butanol and polyhydroxybutyrate will be carried out. In this proposed work, we will determine the techniques and optimize the conditions for pretreating algae, select appropriate enzymes and optimize the conditions for hydrolysis, and screen the appropriate engineering bacteria and optimize the conditions for production of biofuel and biomaterials. If the proposed work is successful, it will provide valuable references for developing a sustainable system to convert algae into bioenergy and biomaterials in an economically efficient manner and open new opportunities for aquaculture farmers in the region as well.
Animal Health Component
40%
Research Effort Categories
Basic
30%
Applied
40%
Developmental
30%
Goals / Objectives
Goals and Objectives: The general goal of this project is to develop a set of scientific methods to use algae byproducts (after extraction of functional food, about 99% of the algae mass left) to produce biomaterials and biofuels (polyhydroxybutyrate (PHB) and ABE). The extraction of some bioactive elements from algae was addressed by our previous studies. Therefore, the specific objectives that will be carried out in a period of five years of the project to accomplish this main goal are as follows: a) pretreatment of raw algae (harvested from pond without further processing) using radio frequency (RF) heating assisted alkaline or acid method; b) enzymatic hydrolysis on glucan (w/v) gained from algae and analysis; c) fermentation with Clostridium beijerinckii 8052 on the hydrolysates from hydrolysis as carbon sources for butanol production and analysis; and d) fermentation with recombinant Escherichia coli strain on the hydrolysates from hydrolysis as carbon sources using the hydrolysates as carbon sources for PHB production and analysis.
Project Methods
MaterialAll algae samples will be collected from newly harvested cultures from various aquaculture systems. Moisture, extractives, ash, cellulose (as glucan), hemicellulose (as xylan) and lignin (acid insoluble lignin and acid soluble lignin) in the raw substrates will be analyzed according to NREL (National Renewable Energy Laboratory -USA) laboratory analytical procedures using the extractive-free samples.Pretreatment of raw algaeA RF heater (SO6B; Strayfield, Berkshire, England) will be employed in this study. This RF heating system worked at a frequency of 27.12 MHz and maximum power output of 6kW. Before pretreatment, algae samples will be soaked in NaOH solution at a certain solid to liquid ratio in a plastic container at room temperature overnight. During the RF heating process, four fiber-optic sensors (UMI, FISO Technologies, Quebec, Canada) will be inserted to keep the system at 90°C with ± 3°C fluctuation for 60 minutes. When sample temperature reached 90°C, the RF heater will be paused for 0.5 min followed by another 1-min RF heating in order to keep the sample at 90°C. This pause-heating pattern is repeated until the predetermined RF heating time is completed.Enzymatic hydrolysis and analysisEnzymatic hydrolysis will be performed in a sodium citrate buffer (pH 4.8) at about 2% glucan (w/v) with Novozym 22C (Novozymes, Franklinton, NC). The enzyme loading used will be 10 FPU/g glucan. The hydrolysis is carried out in a shaker with agitation of 150 rpm at 50 °C for 72 h. Samples will be periodically taken for sugar analysis using an Agilent 1260 Infinity Quaternary LC VL HPLC (Agilent Technologies, Santa Clara, CA) with refractive index detector (RID) following the National Renewable Energy Laboratory (NREL) standard protocol (Selig et al., 2008).Elemental analysis will be performed on a Perkin-Elmer CHNS/O analyzer (model 2400, Seriesto quantify the carbon, hydrogen, nitrogen and sulfur contents in the raw and pretreated materials. Moreover, the supernatant of RF and WB pretreated switchgrass will be analyzed using an Agilent 6890N GC connected with an Agilent 5973 mass-selective detector (MSD) equipped with a DB-1701 column (60m × 0.25 mm, 0.25 μm film thickness).Fermentation for butanol and analysisA laboratory stock of C. beijerinckii 8052 will be routinely stored as spore suspension in sterile double distilled water at 4 oC. Spores are heat-shocked at 80 °C for 10 min, followed by cooling on ice for 5 min. The fermentation solution will consist of the substrate (either biomass hydrolysates or synthetic substrate as control) supplemented with 1% of P2 stock solutions (including mineral, buffer, and vitamin). Prior to inoculation, the pH value of the fermentation broth will be adjusted to 6.8-7.0 with filter-sterilized KOH solution.Butanol fermentation will be carried out in parallel using various carbon sources (supplemented with P2 medium), including algae hydrolysates generated from RF pretreatment (RFH), algae hydrolysates generated from WB pretreatment (WBH), and a control (CON) with mixed sugars and acetic acid at the same levels as in RFH. Cell density and the concentration of fermentation metabolites were monitored through the course of fermentation.ABE, acetic acid, and butyric acid will be quantified using HPLC (Agilent Technologies 1260 series) equipped with an automatic sampler/injector and a RID using an HPX-87H column (Bio- Rad, Hercules, CA, USA). Fermentation samples will be centrifuged at 13,200 rpm for 10 min, and the supernatant were diluted fivefold with distilled water before the HPLC analysis. The 0.005 N solution of H2SO4 was used as the mobile phase at a flow rate of 0.6 mL/min at 45°C.Fermentation for PHB and analysisThe E. coli XL1-blue strain hosting pBHR68 plasmid for PHB production will be used. The pBHR68 plasmid contains the three genes (phaA, phaB, and phaC) needed for PHB synthesis and confers ampicillin resistance. During all the strain cultivation steps, 100 ug/ml ampicillin is supplemented unless otherwise specified. The strain was first grown in Luria-Bertani (LB) medium. Then the overnight culture will be inoculated into standard M9 medium in an orbital shaker operating at 220 rpm and 37 ° C. M9 minimal medium contains 0.6% Na2HPO4, 0.3% KH2PO4, 0.05% NaCl, 0.1% NH4Cl, 0.02% MgSO4, and 0.001% CaCl2. Overnight culture is then used to seed 156 the media for PHB production at the same agitation rate and temperature.The E. coli cell biomass will be harvested by centrifugation at 4,000 rpm and 4°C for 20 min. The pellets will be stored in -80 °C freezer until completely frozen (for more than 12 hours). Frozen samples will be dried using a freeze-dryer (Labconco lyophilizer, Labconco Corporation, Kansas City, MO, USA). PHB quantification will be carried out using 1H nuclear magnetic resonance (NMR) spectrum based on the chloroform-sodium hypochlorite dispersion method with modifications (Linton et al., 2012).