Zhiyou Wen

Producing Docosahexaenoic Acid (DHA)-Rich Algae from Biodiesel-Derived Crude Glycerol : Effects of Impurities on DHA Production and Algal Biomass Composition

Crude glycerol is the primary byproduct of the biodiesel industry. Producing docosahexaenoic acid (DHA, 22:6 n-3) through fermentation of the alga Schizochytrium limacinum on crude glycerol provides a unique opportunity to utilize a large quantity of this byproduct. The objective of this work is to investigate the effects of impurities contained in the crude glycerol on DHA production and algal biomass composition. Crude glycerol streams were obtained from different biodiesel refineries. All of the glycerol samples contained methanol, soaps, and various elements including calcium, phosphorus, potassium, silicon, sodium, and zinc. Both methanol and soap were found to negatively influence algal DHA production; these two impurities can be removed from culture medium by evaporation through autoclaving (for methanol) and by precipitation through pH adjustment (for soap). The glycerol-derived algal biomass contained 45-50% lipid, 14-20% protein, and 25% carbohydrate, with 8-13% ash content. Palmitic acid (C16:0) and DHA were the two major fatty acids in the algal lipid. The algal biomass was rich in lysine and cysteine, relative to many common feedstuffs. Elemental analysis by inductively coupled plasma showed that boron, calcium, copper, iron, magnesium, phosphorus, potassium, silicon, sodium, and sulfur were present in the biomass, whereas no heavy metals (such as mercury) were detected in the algal biomass. Overall, the results show that crude glycerol was a suitable carbon source for algal fermentation. The crude glycerol-derived algal biomass had a high level of DHA and a nutritional profile similar to that of commercial algal biomass, suggesting a great potential for using crude glycerol-derived algae in omega-3-fortified food or feed.

Hydrolysis of animal manure lignocellulosics for reducing sugar production.

Converting animal manure into value-added products provides a potential alternative for treatment and disposal of such materials. Lignocellulosics are a major component of animal manure and represent an undeveloped bioresource. In this work, a process was developed for hydrolyzing manure lignocellulosics into fermentable sugars. When raw dairy manure was pre-treated with 3% sulfuric acid at 110 degrees C for 1 h, hemicellulose was completely degraded into mainly arabinose, galactose and xylose. The pretreated materials were then treated with cellulolytic enzymes, Celluclast-1.5L and Novozyme-188, to hydrolyze the cellulose. The optimal enzyme loadings were identified as 13 FPU cellulase/g substrate and 5 IU beta-glucosidase/g substrate. The optimal temperature and pH were determined to be 46 degrees C and 4.8, respectively. A substrate concentration of 50 g/l favored both glucose concentration (in hydrolysate) and glucose yield (based on per 100 g manure). It was also found that a reduced particle size of 590-mum resulted in a high glucose yield with further decreases in particle size not increasing the yield. For each particle size investigated, the addition of 2% tween-80 resulted in at least 20% improvement in glucose yield. The optimized hydrolysis process achieved a glucose yield of 11.32 g/100 g manure, which corresponded to about 40% cellulose conversion.

Continuous culture of the microalgae Schizochytrium limacinum on biodiesel-derived crude glycerol for producing docosahexaenoic acid

Crude glycerol is a major byproduct of the biodiesel industry; previous research has proved the feasibility of producing docosahexaenoic acid (DHA, 22:6 n-3) through fermentation of the algae Schizochytrium limacinum on crude glycerol. The objective of this work is to investigate the cell growth kinetics, substrate utilization efficiency, and DHA production of the algae through a continuous culture. Steady-state biomass yield, biomass productivity, growth yield on glycerol, specific glycerol consumption rate, and fatty acid composition were investigated within the range of dilution rate (D) from 0.2 to 0.6 day(-1), and the range of feed crude glycerol concentration (S(0)) from 15 to 120 g/L. The maximum specific growth rate was determined as 0.692 day(-1). The cells had a true growth yield of 0.283 g/g but with a relatively high maintenance coefficient (0.2216 day(-1)). The highest biomass productivity of 3.88 g/L-day was obtained at D=0.3 day(-1) and S(0)=60 g/L, while the highest DHA productivity (0.52 g/L-day) was obtained at D=0.3 day(-1) and S(0)=90 g/L due to the higher DHA content at S(0)=90 g/L. The biomass and DHA productivity of the continuous culture was comparable to those of batch culture, while lower than the fed-batch culture, mainly because of the lower DHA content obtained by the continuous culture. Overall, the results show that continuous culture is a powerful tool to investigate the cell growth kinetics and physiological behaviors of the algae growing on biodiesel-derived crude glycerol.

Use of Biodiesel-Derived Crude Glycerol for Producing Eicosapentaenoic Acid (EPA) by the Fungus Pythium irregulare

Crude glycerol is a major byproduct for the biodiesel industry. Producing value-added products through microbial fermentation on crude glycerol provides opportunities to utilize a large quantity of this byproduct. The objective of this study is to explore the potential of using crude glycerol for producing eicosapentaenoic acid (EPA, 20:5 n-3) by the fungus Pythium irregulare . When P. irregulare was grown in medium containing 30 g/L crude glycerol and 10 g/L yeast extract, EPA yield and productivity reached 90 mg/L and 14.9 mg/L x day, respectively. Adding pure vegetable oils (flaxseed oil and soybean oil) to the culture greatly enhanced the biomass and the EPA production. This enhancement was due to the oil absorption by the fungal cells and elongation of shorter chain fatty acids (e.g., linoleic acid and alpha-linolenic acid) into longer chain fatty acid (e.g., EPA). The major impurities contained in crude glycerol, soap and methanol, were inhibitory to fungal growth. Soap can be precipitated from the liquid medium through pH adjustment, whereas methanol can be evaporated from the medium during autoclaving. The glycerol-derived fungal biomass contained about 15% lipid, 36% protein, and 40% carbohydrate, with 9% ash. In addition to EPA, the fungal biomass was also rich in the essential amino acids lysine, arginine, and leucine, relative to many common feedstuffs. Elemental analysis by inductively coupled plasma showed that aluminum, calcium, copper, iron, magnesium, manganese, phosphorus, potassium, silicon, sodium, sulfur, and zinc were present in the biomass, whereas no heavy metals (such as mercury and lead) were detected. The results show that it is feasible to use crude glycerol for producing fungal biomass that can serve as EPA-fortified food or feed.

Effects of hemicellulose and lignin on enzymatic hydrolysis of cellulose from dairy manure

This study focused on the effect of hemicellulose and lignin on enzymatic hydrolysis of dairy manure and hydrolysis process optimization to improve sugar yield. It was found that hemicellulose and lignin in dairy manure, similar to their role in other lignocellulosic material, were major resistive factors to enzymatic hydrolysis and that the removal of either of them, or for best performance, both of them, improved the enzymatic hydrolysis of manure cellulose. This result combined with scanning electron microscope (SEM) pictures further proved that the accessibility of cellulose to cellulase was the most important feature to the hydrolysis. Quantitatively, fed-batch enzymatic hydrolysis of fiber without lignin and hemicellulose had a high glucose yield of 52% with respect to the glucose concentration of 17 g/L at a total enzyme loading of 1300 FPU/L and reaction time of 160 h, which was better than corresponding batch enzymatic hydrolysis.

Kinetic modeling of enzymatic hydrolysis of cellulose in differently pretreated fibers from dairy manure

A kinetic model incorporating dynamic adsorption, enzymatic hydrolysis, and product inhibition was developed for enzymatic hydrolysis of differently pretreated fibers from a nitrogen‐rich lignocellulosic material—dairy manure. The effects of manure proteins on the enzyme adsorption profile during hydrolysis have been discussed. Enzyme activity, instead of protein concentration, was used to describe the enzymatic hydrolysis in order to avoid the effect of manure protein on enzyme protein analysis. Dynamic enzyme adsorption was modeled based on a Langmiur‐type isotherm. A first‐order reaction was applied to model the hydrolysis with consideration being given for the product inhibition. The model satisfactorily predicted the behaviors of enzyme adsorption, hydrolysis, and product inhibition for all five sample manure fibers. The reaction conditions were the substrate concentrations of 10–50 g/L, enzyme loadings of 7–150 FPU/g total substrate, and the reaction temperature of 50°C. Biotechnol. Bioeng. 2008;101: 441–451.

Simultaneous production of nisin and lactic acid from cheese whey: optimization of fermentation conditions through statistically based experimental designs.

A biorefinery process that utilizes cheese whey as substrate to simultaneously produce nisin, a natural food preservative, and lactic acid, a raw material for biopolymer production, was studied. The conditions for nisin biosynthesis and lactic acid coproduction by Lactococcus lactis subsp. lactis (ATCC 11454) in a whey-based medium were optimized using statistically based experimental designs. A Plackett-Burman design was applied to screen seven parameters for significant factors for the production of nisin and lactic acid. Nutrient supplements, including yeast extract, MgSO4, and KH2PO4, were found to be the significant factors affecting nisin and lactic acid formation. As a follow-up, a central-composite design was applied to optimize these factors. Second-order polynomial models were developed to quantify the relationship between nisin and lactic acid production and the variables. The optimal values of these variables were also determined. Finally, a verification experiment was performed to confirm the optimal values that were predicted by the models. The experimented results agreed well with the model prediction, giving a similar production of 19.3 g/L of lactic acid and 92.9 mg/L of nisin.

Value-Added Chemicals from Animal Manure

The objective of the project proposed by Washington State University (WSU) and Pacific Northwest National Laboratory (PNNL) was to develop technology for the utilization of animal manures as feedstocks to produce value-added products. These included medium-volume commodity chemicals such as glycols or diols and protein-based products such as chemicals or feed supplements. The research focused on two aspects of this approach including the analysis and treatment of the feedstock to produce intermediate chemical precursors and the aqueous phase conversion of these intermediates to chemicals and other value-added products.

Nonfeed Application of Rendered Animal Proteins for Microbial Production of Eicosapentaenoic Acid by the Fungus Pythium irregulare

Rendered animal proteins are well suited for animal nutrition applications, but the market is maturing, and there is a need to develop new uses for these products. The objective of this study is to explore the possibility of using animal proteins as a nutrient source for microbial production of omega-3 polyunsaturated fatty acids by the microalga Schizochytrium limacinum and the fungus Pythium irregulare. To be absorbed by the microorganisms, the proteins needed to be hydrolyzed into small peptides and free amino acids. The utility of the protein hydrolysates for microorganisms depended on the hydrolysis method used and the type of microorganism. The enzymatic hydrolysates supported better cell growth performance than the alkali hydrolysates did. P. irregulare displayed better overall growth performance on the experimental hydrolysates compared to S. limacinum. When P. irregulare was grown in medium containing 10 g/L enzymatic hydrolysate derived from meat and bone meal or feather meal, the performance of cell growth, lipid synthesis, and omega-3 fatty acid production was comparable to the that of culture using commercial yeast extract. The fungal biomass derived from the animal proteins had 26-29% lipid, 32-34% protein, 34-39% carbohydrate, and <2% ash content. The results show that it is possible to develop a nonfeed application for rendered animal protein by hydrolysis of the protein and feeding to industrial microorganisms which can produce omega-3 fatty acids for making omega-3-fortified foods or feeds.

Production of Cellulase/β-Glucosidase by the Mixed Fungi Culture of Trichoderma reesei and Aspergillus phoenicis on Dairy Manure

A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L of manure (dry basis [w/w]), 2 g/L KH2PO4, 2 mL/L of Tween-80, and 2mg/L of CoCl2. However, β-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the β-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL) and β-glucosidase (0.64 IU/mL). The ratio of β-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced glucose was significantly (p<0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei.