Wanying Yao

Biogas and CH4 productivity by co-digesting swine manure with three crop residues as an external carbon source

Co-digesting swine manure with three agricultural residues, i.e., corn stalks, oat straw, and wheat straw, to enhance biogas productivity was investigated in this study. A 3x3 experimental design with duplicates was adopted (3 crop residuesx3 carbon/nitrogen ratios) to examine the improvement of batch digestion in terms of biogas volume produced, CH(4) content in the biogas, and net CH(4) volume. The crop residues were first cut into small sections and then ground into fine particles smaller than 40 mesh size (0.422mm) before being added to digesters. All the digesters were run simultaneously under controlled temperature at 37+/-0.1 degrees C. The length of experiment was 25days. The results showed that all crop residues significantly increased biogas production and net CH(4) volume at all C/N ratios, among which corn stalks performed the best with increase in daily maximum biogas volume by 11.4-fold as compared to the control, followed by oat straw (8.45-fold) and wheat straw (6.12-fold) at the C/N ratio of 20/1, which was found to be the optimal C/N ratio for co-digestion in the present study. In addition, corn stalks achieved the highest CH(4) content in the biogas ( approximately 68%), which was about 11% higher than that of oat straw ( approximately 57%), whereas wheat straw and the control both had produced biogas with approximately 47% CH(4) content. Wheat straw demonstrated a lower biogas productivity than corn stalks and oat straw even it had a higher carbon content (46%) than the latter two residues (39%).

In vitro enzymatic conversion of γ-aminobutyric acid immobilization of glutamate decarboxylase with bacterial cellulose membrane (BCM) and non-linear model establishment

The work investigated the properties and feasibility of using bacterial cellulose membrane (BCM) as a new and environmental friendly support carrier to immobilize glutamate decarboxylase (GAD) (a unique enzyme in the conversion of γ-aminobutyric acid (GABA) production). During cultivation, the porosities of BCM decreased successively with more extended fibrils piling above one another in a criss-crossing manner thus forming condensed and spatial structure. The BCM with this ultrafine network structure was found to immobilize GAD best via covalent binding because of the highest efficiency of immobilization (87.56% of the enzyme was bonded) and a good operational stability. And the covalent binding efficiency (amount of enzyme immobilized versus lost) was closely related to the porosity or the inner network of the BCM, not to the surface area. The capacity per surface area (mg/cm(2)) increased from 1.267mg/cm(2) to 3.683mg/cm(2) when the porosity of BCM ranged from 49% to 73.80%, while a declining trend of the loss of GAD specific activity (from 29.30%/cm(2) to 7.38%/cm(2)) was observed when the porosity increased from 49.9% to 72.30%. Two non-linear regression relationships, between the porosity and loading capacity and between porosity and enzyme activity loss, were empirically modeled with the determination of coefficient R(2) of 0.980 and 0.977, respectively. Finally, the established in vitro enzymatic conversion process demonstrated 6.03g/L of GABA at 0.10mol/L Glu, 60min of retention time and 160mL of suspension volume after the 1st run and a loss of 4.15% after the 4th run. The productivity of GABA was 6.03gL(-1)h(-1), higher than that from other reported processes.

Utilization of protein extract from dairy manure as a nitrogen source by Rhizopus oryzae NRRL-395 for l-lactic acid production.

Six levels of crude protein (0.21, 0.42, 0.84, 1.68, 2.52, and 3.36g/L) and six levels of protein hydrolysates from dairy manure, defined by degree of hydrolysis (DH, 6.9%, 17.2%, 25.9%, 33.8%, 36.1%, and 36.7%), were investigated as the nitrogen source for production of l-lactic acid by Rhizopus oryzae NRRL-395 with respect to the influence of nitrogen source on l-lactic acid yield and the correlation with biomass yield and mycelia morphology. Increases in crude protein from 0.21 to 1.68g/L led to an increase in l-lactic acid concentration in the culture media from 6.48 to 57.7g/L. However, further increases beyond 1.68g/L did not present continuing increases in l-lactic acid yields. The highest biomass yield was obtained at a crude protein nitrogen concentration of 2.52g/L. Hydrolysates with high DH resulted in high yields of l-lactic acid and biomass. At a nitrogen level of 0.42g/L (hydrolysates) with DH ranging from 33.8% to 36.7%, the l-lactic acid yield of 0.53-0.56g/g of glucose was achieved, coupled with a 13-14% yield of fungal biomass.

Anaerobic digestion of dairy manure influenced by the waste milk from milking operations

It is not uncommon that a significant amount of milk from milking operations is discharged to manure digesters on dairy farms. To understand the effect of milk on the digester performance, experiments using batch digesters (500-mL flasks) were carried out in this study to co-digest milk and dairy manure at different milk levels for biogas production and pollutant reduction, and a total of 8 treatments were examined [i.e., control (without milk) and 1, 3, 5, 7, 9, 14, and 19% milk additions]. The temperature for all digesters was maintained at 37±0.5°C throughout the experimental period, which was 28 d. The results showed that co-digesting milk with dairy manure could increase biogas productivity, with the percent cumulative biogas volume increased by 5.6, 16.3, 26.5, 40.8, 50.2, 79.9, and 103.8%, as compared with the control, for milk addition of 1, 3, 5, 7, 9, 14, and 19% (vol/vol), respectively. However, the CH(4) content in the biogas decreased slightly as the milk content increased (from 66.5% for the control to 63.5% for 19% milk treatment), implying that the added milk could promote CO(2) production. To avoid that, the milk content in the manure should be controlled below 3%. A linear relationship for the total biogas volume produced with the milk content in the manure was revealed, with a correlation coefficient of 0.99. An improved removal efficiency of chemical oxygen demand was observed for milk-treated digesters. Good linear regressions between the total biogas production and the percent chemical oxygen demand decrease and the substrate carbon/nitrogen ratio were also obtained (correlation coefficients: 0.93 and 0.99, respectively). Besides, co-digestion of dairy manure and milk was found to improve substrate solids breakdown, but had little effect on percent volatile fatty acid decrease. In summary, the waste milk co-digested with dairy manure may not cause negative effects on anaerobic digester performance.

Development and optimization of a culture medium for L-lactic acid production by Rhizopus oryzae using crude protein from dairy manure as a nitrogen source.

Experiments were conducted using the crude protein in fresh dairy manure as the nitrogen source in the culture media for Rhizopus oryzae to produce L-lactic acid. Two uniform design experiments were carried out with one for optimizing seed culture while the other for best percent L-lactic acid production. Multiple linear regression and ANOVA analyses were employed to determine the most significant media components. Data from the first uniform design U6 (62× 3), in which the experimental factors involved included nitrogen concentration (crude protein), spore concentration, and treatment duration, showed that the levels of these components in the optimal condition for the seed culture medium were 2.1 g/L nitrogen, 16 hour culture time, and 105 spore concentration. The biomass weight in the seed medium developed in this study reached 1.32 g/L, which was 48.3% higher than that of the control. The combination of culture time and nitrogen concentration was found to be most significant in influencing the biomass yield. In the second uniform design experiment, flask culture with five factors (glucose, nitrogen from dairy manure, ZnSO4, KH2PO4, and MgSO4) at eight levels was examined using the uniform design table U8 (85) with the content of L-lactic acid as the evaluating response Y. The results showed that ZnSO4 had the most influence on L-lactic acid yield, followed by nitrogen and KH2PO4. The optimum culture medium in terms of lactic acid production consisted of 240 g/L glucose, 1.26 g/L crude protein, 1.05 g/L KH2PO4, and 0.25 g/L MnSO4, which could achieve a yield of 59.57% (7.1% higher than the control).

L-lactic acid fermentation by Rhizopus oryzae using dairy manure as a nitrogen source.

Experiments were conducted to investigate the possibility of using fresh dairy manure as the nitrogen source in a culture medium to produce L-lactic acid by Rhizopus oryzae NRRL 395. The parameters of the culture medium, including glucose (120 vs. 240 g L-1), nitrogen (1.0 vs. 3.0 g L-1, from dairy manure), KH2PO4 (0.65 vs. 1.3 g L-1), MgSO4 (0.25 vs. 0.5 g L-1), ZnSO4 (0.0 vs. 0.05 g L-1), spore culture time (3 vs. 6 h), and the inoculum dosage (6% vs. 12%, v/v), were first examined using the Plackett-Burman design to single out the most important ones to reduce the number of experiments in the optimization stage while still maintaining the statistical validity of the experimental results. In the optimization stage, experiments featuring a two-level, four-factor factorial design (24) for the four most important components, i.e., glucose, nitrogen, KH2PO4, and MgSO4, were carried out, and the results were subjected to ANOVA to determine the optimum levels for each component, within the ranges used in this study, in the culture medium based on the percent L-lactic acid yield. The results showed that glucose, nitrogen, KH2PO4, and MgSO4 concentrations had the strongest impact on percent L-lactic acid yields by Rhizopus oryzae NRRL 395 (degrees of confidence were 93.08%, 96.11%, 89.59%, and 82.2%, respectively). The ANOVA revealed that glucose, nitrogen, and KH2PO4 demonstrated a significant main effect on the yield of L-lactic acid (p < 0.0004). In addition, the interactions of glucose, nitrogen, KH2PO4, and MgSO4 were also found to be significant at p < 0.05. The main effects, as well as the interactions, were summarized by a polynomial regression equation, which was found to be able to sufficiently describe the L-lactic acid production observed in this study (p < 0.0002). The equation was examined against the experimental data, resulting in a coefficient of determination (R2) of 0.9436 for the linear regression relationship between the predicted and observed values. Finally, the optimum culture medium composition with respect to the four major components studied using dairy manure as a base substrate for L-lactic acid generation by Rhizopus oryzae was found to be 3 g L-1 nitrogen, 120 g L-1 glucose, 1.3 g L-1 KH2PO4, and 0.5 g L-1 MgSO4, and the corresponding percent L-lactic acid yield was 29.1% (measured) and 30.76% (predicted), based on the experimental conditions used in this study.

Bioconversion of the Nutrients in Dairy Manure for L-lactic acid Production by Rhizopus Oryzae

Six levels of crude protein (0.21 to 3.36 g/L) were investigated as the nitrogen source for production of L-lactic acid by Rhizopus oryzae with respect to the influence on L-lactic acid yield, biomass and mycelia morphology. Increases in crude protein from 0.21 to 1.68 g/L led to an increase in L-lactic acid concentration from 6.48 to 57.7 g/L. However, further increases beyond 1.68 g/L did not present continuing increases in L-lactic acid yields.

One-step purification of glutamate decarboxylase from E. coli using aqueous two-phase system.

The aqueous two-phase system (ATPS), formed when two polymers or one polymer and one salt are mixed together at appropriate concentrations, is a clean technique to separate and purify biomolecules. ATPS has many advantages over traditional downstream process, especially on the integration of concentration, extraction, and partial purification if properly optimized, which reduce multiple processing steps involved hence in improving the yields and costs of the recovery process. Here, we describe an integrated purification process of glutamate decarboxylase (GAD) from E. coli cell extract using the established ATPS that consists of polyethylene glycol (PEG) and sodium sulfate.

Enhanced Production of Glutamate Decarboxylase by Batch, Fed-Batch, and Repeated Batch Cultivations of Escherichia coli

In order to obtain high yields of glutamate decarboxylase (GAD) to satisfy the growing market for gamma-aminobutyric acid (GABA), production of GAD by Escherichia coli was examined in batch, fed-batch, and repeated batch cultures. In fed-batch mode, the yield of dry cell mass was enhanced, which is beneficial for GAD production, and corresponded to 155.57% improvement of GAD production as compared to batch cultivation. However, extending the culture time to more than 52.5 h resulted in loss of cell mass and decreased GAD activity. Repeated batch culture demonstrated increases in average cell mass and GAD productivity (around 0.27 g L-1 h-1 and 1.8 U mL-1 h-1) in five cycles. An overall faster fermentation with a steady increase in yield (50.6% to 80.6%) occurred in the first three cycles by repeatedly replacing a portion of culture with fresh medium. Nevertheless, long-term operation (after the fifth batch) is not beneficial for cell growth or GAD production. Growth-associated characteristics of GAD production (GAD yield increased or decreased as the amount of cell mass altered) were observed in different cultivation modes of E. coli. The importance of this work was to first investigate various cultivation modes for GAD yield enhancement, which will serve as important research references for industrial purposes and fill in the research gap in comprehensively elucidating the performance of different feeding strategies with respect to GAD yield, productivity, by-product accumulation, process stability, and incubation cycle.

Biological and chemical phosphorus fractionalization in swine manure under aeration

In this work, the dynamic responses of different phosphorous factions in swine manure to aeration treatment were investigated and profiled to provide insight on potential ways to improve the biological phosphorus removal process. Batch reactors fabricated from clear acrylic columns were filled with fresh swine manure containing a 4.6 % solids content, which was aerated continuously for 15 days at an airflow rate of 2 L/min. The results indicate that the treatment can reduce soluble phosphorus (P) by about 78 % after only one-day aeration due largely to chemical precipitations. At the end of the experiment, the average soluble inorganic P level was reduced by 12 % (from 91 down to 79 %), while the average soluble organic P was increased by 13 % (from 9 to 22 %). The biomass P (DNA/RNA/poly-P) was increased by 24 % in the first three days, but only by 14 % in the rest 12 days of aeration. Also increased by aeration was the lipid P (47 %) but not the protein P. The data reveal that the current aeration rate cannot maintain a stable oxygen level [represented by the oxidation-reduction potential (ORP)] in the treated manure, evidenced by the decrease in ORP from 250 mV at the beginning to almost zero at the conclusion of the experiment, which is considered the major factor hindering the growth of aerobes, including the phosphorus-accumulating organisms (PAOs). Therefore, it may be concluded that continuous aeration of swine manure at a constant rate will not guarantee the supply of sufficient oxygen to the growth of PAOs. On another front, it was observed that too much aeration might negatively impact the overall P removal by increasing the release of soluble organic P from dead cells.