Effect of enzymes on anaerobic digestion of primary sludge and septic tank performance

Enzyme additives are believed to improve septic tank performance by increasing the hydrolysis and digestion rates and maintaining a healthy microbial population. Previous studies reported mixed results on the effectiveness of enzymes on mesophilic and thermophilic digestion, and it is not clear whether enzymes would be effective under septic tank conditions where there is no heating or mixing, quantities of enzymes added are small, and they can be washed out quickly. In this study, batch reactors and continuous-flow reactors designed and operated as septic tanks were used to evaluate whether enzymatic treatment would increase the hydrolysis and digestion rates in primary sludge. Total solids, volatile solids, total suspended solids, total and soluble chemical oxygen demand, concentrations of protein, carbohydrate, ammonia and volatile acids in sludge and effluent samples were measured to determine the differences in digestion rates in the presence and absence of enzymes. Overall, no significant improvement was observed in enzyme-treated reactors compared with the control reactors.     

Neutral fat hydrolysis and long-chain fatty acid oxidation during anaerobic digestion of slaughterhouse wastewater

Neutral fat hydrolysis and long-chain fatty acid (LCFA) oxidation rates were determined during the digestion of slaughterhouse wastewater in anaerobic sequencing batch reactors operated at 25 degrees C. The experimental substrate consisted of filtered slaughterhouse wastewater supplemented with pork fat particles at various average initial sizes (D(in)) ranging from 60 to 450 microm. At the D(in) tested, there was no significant particle size effect on the first-order hydrolysis rate. The neutral fat hydrolysis rate averaged 0.63 +/- 0.07 d(-1). LCFA oxidation rate was modelled using a Monod-type equation. The maximum substrate utilization rate (kmax) and the half-saturation concentration (Ks) averaged 164 +/- 37 mg LCFA/L/d and 35 +/- 31 mg LCFA/L, respectively. Pork fat particle degradation was mainly controlled by LCFA oxidation rate and, to a lesser extent, by neutral fat hydrolysis rate. Hydrolysis pretreatment of fat-containing wastewaters and sludges should not substantially accelerate their anaerobic treatment. At a D(in) of 450 microm, fat particles were found to inhibit methane production during the initial 20 h of digestion. Inhibition of methane production in the early phase of digestion was the only significant effect of fat particle size on anaerobic digestion of pork slaughterhouse wastewater. Soluble COD could not be used to determine the rate of lipid hydrolysis due to LCFA adsorption on the biomass.     

Mathematical modeling of non-ideal mixing continuous flow reactors for anaerobic digestion of cattle manure

Most conventional digesters used for animal wastewater treatment include continuously stirred-tank reactors. While imperfect mixing patterns are more common than ideal ones in real reactors, anaerobic digestion models often assume complete mixing conditions. Therefore, their applicability appears to be limited. In this study, a mathematical model for anaerobic digestion of cattle manure was developed to describe the dynamic behavior of non-ideal mixing continuous flow reactors. The microbial kinetic model includes an enzymatic hydrolysis step and four microbial growth steps, together with the effects of substrate inhibition, pH and thermodynamic considerations. The biokinetic expressions were linked to a simple two-region liquid mixing model, which considered the reactor volume in two separate sections, the flow-through and the retention regions. Deviations from an ideal completely mixed regime were represented by changing the relative volume of the flow-through region (a) and the ratio of the internal exchange flow rate to the feed flow rate (b). The effects of the hydraulic retention time, the composition of feed, the initial conditions of the reactor and the degree of mixing on process performance can be evaluated by the dynamic model. The simulation results under different conditions showed that deviations from the ideal mixing regime decreased the methane yield and resulted in a reduced performance of the anaerobic reactors. The evaluation of the impact of the characteristic mixing parameters (a) and (b) on the anaerobic digestion of cattle manure showed that both liquid mixing parameters had significant effects on reactor performance.     

Use of thermitase in the cultivation of embryonal mouse fibroblasts

Thermitase has been investigated as a means for obtaining single cells from tissue material, for enzymatic detachment of cultivated cells from the substrate and rarification of cells with subsequent passaging of mouse embryonic fibroblasts. The action of this enzyme was compared with that of "trypsin for cell cultivation". Tissue digestion showed that thermitase at 1/50 of the enzyme concentration needed with trypsin, was 1.5 fold more effective in yielding single cells. In cell cultivation thermitase is able to detach cells from the substrate at the same low concentration of 0.05 mg enzyme/ml and to give sufficient rarification, without the need of adding complexing agents. Rate of attachment, cell form and multiplication in subcultures corresponded to those after application of trypsin. The best results in cell detachment and rarification and the most uniform cell morphology were obtained with thermitase at a concentration of 0.025 mg enzyme/ml under addition of 4 mM of complexing agent. At that, thermitase proved 50 fold more effective than trypsin. Another advantage of thermitase is its better storage quality at 4 degrees C in dissolved form.     

Enzyme-substrate interactions in the hydrolysis of peptide substrates by thermitase, subtilisin BPN', and proteinase K

Peptide substrates of the general structure acetyl-Alan (n = 2-5), acetyl-Pro-Ala-Pro-Phe-Alan-NH2 (n = 0-3), and acetyl-Pro-Ala-Pro-Phe-AA-NH2 (AA = various amino acids) were synthesized and used to investigate the enzyme-substrate interactions of the microbial serine proteases thermitase, subtilisin BPN', and proteinase K on the C-terminal side of the scissile bond. The elongation of the substrate peptide chain up to the second amino acid on the C-terminal side (P'2) enhances the hydrolysis rate of thermitase and subtilisin BPN', whereas for proteinase K an additional interaction with the third amino acid (P'3) is possible. The enzyme subsite S'1 specificity of the proteases investigated is very similar. With respect to kcat/Km values small amino acid residues such as Ala and Gly are favored in this position. Bulky residues such as Phe and Leu were hydrolyzed to a lower extent. Proline in P'1 abolishes the hydrolysis of the substrates. Enzyme-substrate interactions on the C-terminal side of the scissile bond appear to affect kcat more than Km for all three enzymes.     

Optimization of protein extraction from Gelidiella acerosa by carbohydrases using response surface methodology

In this study, application of response surface methodology for enzymic pretreatment optimization of Gelidiella acerosa was investigated in order to improve the extraction of algal proteins using Viscozyme L and Celluclast 1.5L. The total protein, soluble proteins and reducing sugar recovery in the water‐soluble fraction were studied in relation to the hydrolysis time, type and concentration of the enzymes. Enzymatic digestion appeared to be an effective treatment for protein extraction. While enzyme hydrolysis by Celluclast 1.5L was able to facilitate the protein extraction, it was a relatively inefficient way to improve protein extraction yield, in comparison with Viscozyme L. The optimum conditions for protein extraction was found to be hydrolysis by 2.8 μL mL^?1 of Viscozyme L for 12 h.     

A novel organic-inorganic hybrid monolith for trypsin immobilization

In proteomics, attention has focused on various immobilized enzyme reactors (IMERs) for the realization of high throughput digestion. In this report, a novel organic-inorganic hybrid monolith based IMER was prepared in a 100 μm i.d. capillary with 3-glycidoxypropyltrimethoxysilane (GLYMO) as the monomer and tetraethoxysilane (TEOS) as the crosslinker. Trypsin immobilization was achieved via the reaction between vicinal diol groups, which were obtained from hydrolysis of epoxy groups, and the amino groups of trypsin. Bovine serum albumin was digested thoroughly by this IMER in 47 s. After micro-reverse phase liquid chromatography-tandem mass spectrometry (μRPLC-MS/MS) analysis and database searching, beyond 35% sequence coverage was obtained, and the result was comparable to that of 12 h in solution digestion. The present IMER has potential for high throughput digestion.     

Influence of Guar Gum on the In Vitro Starch Digestibility—Rheological and Microstructural Characteristics

The present study investigates the effect of guar gum on the digestibility of a waxy maize starch in vitro under simulated gastric and intestinal conditions. A detailed rheology and confocal scanning laser microscopy of the digesta were performed in order to study the possible mechanisms of interactions involved during in vitro hydrolysis of starch. No starch hydrolysis was observed under simulated gastric conditions, whereas more than 90% hydrolysis was observed at the end of digestion under simulated intestinal conditions. In the presence of guar gum, the starch hydrolysis was reduced by nearly 25% during the first 10 min and by 15% at the end of in vitro intestinal digestion. The rheological behavior of the digesta was significantly affected in the presence of the gum. The viscosity of digesta decreased during intestinal digestion; however, the extent of decrease was quite low in the presence of guar gum. The consistency index increased and flow behavior index of digesta decreased in the presence of gum after 1 min of intestinal digestion. All the samples (digested or undigested) displayed a pseudoplastic behavior as their apparent viscosity (η _a) decreased with an increase in shear rate. A negative correlation between the starch hydrolysis (%) and storage modulus for the starch sample without gum and a positive correlation for the starch sample with gum were found. Large granule remnants observed through confocal micrographs have shown that the solubilization of starch granule remnants during in vitro digestion is significantly reduced in the presence of gum.     

Use of microbial peptide inhibitors for characterization of the substrate specificity of thermitase,a thermostable serine protease fromThermoactinomyces vulgaris

Seven microbial peptide inhibitors—chymostatin, antipain, elastatinal, leupeptin, pepstatin, bestatin, and phosphoramidon—were tested for their efficiency to inhibit thermitase, a thermostable serine protease fromThermoactinomyces vulgaris. Chymostatin and antipain were the most effective inhibitors, with K_i values of 7×10^–8 M and 2×10^–7 M, respectively. Except for leupeptin, all inhibitors resist hydrolysis by thermitase. Leupeptin, however, is cleaved by thermitase between the two leucylresidues. Further, a close relationship in specificity between thermitase and subtilisin BPN and their distinct discrimination from elastase specificity was demonstrated by using these inhibitors.     

Formation,Stability and In Vitro Digestion of β-carotene in Oil-in-Water Milk Fat Globule Membrane Protein Emulsions

The formation, stability and in vitro digestion of milk fat globule membrane (MFGM) proteins stabilized emulsions with 0.2 wt% β-carotene were investigated. The average particle size of β-carotene emulsions stabilized with various MFGM proteins levels (1%, 2%, 3%, 4%, 5% wt%) decreased with the increase of MFGM proteins levels. When MFGM proteins concentration in emulsions is above 2%, the average particle size of β-carotene emulsions is below 1.0 μm. A quite stable emulsion was formed at pH 6.0 and 7.0, but particle size increased with decrease in acidity of the β-carotene emulsion. β-carotene emulsions stabilized with MFGM proteins were stable with a certain salt concentrations (0–500 mMNaCl). β-carotene emulsions were quite stable to aggregation of the particles at elevated temperature and time (85 °C for 90 min). At the same time, β-carotene emulsions were stable against degradation under heat treatment conditions. In vitro digestion of β-carotene emulsion showed the mean particle size of β-carotene emulsions stabilized with MFGM proteins in the simulated stomach conditions and intestinal conditions is larger than that of initial emulsions and simulated mouth conditions. Confocal laser scanning microscopy of β-carotene MFGM proteins emulsions also showed the corresponding results to different vitro digestion model. There was a rapid release of free fatty acid (FFA) during the first 10 min and after this period, an almost constant 70% digestion extent was reached. Approximately 80% of β-carotene was released within 2 h of incubation under the simulated intestinal fluid. These results showed that MFGM protein can be used as a good emulsifier in emulsion stabilization, β-carotene rapid release as well as lipophilic bioactive compounds delivery.     

Stomach digestion and its effect upon protein hydrolysis in the intestine of rainbow trout (Salmo gairdneri Richardson)

• 1.1. The in vitro digestion of soya protein by pancreatic proteases of the trout was measured following various conditions of stomach digestion.
• 2.2. Peptic hydrolysis results in an increase of small peptides.
• 3.3. Acid treatment and peptic proteolysis do not affect the degradation of insoluble proteins or the formation of free amino acids during intestinal digestion, but they do lead to a significant shift from soluble polypeptides to di- and oligopeptides.

     

Kinetic studies on urea hydrolysis by immobilized urease in a batch squeezer and flow reactor

The immobilization of urease on the reticulated polyurethane foam, and the kinetic phenomenon of urea hydrolysis by the resulting immobilized urease in both batch squeezer and circulated flow reactors were studied. Urease was immobilized with bovine serum albumin and glutaraldehyde on polyurethane foam support of 7 to 15 mum thickness. The residual apparent activity of urease after immobilization was about 50%. The good hydrodynamic property and flexibility of polyurethane foam were retained in solution after immobilization. A modified biofilm reactor model was used to describe the kinetic phenomenon of urea hydrolysis in both batch squeezer and circulated flow reactors. The characteristic parameters of the reactor model for both bioreactors were obtained by combining the Rosenbrock optimization method, the Rungs-Kutta method, and the Newton-Raphson method. The best-fit results were in good agreement with the experimental data. This study suggests another application of polyurethane foam in enzyme immobilization and immobilized enzyme reactors, which offers potential for practical applications in various bioreactors. (c) 1992 John Wiley & Sons, Inc.     

ADP-ribosylation induced changes in the conformation of the chromatin of the brain of developing rats

Conformational changes in the chromatin of the cerebral hemisphere of 3-, 14- and 30-day old developing rats were studied before and after its ADP-ribosylation using DNase I and micrococcal nuclease (MNase). The rate and extent of digestion of chromatin by DNase I are the highest at 3-day and decline progressively thereafter. The rate and extent of digestion by MNase do not change during development. ADP-ribosylation of chromosomal proteins was carried out by incubating nuclei with NAD+ for 30 min and was followed by endonuclease digestion. Both the rate and extent of digestion by DNase I and MNase were enhanced after ADP-ribosylation which was the maximum for 3-day rats.     

Distributed model of solid waste anaerobic digestion: effects of leachate recirculation and pH adjustment

A distributed model of solid waste digestion in a 1-D bioreactor with leachate recirculation and pH adjustment was developed to analyze the balance between the rates of polymer hydrolysis/acidogenesis and methanogenesis during the anaerobic digestion of municipal solid waste (MSW). The model was calibrated on previously published experimental data generated in 2-L reactors filled with shredded refuse and operated with leachate recirculation and neutralization. Based on model simulations, both waste degradation and methane production were stimulated when inhibition was prevented rapidly from the start, throughout the reactor volume, by leachate recirculation and neutralization. An optimal strategy to reduce the time needed for solid waste digestion is discussed.     

Effects of ammonia on hydrolysis of proteins and lipids from fish residues

This study investigated the influence of ammonia on the hydrolysis rates of proteins and lipids in fish residues under mesophilic anaerobic incubation at a neutral pH. The hydrolysis kinetics of the fish residues, which contained primarily proteins and lipids, were examined at initial ammonia concentrations of 0–16 g N l⁻¹. Carbon hydrolysis was suppressed more by ammonium in the acidogenesis phase than in the acidogenesis/methanogenesis period of a single-stage anaerobic digestion. Conversely, hydrolysis of compounds containing nitrogen was similarly suppressed by ammonia during acidogenesis and acidogenesis/methanogenesis phases of a single-stage anaerobic digestion. Parameter uncertainty analysis demonstrated that the proteins fraction in the fish residues was entirely biodegradable. Model fitting demonstrated that two fractions of lipid substrates exist, namely, easy and hard to biodegrade with hydrolysis rates that were affected differently by ammonia content.     

Sensitivity of Coxiella burnetii peptidoglycan to lysozyme hydrolysis and correlation of sacculus rigidity with peptidoglycan-associated proteins

The protease-resistant proteins associated with the peptidoglycan (PG) of the phase I small-cell variant Coxiella burnetii were either partially released from the PG by boiling the PG-protein complex (PG-PC) in sodium dodecyl sulfate containing 2-mercaptoethanol and EDTA or totally released by 1 N NaOH hydrolysis at 23 degrees C. An 18,300-dalton protein was released from the PG-PC under reducing conditions, whereas 1 N NaOH treatment extracted PG-associated proteins without apparent dissolution of the PG. Purified PG was composed of muramic acid, glucosamine, glutamic acid, alanine, and meso-diaminopimelic acid in a molar ratio of 0.9:0.9:1.0:1.4:1.0. Lysozyme hydrolysis of cell walls, PG-PC, and purified PG caused an increase in reducing groups which correlated with roughly 60 to 100% digestion of disaccharides. There was no significant decrease in turbidity during lysozyme hydrolysis of cell walls and PG-PC; however, hydrolysis of purified PG caused about 90% decrease in turbidity. Approximately 60% of the meso-diaminopimelic acid groups of PG were not susceptible to dinitrophenylation, thus, demonstrating an apparent contribution of PG-associated proteins, rather than cross-linkage between peptides, to sacculus rigidity of cell wall and PG-PC. This association of PG and protease-resistant covalently bound proteins may be important structural and functional determiners of resistance to both environmental conditions and intracellular digestion of C. burnetii by eucaryotic cells.     

Characteristics of carbohydrate degradation and the rate-limiting step in anaerobic digestion

The characteristics of the degradation of cellulose, soluble starch, and glucose in the acidogenic phase and the effects of the substrate loading rate and biological solids retention time on the methanogenic phase of anaerobic digestion were investigated. The results obtained from continuous experiments using laboratory-scale anaerobic chemostat reactors elucidated the true rate-limiting step of anaerobic digestion. The specific rate of substrate utilization decreased in the following order: glucose, soluble starch, acetic acid, and cellulose. The rate of the hydrolysis of cellulose was so low that this was shown to be the rate-limiting step in overall anaerobic digestion. Among methanogenic bacteria Methanosarcina would provide a higher substrate utilization rate than Methanothrix, and the maximum allowable substrate loading rate in the methanogenic phase was 11.2 g acetic acid/L day.     

Adsorption and recovery of cellulases during hydrolysis of newspaper

The adsorption of cellulases from Trichoderma viride was studied during the hydrolysis of newspaper. By measuring individual enzyme activities it was found that in the early stage of hydrolysis enzyme components showing C_xA; were adsorbed preferentially to those showing C₁A; afterwards ths situation was inverted. Electrophoretic resolution of proteins in hydrolysates showed a continuous decrease of enzyme proteins in solution, and furthermore suggested that the enzymes once adsorbed remained immobilized on the substrate (even after extensive digestion). Experiments to recover the enzyme that had remained in solution after typical hydrolysis showed a potential saving of up to 40%.     

Combination of online enzyme digestion with stable isotope labeling for high‐throughput quantitative proteome analysis

Various enzyme reactors and online enzyme digestion strategies have been developed in recent years. These reactors greatly enhanced the detection sensitivity and proteome coverage in qualitative proteomics. However, these devices have higher rates of miscleavage in protein digestion. Therefore, we investigated the effect of online enzyme digestion on the quantification accuracy of quantitative proteomics using chemical or metabolic isotope labeling approaches. The incomplete digestion would introduce some unexpected variations in comparative quantification when the samples are digested and then chemically isotope labeled in different aliquots. Even when identical protein aliquots are processed on these devices using post‐digestion chemical isotope labeling and the CVs of the ratios controlled to less than 50% in replicate analyses, about 10% of the quantified proteins have a ratio greater than two‐fold, whereas in theory the ratio is 1:1. Interestingly, the incomplete digestion with enzyme reactor is not a problem when metabolic isotope labeling samples were processed because the proteins are isotopically labeled in vivo prior to their simultaneous digestion within the reactor. Our results also demonstrated that both high quantification accuracy and high proteome coverage can be achieved in comparative proteome quantification using online enzyme digestion even when a limited amount of metabolic isotope labeling samples is used (1683 proteins comparatively quantified from 10⁵ Hela cells).     

Nutritional value of proteins from edible seaweed Palmaria palmata (dulse)

Palmaria palmata (Dulse) is a red seaweed that may be a potential protein source in the human diet. Its protein content, amino acid composition, and protein digestibility were studied with algae collected every month over a 1-year period. Significant variations in protein content were observed according to the season: The highest protein content (21.9 +/- 3.5%) was found in the winter-spring period and the lowest (11.9 +/- 2.0%) in the summer-early autumn period. Most of the essential amino acids were present throughout the year. After 6-hour in vitro digestion in a cell dialysis using porcine pepsin and porcine pancreatin, the digestibility of proteins from Palmaria palmata crude powder, represented by dialyzed nitrogen, was estimated at 29.52 +/- 1.47%. Relative digestibility was 56%, using casein hydrolysis as 100% reference digestibility. In vitro digestibility of proteins extracted in water was analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis using either bovine trypsin, bovine chymotrypsin, pronase from Streptomyces griseus, or human intestinal juice. Dulse proteins were hydrolyzed to a limited extent, which confirmed a rather low digestibility. Hydrolysis rate was higher with trypsin and lower with chymotrypsin compared with the two other enzymatic systems, pronase and intestinal juice, respectively. The association of algal powder and protein extract to casein and bovine serum albumin, respectively, produced a significant decrease in the hydrolysis rate of the standard proteins. In conclusion, the digestibility of Palmaria palmata proteins seems to be limited by the algae non-proteic fraction.