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Increases in fecal coliform bacteria resulting from centrifugal dewatering of digested biosolids.

In many countries, the classification of biosolids for disposal purposes can be based, in part, on fecal coliform levels, with alternative criteria also available based on the stabilization process used, such as anaerobic digestion. The assumption that these alternative criteria provide equivalent protection may be flawed. This paper demonstrates that fecal coliform levels determined after digestion do not always indicate the bacterial levels after the same biosolids have been dewatered by centrifugation. In samples from mesophilic digestion, half had significant increases in coliform numbers (P<0.05) with up to one order of magnitude increase during centrifugation, suggesting coliform regrowth. Thermophilically digested samples had significant increases of several orders of magnitude during dewatering, more likely from reactivation of viable but non-culturable coliforms than from regrowth. In other cases, centrifugation induced coliform regrowth or reactivation upon incubation and storage of dewatered samples, but not digested samples. These 2-3 order of magnitude increases occurred with both 25 and 37 degrees C incubations. Coliform increases continued for up to 5 days, then gradually declined. However, by day 20 coliform numbers were still 2 orders of magnitude greater than when originally sampled. The magnitude of the increases could be due either to regrowth or reactivation, but the nature of the longer-term increases--also seen in biosolids/soil mixtures--suggests regrowth. Differences in numbers between digested and dewatered samples could not be duplicated with high shear processing in lab-scale devices, with nitrogen purging to remove volatile or gaseous constituents, or with redilution using centrate. They could not be attributed to enumeration methods, to interference of Bacillus spp. on apparent coliform counts, or to temperature changes. The increases have practical implications in the use of fecal coliform or alternative criteria to define pathogen content in biosolids.

Centrifugation↗

Nutritional development of feeding strategies in arctic ruminants: digestive morphometry of reindeer, Rangifer tarandus, and muskoxen, Ovibos moschatus.

Reindeer have been classified as intermediate feeders and muskoxen as grazers based on differences in digestive morphology and consumption of fibrous plants. We hypothesized that the digestive morphology of young (< 2 months) reindeer and muskoxen anticipates transitions in diet and determines the feeding strategy of each species at adulthood. We compared structural morphology and rates of cell division in the rumen, abomasum, duodenum and liver of reindeer and muskoxen as neonates (1 day old), during the transition from milk to forage (30-60 days old) and in adults (> 7 yr). Development in utero provided the neonate with a functioning mucosa of the gastric abomasum and duodenal mucosa with high surface enlargement for digestion and absorption of concentrated milks. Transition to forage was preceded by changes in ruminal papillae structure that increased surface area and likely contributed to active fermentation by 60 days of age. The abomasum also increased in acid-secreting parietal cells during the transition to forage, which may enhance digestion of plant and microbial proteins. Rates of cell division also indicated a sustained differentiation of tissue structure during the transitional period. Young arctic ruminants expressed digestive structures that preceded full function, which indicated the ultimate feeding strategy of each species. For example, the rumen of young muskoxen had thick cornified epithelia and muscle layers that would provide ruminal mucosa with better protection from fibrous abrasion and enhance motility of bulky diets. Conversely, young reindeer had more complex papillary shapes in the rumen and more foliate villi in the duodenum, indicating a greater absorptive capacity of these structures than in muskoxen. Ontogenetic programs, therefore, play the primary role for digestive development of reindeer and muskoxen and determine the nutritional strategies of adults.

Journal Article↗

The sulphation pattern in chondroitin sulphate chains investigated by chondroitinase ABC and ACII digestion and reactivity with monoclonal antibodies.

We have used progressive chondroitinase digestion of pig aggrecan in conjunction with ELISA assays and disaccharide analysis to derive information about the pattern of 4- and 6-sulphation in chondroitin sulphate chains. Digestion with chondroitinase ABC resulted in the release of mainly disaccharides from the nonreducing terminal of chondroitin sulphate chains but there was also the release of some tetra- and hexa-saccharides which were degraded to disaccharides with more extensive digestion. Chondroitinase ACII, in contrast, released only disaccharides. Analysis of the disaccharide composition of the intact and digested products at different stages of digestion showed that there was a slight increase in 6-sulphate content of the chains as they were shortened. Reaction of the partially digested proteoglycans with monoclonal antibodies 3-B-3 and 3-D-5 which recognise chains terminating in 6- or 4-sulphated disaccharides, respectively, showed major differences between chondroitinase ABC and ACII products. The results suggested that chondroitinase ABC preferentially cleaved next to 4-sulphated, rather than 6-sulphated disaccharides and this resulted in some oligosaccharides as well as disaccharide being released. Chondroitinase ACII also cleaved an additional disaccharide next to the linkage to protein of chondroitin sulphate, which was not removed by chondroitinase ABC and this disaccharide was mainly nonsulphated.

Aggrecans↗

Computer simulation of high-performance liquid chromatographic separations of peptide and protein digests for development of size-exclusion, ion-exchange and reversed-phase chromatographic methods.

A computer program, called Pro Digest-LC, has been developed which assists scientists in devising methods of size-exclusion, cation-exchange and reversed-phase high-performance liquid chromatography for the analytical separation and purification of biologically active peptides and peptide fragments from enzymatic and chemical digests of proteins. Pro Digest-LC accurately predicts the retention behaviour of peptides of known composition, containing 2-50 amino acid residues, and simulates the elution profiles in all three modes of chromatography. In addition, Pro Digest-LC is a user-friendly program, designed as a teaching aid for both students and researchers in selecting the correct conditions for chromatography, that is, the mode of chromatography, column selection, mobile-phase selection, and has the ability to examine the effects of flow-rate, gradient-rate, and sample size on the separation. We have designed a set of peptide standards for each mode of chromatography to aid the researcher in eliminating non-specific interactions, to standardize retention behaviour on the user's columns, to monitor column performance and to compare packing materials. In the development of each prediction mode, experimental peak heights, peak widths, and retention times from model synthetic peptide standards were incorporated directly into the program and can be used as default values. Pro Digest-LC is an interactive program, in that researchers can run peptide standards on their particular columns and enter the peak width at half-height, peak height, retention time and quantity injected to adjust the simulation to their particular column. The simulated experiments eliminate the time-consuming trial-and-error methods used to suitable separation or purification procedures. The researcher would perform the actual experiment only after predicting the optimized conditions, thereby saving valuable sample and research time. The general concepts of the program along with representative separations of protein digests are displayed.

Animals↗

Potential for chlorate interference in ion chromatographic determination of total nitrogen in natural waters following alkaline persulfate digestion.

Determination of total nitrogen in aqueous samples after thermal potassium peroxydisulfate (persulfate) digestion is a commonly used alternative to the tedious Kjeldahl procedure. When ion chromatography is used to quantify the nitrate formed during digestion, there is a potential for interference from a chlorate peak if the digested sample initially contained chloride in concentrations close to or greater than the concentration of nitrogen. It was determined that this interference can be avoided either by using chromatographic conditions which cleanly resolve the nitrate and chlorate peaks (e.g., the Dionex AG9-HG column) or by using digestion reagent concentrations chosen to maintain a high pH throughout the digestion. The second alternative is not a viable option for investigators using a single digestion for both total nitrogen (TN) and total phosphorus (TP) analysis.

Chlorates↗

Enhanced pepsin digestion: a novel process for purifying antibody F(ab')(2) fragments in high yield from serum.

Enzyme-cleaved antibodies are used widely for the treatment of envenoming. Such products should comprise only 'highly pure' immunoglobulin fragments since Fc or other contaminating protein fragments or their aggregates may lead to side effects. The digestion of ovine antiserum and its purified IgG were investigated using pepsin and trypsin. Trypsin was effective at digesting purified IgG but unsuitable for the direct digestion of serum. In contrast, pepsin was highly effective at digesting all unwanted serum components to low molecular weight (< or =13 kDa) fragments while leaving the approximately 100-kDa F(ab')(2) intact. The optimum pH for pepsin digestion was between 3.25 and 3.50. The effects of salt concentration and pH on the digestion products were investigated by size exclusion chromatography under various conditions, which revealed a pH-dependent aggregation of some of the low molecular weight Fc and non-IgG fragments. These high molecular weight aggregates were not shown by SDS-PAGE. Unwanted low molecular weight fragments could be removed simply by diafiltration with a 30-kDa nominal molecular weight cutoff membrane and piperazine buffer (containing 150 mM NaCl, pH 6), leaving an F(ab')(2) solution contaminated only with some pepsin and a small amount of the aggregated low molecular weight fragments. These highly acidic contaminants were then removed easily using an anion exchange column and the F(ab')(2) produced following a subsequent concentration step was essentially free from pepsin and aggregates with a purity of over 96% and a yield of 19.3 g F(ab')(2)/l serum. This novel, high yield method for processing serum to highly pure F(ab')(2) avoids salt precipitation and centrifugation and should be suitable for large-scale production.

Animals↗

Digestion of host immunoglobulin and activity of midgut proteases in the buffalo fly Haematobia irritans exigua.

The digestion of blood by the buffalo fly (Haematobia irritans exigua) was monitored for 6h at 33 degrees C after a single meal. Following the meal, the concentration of soluble protein within the midgut increased to a peak at 2 hours then decreased steadily over the next 4h. The magnitude of the increase in soluble protein at 2h indicated a release of protein from another source; most likely from lysed red blood cells. The immunoglobulin (IgG) fraction of the blood meal was digested rapidly (50% within one hour of feeding) and fully digested within 4h. This is indicative of its accessibility to digestive enzymes within the midgut. In contrast, when flies had continuous access to blood, the concentration of IgG in the midgut remained at a more constant level. The loss of antigen-binding activity of a specific antibody was more rapid than complete degradation of the IgG, with 70% of binding activity lost within one hour of feeding. The level of trypsin activity in the midgut increased from pre-feeding levels to reach a peak at 2h before returning to basal levels after 6h. The pattern of trypsin activity follows closely that of the concentration of soluble protein in the midgut (r=0.88). The activity of leucine aminopeptidase in the midgut also increased immediately after feeding and remained elevated for 4 h before declining to a basal level after 6h. The rapid digestion of IgG and subsequent loss of antibody activity suggests that for a specific anti-buffalo fly antibody to be effective it would need to be able to either evade the digestive system or induce a rapid response.

Journal Article↗

Aerobic waste activated sludge (WAS) for start-up seed of mesophilic and thermophilic anaerobic digestion.

Since there are very limited numbers of thermophilic anaerobic digesters being operated, it is often difficult to start up a new one using sludge from an existing reactor as a seed. However, for obvious reasons it seems few attempts have been made to compare the start-up performance of thermophilic anaerobic digestion using different sources of seed sludges. The purpose of this study was to evaluate the start-up performance of anaerobic digestion using aerobic waste activated sludge (WAS) from a plant which has no anaerobic digesters and mesophilic anaerobic digested sludge (ADS) as the seed source at both mesophilic (35 degrees C) and thermophilic (55 degrees C) temperatures. In this study, two experiments were conducted. First, thermophilic anaerobic reactors were seeded with WAS (VSS = 4400 mg/L) and ADS (VSS = 14,500 mg/L) to investigate start-up performance with a feed of acetate as well as propionate. The results show that WAS started to produce CH4 soon after acetate feeding without a lag time, while ADS had a lag time of 10 days. When the feed was changed to propionate, WAS removed propionate down to below the detection limit of 10 mg/L, while ADS removed little propionate and produced little CH4. Second, in order to further compare the methanogenic activity of WAS and ADS, both mesophilic and thermophilic reactors were operated. WAS acclimated to anaerobic conditions shortly (< 5 days at both mesophilic and thermophilic) and after acclimating it produced more CH4 per unit amount of seeded VSS than ADS. WAS at mesophilic temperature biodegraded acetate at the same rate as for thermophilic. However WAS at mesophilic temperature biodegraded propionate at a much faster rate than at thermophilic. WAS as the seed source of anaerobic digestion resulted in much better performance than ADS at both mesophilic and thermophilic temperatures for both acetate and propionate metabolism.

Bacteria, Anaerobic↗

Scale-up of anaerobic digestion of the biowaste fraction from domestic wastes.

In the City of Karlsruhe/Germany anaerobic digestion of 7200 ta(-1) of separately collected biowaste has proven its feasibility at an organic loading rate (OLR) of up to 8.5 kg CODm(-3)d(-1). An extension of biowaste collection over the whole city area would increase the amount of biowaste to 12,000 ta(-1), leading to an OLR of the existing anaerobic reactor of up to 15 kg CODm(-3)d(-1). To test, whether the increased amount of biowaste could be stabilized in the existing plant, biowaste suspensions were digested in a laboratory reactor at a maximum OLR, that exceeded the future OLR of the full-scale plant. The laboratory reactor was started with effluent of the full-scale biowaste digester. Like in full-scale, biowaste suspension from the hydropulper was added in a fed-batch mode. The elimination of organic material (measured as COD, chemical oxygen demand) and the volumetric gas production were linearly increasing with the OLR from 4.3 to 19 kg CODm(-3)d(-1). Thus, safe operation of the full-scale plant at an OLR of 15 kg CODm(-3)d(-1) should be possible, leaving still some reserve capacity. To determine the metabolic reserves for fatty acid degradation during digestion at an OLR of 10 kg CODm(-3)d(-1), digester effluent was supplemented with either 40 mmoll(-1) acetate, propionate, i-butyrate or n-butyrate. Results of these batch assays indicated a rapid degradation of all fatty acids and fatty acid conversion rates, that would allow a stable anaerobic fermentation at 15 kg CODm(-3)d(-1)OLR. On the basis of the laboratory results the OLR of the full-scale methane reactor was increased to 15 kg CODm(-3)d(-1). After 7 months, results of full-scale digestion were still consistent with the previously obtained laboratory results.

Bacteria, Anaerobic↗

Generation of hemoglobin peptides in the acidic digestive vacuole of Plasmodium falciparum implicates peptide transport in amino acid production.

Intraerythrocytic malaria parasites avidly consume hemoglobin as a source of amino acids for incorporation into parasite proteins. An acidic organelle, the digestive vacuole, is the site of hemoglobin proteolysis. Early events in hemoglobin catabolism have been well studied. Two aspartic proteases, plasmepsins I and II, and a cysteine protease, falcipain, cleave hemoglobin into peptides. While it has been presumed that hemoglobin peptide fragments are degraded to individual amino acids by exopeptidase activity in the digestive vacuole, this hypothesis lacks experimental support. Incubation of human hemoglobin with P. falciparum digestive vacuole lysate generated a series of discrete peptide fragments with cleavage sites an average of 8.4 amino acids apart. No free amino acids could be detected and there was no evidence of peptide heterogeneity due to exopeptidase trimming. These sites correspond to points of cleavage previously established for plasmepsin I, plasmepsin II, and falcipain as well as some novel sites that suggest the existence of an additional endoproteinase. By colorimetric assay, P. falciparum has abundant aminopeptidase activity but this activity is not found in the digestive vacuoles and the parasite lacks detectable carboxypeptidase activity altogether. These data support a model for hemoglobin catabolism wherein small peptides are formed from cleavage of hemoglobin by the enzymes of the digestive vacuole and then are transported through the membrane of the digestive vacuole to the cytoplasm. There, exopeptidase activity converts the peptides to individual amino acids for parasite growth and maturation.

Amino Acids↗

Hydrogen peroxide induces rapid digestion of oligodendrocyte chromatin into high molecular weight fragments.

High molecular weight (HMW) fragmentation of nuclear chromatin was studied in cultured rat oligodendrocytes (OL) exposed to hydrogen peroxide (H2O2). Intact genomic DNA was isolated by agarose embedding, and analyzed by field inversion gel electrophoresis, with and without S1 endonuclease digestion to detect and discriminate between single and double stranded fragmentations, respectively. The exposure of OL to H2O2 resulted in a very rapid degradation of chromosomal DNA into HMW fragments that reflect native chromatin structure. Hence, within 10 min after the addition of 1 mM H2O2, a discrete pool representing approximately 45% of the nuclear chromatin underwent single strand digestion into >400 kb fragments likely at AT-rich matrix attachment regions. Subsequent accumulation of single stand breaks at these regions led to bifilar scission. Ultimately, chromatin within this susceptible pool was cleaved at remaining matrix attachment regions into 50-200 kb fragments. Chromatin digestion could be elicited with H2O2 concentrations as low as 50 microM. After the removal of H2O2, most >400 kb fragments were religated within 2 h; however, digestion into 50-200 kb fragments was irreversible. The DNA digestion was not accompanied by the degradation of nuclear proteins, i.e., lamins A/C and poly (ADP-ribose) polymerase indicating that chromatin fragmentation is unlikely to be mediated by proteolysis. In conclusion, H2O2 at pathologically relevant concentrations induces a very rapid and extensive digestion of OL chromatin into HMW fragments. Because the chromatin fragmentation is only partly reversible, it may be a decisive factor in committing oxidatively stressed OL to degeneration and/or death.

Animals↗

Evidence for the resistance of thyrotropin-releasing hormone (TRH) and pseudo-hormone, pyroglutamyl histidyl-amphetamine, to degradation by enzymes of the digestive tract in vitro.

TRH and pseudo-hormone (pyro Glu-His-amphetamine) were submitted to the digestion of chymotrypsin and prolidase and independently to the digestion of enzymes of the digestive track: pepsin (stomach), pancreatins (pancreas) and enzymes extracted from the intestinal mucosa (small intestine). Using thin layer chromatography and high voltage electrophoresis techniques to detect enzymic digestion products, only intact TRH and pseudo-hormone were found, indicating that both entities were, under the conditions used, resistant to in vitro digestion by enzymes of the digestive tract.

Amphetamines↗

Temporal analysis of DNA restriction digests by capillary electrophoresis.

We demonstrate a facile means for temporal analysis of DNA restriction enzyme digests by capillary electrophoresis-laser-induced fluorescence (CE-LIF) detection. phi X-174 DNA was digested with HaeIII restriction enzyme under conditions that allowed the monitoring of digestion as it proceeded toward completion. Separation by a polymer solution of methylcellulose in a polyacrylamide coated capillary allowed high resolution and a high degree of reproducibility between sequential runs. At pre-selected time intervals an injection of the digest, directly from the reaction mixture, was made. Sensitive detection was achieved by using ethidium bromide as an intercalation dye and allowing intercalation to occur on-column. It is demonstrated that the course of the digestion (i.e., the creation and diminishing of fragment peaks) can be followed using this methodology. Also demonstrated is the ability to use temporal analysis to determine ideal conditions for producing a single cut within a cloning and expression vector (pET3a-PAI-1) which contains 11 potential restriction endonuclease cleavage sites. This initial attempt to follow a restriction digest on-column not only provides meaningful information for the biochemical researcher, but also furthers the use of CE a diagnostic tool for the biochemical laboratory.

Bacteriophage phi X 174↗

Importance of complete DNA digestion in minimizing variability of 8-oxo-dG analyses.

Estimates of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA vary at least one order of magnitude using different quantitative methods or even the same method. Our hypothesis is that an incomplete DNA hydrolysis to nucleosides by the conventional nuclease P1 (NP1) and alkaline phosphatase (AP) digestion system plays an important role in contributing to the variability of measurements using HPLC coupled with UV and electrochemical (EC) detection. We show here that factors, such as the amount of DNA, choice of enzymes, their activities, and incubation time, can affect DNA digestion and, thus, cause variability in 8-oxo-dG levels. The addition of DNase I and phosphodiesterases I and II to the NP1 + AP system improves the DNA digestion by completely releasing normal nucleosides and 8-oxo-dG, thereby reducing the interday variations of 8-oxo-dG levels. Diethylenetriamine pentaacetic acid (DTPA), an iron chelator, prevented background increases of 8-oxo-dG during DNA digestion, as well as during the waiting period in the autosampler when a batch of DNA samples is analyzed by HPLC. After optimization of the DNA digestion conditions, the interday variability of 8-oxo-dG measurements using commercially available salmon testes DNA (ST DNA) were 26% over a period of 2 years. Under these optimal conditions, our laboratory variability may contribute as little as 13% to the overall variability as shown by assessment of oxidative DNA damage in a population of smokers. Based on our results, we believe that the modified DNA digestion conditions will provide much more accurate 8-oxo-dG determinations and, thus, more reliable estimates of cancer risk.

8-Hydroxy-2'-Deoxyguanosine↗

Effects of ammonia nitrogen of H2 and CH4 production during anaerobic digestion of dairy cattle manure.

A number of researchers have verified the inhibitory effects of elevated H2 concentrations on various anaerobic fermentation processes. The objective of this work was to investigate the potential for using hydrogen gas production to predict upsets in anaerobic digesters operating on dairy cattle manure. In an ammonia nitrogen overload experiment, urea was added to the experimental digesters to obtain increased ammonia concentrations (600, 1,500, or 3,000 mg N/l). An increase in urea concentration resulted in an initial cessation of H2 production followed by an increase in H2 formation. Additions of 600, 1,500, or 3,000 mg N/l initially resulted in the reduction of biogas H2 concentrations. After 24 h, the H2 concentration increased in the 600 and 1,500 mg N/l digesters, but production remained inhibited in the 3,000 mg N/l digesters. Both methane and total biogas production decreased following urea addition. Volatile solids reduction also decreased during these periods. The digester effluent pH and alkalinity increased due to the increased NH4 formed with added urea. Based on these results, changes in H2 concentration could be a useful parameter for monitoring changes due to increased NH3 in dairy cattle manure anaerobic digesters.

Ammonia↗

A novel fed-batch digestion system for biomethanation of plant biomasses.

Plant biomasses, which in the absence of adequate pretreatment pose serious operational problems in biogas production using conventional domestic flow-through digesters, can be successfully digested in a novel fedbatch digestion system that produces a steady rate of biogas. Basically, the system is a batch digestion operated with a regular input of a calculated amount of feed based on first order decay kinetics in order to maintain a regular biogas production rate. For nearly three years the system was tested in a laboratory-scale fed-batch digester (10 l) using dried water hyacinth as feed providing the desired biogas production rate. A field-scale domestic digester of masonry construction with a working volume of 10 m3 was designed and tested for about 9 months by feeding a mixture of dried water hyacinth or banana stem along with sugarcane press mud, yielding an average biogas production of 90-100% of the expected rate calculated on the basis of the feed rate.

Journal Article↗

C-terminal ladder sequencing via matrix-assisted laser desorption mass spectrometry coupled with carboxypeptidase Y time-dependent and concentration-dependent digestions.

The utility of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the analysis of C-terminal peptide ladders from carboxypeptidase Y (CPY) digestions is discussed. MALDI analysis of aliquots of an optimized time-dependent CPY digestion of ACTH 7-38 fragment allowed for the sequence of the first 19 amino acids from the C-terminus to be determined in 25 min of digestion time. A strategy for performing parallel concentration-dependent digestions on the MAL-DI plate is proven to be superior to the time-dependent approach as the method development time and practical amounts of both peptide and enzyme consumed are reduced significantly. The on-plate approach offered the same sequence information from the ACTH 7-38 fragment and was used to digest 22 peptides of various amino acid composition, size, charge, and polarity. Of the 22 peptides digested on-plate, sequence information was derived from 19 of them. A statistical analysis strategy for ladder sequencing utilizing t-statistics is offered as a method for placing confidence intervals on residue assignments.

Adrenocorticotropic Hormone↗

Microwave digestion of environmental and natural waters for selenium speciation.

A microwave preparation procedure is proposed for selenium speciation in natural and drinking waters. Different chemical reagents were tested, and the conditions for Se speciation were optimized. The effect of the different reagents on various oxidation states of selenium under microwave digestion conditions was investigated. Most of the Se(-II) was converted to selenite when digested with HNO3 and <20% to selenate. The digestion with H2O2/H2SO4 can change most Se species into Se(IV). The concentration of Se(IV) in the samples was then determined by HPLC with a fluorescence detector after derivatization with 2,3-diamino-naphthalene (DAN). The microwave preparation procedure allows Se speciation in water samples. Se(IV) was determined after concentrating the sample under nitrogen protection. The amount of Se(IV) and Se(VI) was measured by adding an equal volume of concentrated hydrochloric acid to water sample to reduce Se(VI) to Se(IV). Then the amount of Se(VI) can be calculated by subtraction. The total selenium can be determined after digestion with H2O2/H2SO4, or after digestion with HNO3 followed by reduction with concentrated hydrochloric acid. Selenium (-II, 0) was calculated by subtracting inorganic Se(IV+VI) from the total. Detection limits of 0.0066 ng and 0.0096 ng Se were obtained for HNO3 and H202/H2SO4 as digestion reagents, respectively. The total Se in the four water samples tested range from 0.20 to 0.90 microg L(-1). Among them the dominant form was Se(VI) with the exception of pond waters where Se(-II) predominated.

Chromatography, High Pressure Liquid↗