Histochemical and ultrastructural observations on feeding and digestion in Reighardia sternae (Pentastomida: Cephalobaenida)

Histological, histochemical and ultrastructural methods have been used to study gut structure and function in the cephalobaenid pentastomid Reighardia sternae (Diesing, 1864).
R. sternae feeds exclusively on blood. Haemolysis of ingested erythrocytes is thought to be initiated by non-specific esterase secreted by cells lining the posterior oesophagus. Only one other enzyme, acid phosphatase, was demonstrated; its activity is confined to the microvillar layer of the gastrodermis.
Digestion appears to be largely extracellular, as large volumes of haematin form in the gut lumen after haemolysis. However, this is supplemented by a significant amount of intracellular digestion: accumulations of iron-positive granules occur in approximately 20% of gastrodermal cells.
All gastrodermal cells are alike, and all can undertake the same intracellular digestive process. This is characterized by a number of morphologically distinct stages which have been studied using the electron microscope. The iron-positive particles distinguished under the light microscope were easily identifiable ultrastructurally. Spherical, weakly iron-positive bodies form within the cisternae of the endoplasmic reticulum. Phagosomes containing haemoglobin, or some breakdown product of haemoglobin, apparently bud off from the termini of a labyrinth system of tubules under the microvilli and fuse with the ER to form these phago-lysosomes. Intensely iron-positive heterolysosomes, also present in the cytoplasm of these cells, accumulate haemosiderin particles which are thought to be released into the cytoplasm during phago-lysosomal digestion.
The ensuing complex changes in cell morphology are, apparently, related to the elimination of spent phago-lysosomes. It has been suggested that the purpose of intracellular digestion is to provide a specific metabolite which the extracellular degradation of haemoglobin cannot supply.     

Haemosiderin and tissue damage

High levels of haemosiderin occur in iron overload syndromes such as idiopathic haemochromatosis or secondary iron overload in thalassaemic patients; haemosiderin is the predominant iron-storage compound in such cases. It consists of a large aggregate of FeOOH cores, many of which have an incomplete shell of protein, and is probably derived from ferritin by lysosomal proteolysis. In addition, some chemical degradation of the ferritin cores appears to occur on conversion to haemosiderin. Other biochemical components are phosphate and magnesium, which may be adsorbed to the core surface, and perhaps certain lipids. Haemosiderin may have a central role, either directly or indirectly, in iron cytotoxicity and therefore the chemistry and biochemistry of this material warrants further study.     

Biochemical studies of the iron cores and polypeptide shells of haemosiderin isolated from patients with primary or secondary haemochromatosis

Haemosiderin isolated from different iron-loading syndromes, primary haemochromatosis (PHC) and secondary haemochromatosis (SHC) biochemically exhibited differences in both their iron core and peptide composition. The rate of release of iron from PHC haemosiderin to oxalate was 3-fold greater than that from SHC haemosiderin. The major peptides separated by SDS-PAGE showed a major band at Mr 20,000 for PHC haemosiderin and at Mr 15,000 for SHC haemosiderin.     

Morphological aspects of blood digestion in a parasitic mite Bakericheyla chanayi

All life stages of B. chanayi (Acariformes: Cheyletidae) are characterized by occasional bloodsucking and a long period of digestion. No newly engorged mites were found during the period of their host birds' migration. The fine structure of the digestive tract of a blood-feeding acariform mite is described for the first time. The anterior midgut (AMG) is a place of blood digestion, while the posterior midgut (PMG) is involved in nitrogen metabolism forming guanine crystals as the main end-product. The AMG epithelium consists of digestive cells that probably arise from mitotically active basal cells with high synthesizing activity.As observed in ticks, blood digestion is accompanied by the formation of huge endosomes that serve as places of storage and sorting of ingested material. Digestive cells show different types of endocytotic activity as well as various late endosomes, which implies different subcellular pathways for different blood components. In both midgut regions, elimination of the excretory material occurs by apocrine secretion or by discharging of apical cell fragments (loaded with lysosomes) into the gut lumen. The formation of guanine granules occurs inside the lysosomes of PMG epithelial cells thus having much in common with intracellular digestion. Peculiarities of intracellular blood digestion were analyzed according to the modern hypothesis of endocytosis and compared to what is known in ticks.     

Anaerobic digestion of microalgal biomass with ultrasonic disintegration

The use of photosynthetic microalgae for nutrient removal and biofuel production has been widely discussed. Anaerobic digestion of waste microalgal biomass to produce biogas is a promising technology for bioenergy production. However, the methane yield from this anaerobic process was limited because of the hard cell wall of Chlorella vulgaris. The use of ultrasound has proven to be successful at improving the disintegration and anaerobic biodegradability of Chlorella vulgaris. Ultrasonic pretreatment in the range of 5–200 J ml⁻¹ was applied to waste microalgal biomass, which was then used for batch digestion. Ultrasound techniques were successful and showed higher soluble COD at higher applied energy. During batch digestion, cell disintegration due to ultrasound increased in terms of specific biogas production and the degradation rate. Compared to the untreated sample, the specific biogas production was increased in the ultrasound-treated sample by 90% at an energy dose of 200 J ml⁻¹. For the disintegrated samples, volatile solids reduction was also increased according to the energy input and degradation. These results indicate that the hydrolysis of microalgal cells is the rate-limiting step in the anaerobic digestion of microalgal biomass.     

白腐菌胞外提取液与纤维素酶对玉米秸秆的协同酶解研究

通过评价几株白腐菌固体发酵不同时间的胞外提取液与纤维素酶同时加入对玉米秸秆酶解产糖的影响,得到一个较高糖化效果的胞外降解体系,并对其相关影响因素进行研究,进而初探其协同酶解的机制.结果表明,Echinodontium taxodii 2538固体培养5 d的胞外提取液与纤维素酶形成的胞外降解体系的协同酶解效果较好,酸处...     

Ammonia inhibition in anaerobic digestion: A review

Even though ammonia is an essential nutrient for bacterial growth, it may inhibit methanogenesis during anaerobic digestion process if it is available at high concentrations. Therefore, ammonia is regarded as a potential inhibitor during anaerobic digestion, particularly when dealing with complex type of substrates such as manure or the organic fraction of municipal solid waste (OFMSW). Ammonia is produced through biological degradation of nitrogenous matter. Ammonium ion (NH₄⁺) and free ammonia (NH₃) are the two principal forms of inorganic ammonia nitrogen. Both forms can directly and indirectly cause inhibition in an anaerobic digestion system. Particularly, free ammonia (FAN) is a powerful inhibitor in an anaerobic digester above threshold concentrations. Process inhibition is related to the particular characteristics of the substrate to be anaerobically digested, pH, process temperature (mesophilic or thermophilic), type of the seed sludge (inoculum), the reactor configuration and to the concentrations of ammonium and ammonia. In this paper, ammonia inhibition in anaerobic digestion systems and the recovery efforts after inhibition are discussed. Furthermore, the impacts of ammonia inhibition on the microbial population available in anaerobic digesters, namely bacteria and Archaea, are also evaluated in detail.     

Biochemical studies on the isolation and characterization of human spleen haemosiderin.

Haemosiderin was isolated from thalassaemic human spleens by centrifugation through concentrated KI solutions. A method for solubilizing haemosiderin was developed which leaves the iron oxyhydroxide cores and constituent polypeptides intact, facilitating further purification and analysis. Purified haemosiderin contained no detectable haem, trace amounts of carbohydrate, and iron and phosphorus in a molar ration of 6:1; much of the phosphate may be present as core-adsorbed. Several lipids were present, but it is not certain whether these are contaminants or components of the haemosiderin granules. In all preparations examined, a characteristic group of six to seven peptides of apparent Mr 12 900-17 800 were found, with a major band at Mr 14 500 and, in addition, a minor component of Mr 42 000; these peptides co-chromatographed with the cores. Negatively stained electron micrographs suggest that these peptides form an incomplete shell about the cores, consistent with the view that haemosiderin is a proteolytic product of ferritin.     

Determination of haemoglobin in birds by a modified alkaline haematin (D-575) method

A recently published method for measuring human haemoglobin based on alkaline haematin (Zander et al., Clin. chem. Acta 136, 83-93, 1984) has been adopted for bird samples. The new method yields comparable haemoglobin values with that of a previously used alkaline haematin method. Levels of haemoglobin estimated using alkaline haematin were higher than for cyanhaemiglobin, the reference method for human haemoglobin. This difference is due to the loss of haemoglobin in the cyanhaemiglobin procedure due to insolubility. The values for haemoglobin found by the alkaline haematin method did not vary significantly between a range of bird species. The method overcomes some important deficiencies of the cyanhaemiglobin method, in particular, problems of turbidity and quality control assessment.     

The tidal rhythm of feeding and digestion in the pacific oyster, Crassostrea gigas (Thunberg)

A study has been made of the processes of extra- and intracellular digestion in the Pacific oyster, Crassostrea gigas (Thunberg) over two 24-h cycles in winter and summer.The results show that the digestive processes are discontinuous and related to the tidal cycle. Variations in tidal height resulting from a diurnal inequality of the tide affect both the relative dissolution of the crystalline style and the relative lengths of the constituent phases of the intracellular digestive process in the digestive diverticula. On a seasonal basis the style is present for a greater length of time in winter and, conversely, remains dissolved longer in summer. A seasonal variation in the structure of the digestive tubules has also been found.The results confirm conclusions reached earlier that the processes of extracellular digestion in the stomach and intracellular digestion in the digestive diverticula of intertidal bivalves are both discontinuous, alternate, and irrevocably interrelated since breakdown of the digestive cells of the digestive diverticula following intracellular digestion results in the dissolution of the crystalline style. The arrival of food in the stomach has a minimal effect upon the style. Moreover, the cytological structure of the digestive diverticula of C. gigas undergoes a series of synchronized changes related to the tidal cycle.     

The effect of enzyme addition on anaerobic digestion of JoseTall Wheat Grass

The effects of the addition of enzyme products containing cellulase, hemicellulase, and β-glucosidase to anaerobic digestion systems were studied using JoseTall Wheat Grass (wheat grass) as a model substrate. Anaerobic digestion tests were performed using batch reactors operated at 50 °C. The application of enzyme products in three digestion configurations were simulated and investigated: (1) enzyme addition to a single-stage digester, (2) pre-treatment of wheat grass with enzymes followed by a single-stage anaerobic digestion, and (3) enzyme addition to the first stage (hydrolysis and acidification) of a two-stage digestion system. The enzyme products showed positive effects on the solubilization of wheat grass when used alone to treat the wheat grass. However, no significant differences in biogas and methane yields, and volatile solids reduction resulted when the enzyme products were tested in the anaerobic digestion systems. This reveals that the microorganisms present in the inoculum were effective in carrying out the digestion of wheat grass. The types of microorganisms present in the inoculum were identified using 16S rRNA sequence analysis. A comparison of the sequences between the different inocula revealed that the prevalent operational taxonomic units were similar, but that the acidified inoculum contained a higher percentage of the species Thermotogae.     

THE UPTAKE OF IRON IN RABBIT SYNOVIAL TISSUE FOLLOWING INTRA-ARTICULAR INJECTION OF IRON DEXTRAN : A Light and Electron Microscope Study

Iron dextran (molecular weight 7,000) diffuses rapidly from the joint cavity through the synovium, along lymphatics and extracellular tissue spaces; articular cartilage is impermeable to iron dextran. There is also rapid cellular uptake by synovial lining cells, particularly of the vacuolar type; endoplasmic reticulum-containing lining cells rarely take up iron dextran. Cellular uptake is probably effected by pseudopodial folds projecting from the cell surface and enclosing extracellular material. Cells containing iron may degenerate and be ingested by phagocytes, and this may account for the concentration of iron in a smaller proportion of cells on or below the synovial surface in the later stages. At 6 to 18 hours after injection there is a mild inflammatory reaction and some synovial proliferation; from this stage onwards intracellular iron occurs in the form of haemosiderin. Granules of haemosiderin are present in the synovium 3 months after injection and possibly longer.     

Degradation of aflatoxin B1 during anaerobic digestion and its effect on process stability

The effect of aflatoxin B1 (AFB1) on an anaerobic digestion process (AD) was studied. Batch anaerobic digestion trials were performed with both non-contaminated AFB1 corn grain (Control A) and contaminated-AFB1 corn grain at different doses (AFB1 contents of 0.54, 66.2, and 110 μg kg⁻¹ wet weight). Both cumulative biogas production and the degradation rate of AFB1 were studied. Results indicated that no adverse effects on AD were detected during the processes which could be attributed to the presence of AFB1. AFB1 degradation ranged from 69% to 87% of the total initial AFB1 content.Anaerobic digestion trials using Completely Stirred Tank Reactors (CSTR) were also carried out, comparing the biogas production of a mix of contaminated corn grain plus pig slurry (AFB1 content of 7.2 μg kg⁻¹ wet weight) with a mix of non-contaminated corn grain plus pig slurry (Control B). No adverse effect of AFB1 on biogas production was detected. The CSTR trial resulted in an average degradation of AFB1 of 42%. The further storage of the digestate for 30 days resulted in an overall degradation (CSTR plus storage) of AFB1 of 61% of the starting content.     

Studies on haemosiderin and ferritin from iron-loaded rat liver

Summary Haemosiderin has been isolated from siderosomes and ferritin from the cytosol of livers of rats iron-loaded by intraperitoneal injections of iron-dextran. Siderosomal haermosiderin, like ferritin, was shown by electron diffraction to contain iron mainly in the form of small particles of ferrihydrite (5Fe₂O₃ · 9H₂O), with average particle diameter of 5.36±1.31 nm (SD), less than that of ferritin iron-cores (6.14±1.18 nm). Mössbauer spectra of both iron-storage complexes are also similar, except that the blocking temperature,T _B, for haemosiderin (23 K) is lower than that of ferritin (35 K). These values are consistent with their differences in particle volumes assuming identical magnetic anisotropy constants. Measurements of P/Fe ratios by electron probe microanalysis showed the presence of phosphorus in rat liver haemosiderin, but much of it was lost on extensive dialysis. The presence of peptides reacting with anti-ferritin antisera and the similarities in the structures of their iron components are consistent with the view that rat liver haemosiderin arises by degradation of ferritin polypeptides, but its peptide pattern is different from that found in human-thalassaemia haemosiderin. The blocking temperature, 35 K, for rat liver ferritin is near to that reported, 40 K, for human-thalassaemia spleen ferritin. However, the haemosiderin isolated from this tissue, in contrast to that from rat liver, had aT _B higher than that of ferritin. The iron availability of haemosiderins from rat liver and human-thalassaemic spleen to a hydroxypyridinone chelator also differed. That from rat liver was equal to or greater, and that from human spleen was markedly less, than the iron availability from either of the associated ferritins, which were equivalent. The differences in properties of the two types of haemosiderin may reflect their origins from primary or secondary iron overload and differences in the duration of the overload.     

Transition of microbial communities during the adaption to anaerobic digestion of carrot waste

In this study a microbial community suitable for anaerobic digestion of carrot pomace was developed from inocula obtained from natural environmental sources. The changes along the process were monitored using pyrosequencing of the 16S rRNA gene. As the community adapted from a diverse natural community to a community with a definite function, diversity decreased drastically. Major bacterial groups remaining after enrichment were Bacilli (31-45.3%), Porphyromonadaceae (12.1-24.8%) and Spirochaetes (12.5-18.5%). The archaeal population was even less diverse and mainly represented by a single OTU that was 99.7% similar to Methanosarcina mazei. One enrichment which failed to produce large amounts of methane had shifts in the bacterial populations and loss of methanogenic archaea.     

Methionine transport in the malaria parasite Plasmodium falciparum

The intraerythrocytic malaria parasite, Plasmodium falciparum, derives amino acids from the digestion of host cell haemoglobin. However, it also takes up amino acids from the extracellular medium. Isoleucine is absent from adult human haemoglobin and an exogenous source of isoleucine is essential for parasite growth. An extracellular source of methionine is also important for the normal growth of at least some parasite strains. In this study we have characterised the uptake of methionine by P. falciparum-infected human erythrocytes, and by parasites functionally isolated from their host cells by saponin-permeabilization of the erythrocyte membrane. Infected erythrocytes take up methionine much faster than uninfected erythrocytes, with the increase attributable to the flux of this amino acid via the New Permeability Pathways induced by the parasite in the erythrocyte membrane. Having entered the infected cell, methionine is taken up by the intracellular parasite via a saturable, temperature-dependent process that is independent of ATP, Na⁺ and H⁺. Substrate competition studies, and comparison of the transport of methionine with that of isoleucine and leucine, yielded results consistent with the hypothesis that the parasite has at its surface one or more transporters which mediate the flux into and out of the parasite of a broad range of neutral amino acids. These transporters function most efficiently when exchanging one neutral amino acid for another, thus providing a mechanism whereby the parasite is able to import important exogenous amino acids in exchange for surplus neutral amino acids liberated from the digestion of host cell haemoglobin.     

Ultrasonic pretreatment for thermophilic aerobic digestion in industrial waste activated sludge treatment

In order to enhance the degradation efficiency of waste activated sludge (WAS) by thermophilic aerobic digestion, an ultrasonic pretreatment was examined. It was observed that ultrasonic pretreatment increased the solubilization of organic matter in the WAS and that the solubilization ratio of the organics increased during the first 30 min but did not extensively increase thereafter. Therefore, a pretreatment time of 30 min was determined to be the economical pretreatment time from the experimental results. From the digestion experiments, which was conducted using the WAS collected from an oil refinery plant in Inchon, Korea, investigating the effects of an ultrasonic pretreatment on thermophilic aerobic digestion, it was confirmed that the proposed ultrasonic pretreatment was effective at enhancing the release of the cellular components in WAS and the degradation of released components in the thermophilic aerobic digestion.     

A titration approach to identify the capacity for starch digestion in milk-fed calves

Calf milk replacers (MR) commonly contain 40% to 50% lactose. For economic reasons, starch is of interest as a lactose replacer. Compared with lactose, starch digestion is generally low in calves. It is, however, unknown which enzyme limits the rate of starch digestion. The objectives were to determine which enzyme limits starch digestion and to assess the maximum capacity for starch digestion in milk-fed calves. A within-animal titration study was performed, where lactose was exchanged stepwise for one of four starch products (SP). The four corn-based SP differed in size and branching, therefore requiring different ratios of starch-degrading enzymes for their complete hydrolysis to glucose: gelatinised starch (α-amylase and (iso)maltase); maltodextrin ((iso)maltase and α-amylase); maltodextrin with α-1,6-branching (isomaltase, maltase and α-amylase) and maltose (maltase). When exceeding the animal’s capacity to enzymatically hydrolyse starch, fermentation occurs, leading to a reduced faecal dry matter (DM) content and pH. Forty calves (13 weeks of age) were assigned to either a lactose control diet or one of four titration strategies (n=8 per treatment), each testing the stepwise exchange of lactose for one SP. Dietary inclusion of each SP was increased weekly by 3% at the expense of lactose and faecal samples were collected from the rectum weekly to determine DM content and pH. The increase in SP inclusion was stopped when faecal DM content dropped below 10.6% (i.e. 75% of the average initial faecal DM content) for 3 consecutive weeks. For control calves, faecal DM content and pH did not change over time. For 87% of the SP-fed calves, faecal DM and pH decreased already at low inclusion levels, and linear regression provided a better fit of the data (faecal DM content or pH v. time) than non-linear regression. For all SP treatments, faecal DM content and pH decreased in time (P<0.001) and slopes for faecal DM content and pH in time differed from CON; P<0.001 for all SP), but did not differ between SP treatments. Faecal DM content of SP-fed calves decreased by 0.57% and faecal pH by 0.32 per week. In conclusion, faecal DM content and pH sensitively respond to incremental inclusion of SP in calf MR, independently of SP characteristics. All SP require maltase to achieve complete hydrolysis to glucose. We therefore suggest that maltase activity limits starch digestion and that fermentation may contribute substantially to total tract starch disappearance in milk-fed calves.     

Development of continuous microwave-assisted protein digestion with immobilized enzyme

In this study, an easy and efficiency protein digestion method called continuous microwave-assisted protein digestion (cMAED) with immobilized enzyme was developed and applied for proteome analysis by LC–MSⁿ. Continuous microwave power outputting was specially designed and applied. Trypsin and bromelain were immobilized onto magnetic micropheres. To evaluate the method of cMAED, bovine serum albumin (BSA) and protein extracted from ginkgo nuts were used as model and real protein sample to verify the digestion efficiency of cMAED. Several conditions including continuous microwave power, the ratio of immobilized trypsin/BSA were optimized according to the analysis of peptide fragments by Tricine SDS–PAGE and LC–MSⁿ. Subsequently, the ginkgo protein was digested with the protocols of cMAED, MAED and conventional heating enzymatic digestion (HED) respectively and the LC–MSⁿ profiles of the hydrolysate was compared. Results showed that cMAED combined with immobilized enzyme was a fast and efficient digestion method for protein digestion and microwave power tentatively affected the peptide producing. The cMAED method will be expanded for large-scale preparation of bioactive peptides and peptide analysis in biological and clinical research.     

PHB recovery by hypochlorite digestion of non-PHB biomass

Summary Hypochlorite digestion of bacterial biomass from intracellular poly--hydroxybutyrate (PHB) has not been used on a large scale since it has been reported to severely degrade PHB. In this study, to minimize degradation, the initial biomass concentration, digestion time and pH of the hypochlorite solution were optimized. Consequently, PHB of 95% purity with a weight average molecular weight (M_W) of 600,000 and a polydispersity index (PI) of 4.5 was recovered from biomass initially containing PHB with a M_W of 1,200,000 and a PI of 3.