Integrated production of biogas and protein biomass from piggery manure

Summary An integrated process involving the production of biogas and the recovery of proteins by anaerobic digestion of piggery manure has been studied. the digestion, effected in a 616 L downflow stationary fixed film reactor, resulted in a biogas production rate of approximately 0.86 m³ per m³ reactor per day (35°C, hydraulic retention time 7.2 days). Treatment of the digested effluent by flocculation, decantation and/or sieving yielded a solid biomass with reduced coliform counts and a protein content of about 14%.     

Amino‐functionalized macroporous silica for efficient tryptic digestion in acidic solutions

Amino‐functionalized macroporous silica foam (NH₂‐MOSF) has been developed as a host reactor to realize highly efficient proteolysis in acidic solutions where normal tryptic reactions cannot occur. The digestion protocol consists simply of adding the functionalized NH₂‐MOSF into the protein and trypsin solutions without altering the bulk pH or preloading the enzymes on the materials. With this protocol, digestion of sample fractions from LC can be efficiently realized in the acidic solutions directly. Digestion of a protein fraction extracted from rat liver tissue after LC separation was performed to illustrate this principle, where 103 proteins were successfully identified at pH 3 after 1.5 h of tryptic digestion.     

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).     

Proteomic reactors and their applications in biology

Proteomic analysis requires the combination of an extensive suite of technologies including protein processing and separation, micro-flow HPLC, MS and bioinformatics. Although proteomic technologies are still in flux, approaches that bypass gel electrophoresis (gel-free approaches) are dominating the field of proteomics. Along with the development of gel-free proteomics, came the development of devices for the processing of proteomic samples termed proteomic reactors. These microfluidic devices provide rapid, robust and efficient pre-MS sample procession by performing protein sample preparation/concentration, digestion and peptide fractionation. The proteomic reactor has advanced in two major directions: immobilized enzyme reactor and ion exchange-based proteomic reactor. This review summarizes the technical developments and biological applications of the proteomic reactor over the last decade.     

Open Tubular Lab-On-Column/Mass Spectrometry for Targeted Proteomics of Nanogram Sample Amounts

A novel open tubular nanoproteomic platform featuring accelerated on-line protein digestion and high-resolution nano liquid chromatography mass spectrometry (LC-MS) has been developed. The platform features very narrow open tubular columns, and is hence particularly suited for limited sample amounts. For enzymatic digestion of proteins, samples are passed through a 20 µm inner diameter (ID) trypsin + endoproteinase Lys-C immobilized open tubular enzyme reactor (OTER). Resulting peptides are subsequently trapped on a monolithic pre-column and transferred on-line to a 10 µm ID porous layer open tubular (PLOT) liquid chromatography LC separation column. Wnt/ß-catenein signaling pathway (Wnt-pathway) proteins of potentially diagnostic value were digested+detected in targeted-MS/MS mode in small cell samples and tumor tissues within 120 minutes. For example, a potential biomarker Axin1 was identifiable in just 10 ng of sample (protein extract of ∼1,000 HCT15 colon cancer cells). In comprehensive mode, the current OTER-PLOT set-up could be used to identify approximately 1500 proteins in HCT15 cells using a relatively short digestion+detection cycle (240 minutes), outperforming previously reported on-line digestion/separation systems. The platform is fully automated utilizing common commercial instrumentation and parts, while the reactor and columns are simple to produce and have low carry-over. These initial results point to automated solutions for fast and very sensitive MS based proteomics, especially for samples of limited size.     

High-throughput peptide mass mapping using a microdevice containing trypsin immobilized on a porous polymer monolith coupled to MALDI TOF and ESI TOF mass spectrometers

An enzymatic microreactor with a volume of 470 nL has been prepared by immobilizing trypsin on a 10 cm long reactive porous polymer monolith located in a 100 microm i.d. fused silica capillary. This reactor affords suitable degrees of digestion of proteins even after very short residence times of less than 1 min. The performance is demonstrated with the digestion of eight proteins ranging in molecular mass from 2848 to 77 754. The digests were analyzed using mass spectrometry in two modes: off-line MALDI and in-line nanoelectrospray ionization. The large numbers of identified peptides enable a high degree of sequence coverage and positive identification of the proteins. The extent of sequence coverage decreases as the molecular mass of the digested protein increases.     

Nutrient removal in an A2O-MBR reactor with sludge reduction

In the present study, an advanced sewage treatment process has been developed by incorporating excess sludge reduction and phosphorous recovery in an A2O-MBR process. The A2O-MBR reactor was operated at a flux of 17 LMH over a period of 210 days. The designed flux was increased stepwise over a period of two weeks. The reactor was operated at two different MLSS range. Thermo chemical digestion of sludge was carried out at a fixed pH (11) and temperature (75 °C) for 25% COD solubilisation. The released phosphorous was recovered by precipitation process and the organics was sent back to anoxic tank. The sludge digestion did not have any impact on COD and TP removal efficiency of the reactor. During the 210 days of reactor operation, the MBR maintained relatively constant transmembrane pressure. The results based on the study indicated that the proposed process configuration has potential to reduce the excess sludge production as well as it didn’t detoriated the treated water quality.     

Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor

Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥ 2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.     

A pilot scale anaerobic upflow reactor treating distillery wastewaters

Summary A two-stage pilot reactor has been tested for the anaerobic digestion of distillery wastewater with a COD of-10,000 mg/1. In the first stage (residence time 16–72 hours), carbohydrates are fermented to low molecular weight metabolites. The second stage is an upflow reactor (residence time 14 hours) in which these metabolites are converted to biogas. Overall COD elimination is 84% (BOD, 92%) with biogas production 5–7 times the active volume of the upflow reactor per day. The process withstands temporary stress conditions fairly well. The results indicate that anaerobic treatment in the upflow reactor qualifies as an efficient and low cost method for distillery wastewater treatment.     

Thermophilic anaerobic digestion of Lurgi coal gasification wastewater in a UASB reactor

Lurgi coal gasification wastewater (LCGW) is a refractory wastewater, whose anaerobic treatment has been a severe problem due to its toxicity and poor biodegradability. Using a mesophilic (35 ± 2 °C) reactor as a control, thermophilic anaerobic digestion (55 ± 2 °C) of LCGW was investigated in a UASB reactor. After 120 days of operation, the removal of COD and total phenols by the thermophilic reactor could reach 50-55% and 50-60% respectively, at an organic loading rate of 2.5 kg COD/(m³ d) and HRT of 24 h; the corresponding efficiencies were both only 20-30% in the mesophilic reactor. After thermophilic digestion, the wastewater concentrations of the aerobic effluent COD could reach below 200 mg/L compared with around 294 mg/L if mesophilic digestion was done and around 375 mg/L if sole aerobic pretreatment was done. The results suggested that thermophilic anaerobic digestion improved significantly both anaerobic and aerobic biodegradation of LCGW.     

Trypsin-immobilized fiber core in syringe needle for highly efficient proteolysis

In this report, a core-changeable needle enzymatic reactor was developed for highly efficient proteolysis. A piece of enzyme-immobilized fiber core was inserted into the needle of a syringe pump to form a flow-through bioreactor. The novel in-needle bioreactor could be regenerated by changing its fiber core. The feasibility and performance of the unique bioreactor were demonstrated by the tryptic digestion of BSA and lysozyme and the digestion time was significantly reduced to less than 5 s. The digests were identified by MALDI-TOF MS with sequence coverages comparable to those obtained by the conventional in-solution tryptic digestion. The present in-needle bioreactor provides a promising platform for the high-throughput protein identification.     

Chemically modified, immobilized trypsin reactor with improved digestion efficiency

Tryptic digestion followed by identification using mass spectrometry is an important step in many proteomic studies. Here, we describe the preparation of immobilized, acetylated trypsin for enhanced digestion efficacy in integrated protein analysis platforms. Complete digestion of cytochrome c was obtained with two types of modified-trypsin beads with a contact time of only 4 s, while corresponding unmodified-trypsin beads gave only incomplete digestion. The digestion rate of myoglobin, a protein known to be rather resistant to proteolysis, was not altered by acetylating trypsin and required a buffer containing 35% acetonitrile to obtain complete digestion. The use of acetylated-trypsin beads led to fewer interfering tryptic autolysis products, indicating an increased stability of this modified enzyme. Importantly, the modification did not affect trypsin's substrate specificity, as the peptide map of myoglobin was not altered upon acetylation of immobilized trypsin. Kinetic digestion experiments in solution with low-molecular-weight substrates and cytochrome c confirmed the increased catalytic efficiency (lower K(M) and higher k(cat)) and increased resistance to autolysis of trypsin upon acetylation. Enhancement of catalytic efficiency was correlated with the number of acetylations per molecule. The favorable properties of the new chemically modified trypsin reactor should make it a valuable tool in automated protein analysis systems.     

Optimisation of the anaerobic digestion of agricultural resources

It is in the interest of operators of anaerobic digestion plants to maximise methane production whilst concomitantly reducing the chemical oxygen demand of the digested material. Although the production of biogas through anaerobic digestion is not a new idea, commercial anaerobic digestion processes are often operated at well below their optimal performance due to a variety of factors. This paper reviews current optimisation techniques associated with anaerobic digestion and suggests possible areas where improvements could be made, including the basic design considerations of a single or multi-stage reactor configuration, the type, power and duration of the mixing regime and the retention of active microbial biomass within the reactor. Optimisation of environmental conditions within the digester such as temperature, pH, buffering capacity and fatty acid concentrations is also discussed. The methane-producing potential of various agriculturally sourced feedstocks has been examined, as has the advantages of co-digestion to improve carbon-to-nitrogen ratios and the use of pre-treatments and additives to improve hydrolysis rates or supplement essential nutrients which may be limiting. However, perhaps the greatest shortfall in biogas production is the lack of reliable sensory equipment to monitor key parameters and suitable, parallelised control systems to ensure that the process continually operates at optimal performance. Modern techniques such as software sensors and powerful, flexible controllers are capable of solving these problems. A direct comparison can be made here with, for instance, oil refineries where a more mature technology uses continuous in situ monitoring and associated feedback procedures to routinely deliver continuous, optimal performance.     

Anaerobic digestion of food waste in a hybrid anaerobic solid–liquid system with leachate recirculation in an acidogenic reactor

Recirculation of the leachate in the acidogenic reactor was proposed to enhance anaerobic digestion of food waste in the hybrid anaerobic solid–liquid (HASL) system. Recirculation of the leachate in the acidogenic reactor provided better conditions for extraction of organic matter from the treated food waste and buffering capacity to prevent excessive acidification in the acidogenic reactor. It ensured faster supply of nutrients in the methanogenic reactor in experiment. The highest dissolved COD and VFA concentrations in the leachate from the acidogenic reactor were reached for shorter time and were 16,670 mg/l and 9450 mg/l in control and 18,614 mg/l and 11,094 mg/l in experiment, respectively. Recycling of the leachate in the acidogenic reactor intensified anaerobic digestion of food waste and diminished time needed to produce the same quantity of methane by 40% in comparison with anaerobic digestion of food waste without recirculation.     

Characterization of efficient proteolysis by trypsin loaded macroporous silica

Tryptic digestion of proteins in trypsin loaded porous silica has been shown to be highly efficient. Enzymatic silica-reactors were prepared by immobilizing trypsin into macroporous ordered siliceous foam (MOSF) and into mesoporous SBA-15 silica which has a smaller pore size. The tryptic products from the silica reactors were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and a higher proteolysis efficiency was obtained with MOSF. These results can be well interpreted by a sequential digestion model taking into account the confinement and concentration enrichment of both the substrates and enzymes within the silica pores. Proteins at low concentrations and proteins in urea and surfactant solutions were also successfully digested with the MOSF-based reactor and identified by MS. Considering that the immobilized trypsin could retain its enzymatic activity for weeks, this MOSF reactor provides many advantages compared to free enzyme proteolysis. As a proof-of-concept, the digest of a real complex sample extracted from the cytoplasm of mouse liver tissue using trypsin loaded MOSF yielded better results than the typical in-solution protocol.     

Structural domains of ribosomal protein S8 and their relationship to ribosomal RNA binding

Escherichia coli ribosomal protein S8 has been subjected to mild proteolytic digestion in order to search for structural domains within the protein [1]. A characteristic fragment produced in high yield after chymotrypsin treatment has been located with the protein sequence. Circular dichroism has shown this domain to be rich in α helix. However, the fragment loses its ability to bind to 16 S rRNA as does a similar fragment produced by trypsin cleavage. The intact protein is required for rRNA binding and is highly protected against proteolytic digestion when bound to the RNA.     

High rate anaerobic digestion of piggery manure with polyurethane sponges as support material

Summary The use of polyurethane foam sponges to colonize methanogenic associations for the digestion of piggery manure has been investigated. Fermentors containing polyurethane pads as colonization matrix reached a biogas production rate of ca. 2.0 litres per litre reactor per day (30–33°C), hydraulic retention time 7.5 daysl and a biogas yield of 16 litres per litre piggery manure (7–9% TS). Corresponding control fermentors containing no pads reached a gas production rate of 1.3 litres per litre reactor per day and only about 10 litres biogas per litre piggery manure.     

Rapid protein identification using monolithic enzymatic microreactor and LC-ESI-MS/MS

A monolithic enzymatic microreactor was prepared in a fused-silica capillary by in situ polymerization of acrylamide, glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, followed by ammonia solution treatment, glutaraldehyde activation and trypsin modification. The choice of acrylamide as co-monomer was found useful to improve the efficiency of trypsin modification, thus, to increase the enzyme activity. The optimized microreactor offered very low back pressure, enabling the fast digestion of proteins flowing through the reactor. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at high flow rate. The digests were then characterized by CE and HPLC-MS/MS with the sequence coverage of 57.7%. The digestion efficiency was found over 230 times as high as that of the conventional method. In addition, for the first time, protein digestion carried out in a mixture of water and ACN was compared with the conventional aqueous reaction using MS/MS detection, and the former solution was found more compatible and more efficient for protein digestion.     

Performance of a fixed-bed reactor packed with carbon felt during anaerobic digestion of cellulose

The anaerobic digestion of cellulose was assessed in batch and semi-continuous studies using a carbon felt fixed-bed reactor. In the batch operation, the volatile solids reduction (%) and the cumulative methane production during the mesophilic and thermophilic digestion were 52.2% and 15.9%, 96.7 and 49.2 ml/g-total solid fed, respectively. After 99 days of semi-continuous mesophilic digestion, the degradation of cellulose reached its highest level of 67.6% at the hydraulic retention time of 9 days. The methane production and methane concentration of biogas from the bioreactor were maintained at a steady state. The fixed-bed reactor with carbon felt would be suitable for the efficient anaerobic digestion of cellulose. The biomass distribution in the reactor was, in the liquid phase 0.73 g/l-reactor, in the felt 1.59 g/l-reactor, and on the felt surface 9.86 g/l-reactor, which indicated that most of the microbes were immobilized on the carbon felt fixed-bed in the reactor.     

Two-phase anaerobic fermentation of liquid swine waste to methane

Two-phase anaerobic digestion of liquid swine manure has been developed with options for single-cell protein (SCP) or methane production. In the acidogenic phase at two to four days retention time, and 2.5-7.0% dry matter (DM) concentration, 8-46% of the volatile solids was solubilized. Maximum reactor capacity was 3.86 g/L at 7.0% DM concentration, but optimal operation was achieved at 4.5% DM concentration at four days retention time. The second methanogenic phase was operated continuously and had a maximum specific methane production rate of 0.70 L/L day at 12 days retention time. With recirculation, the rate was 1.16 L/L day at 8.5 days retention time with 52.7% conversion of organic matter. Maximum digestibility was 66% of the lignin free organic matter.