Structure of a cellulose degrading bacterial community during anaerobic digestion

It is widely accepted that cellulose is the rate-limiting substrate in the anaerobic digestion of organic solid wastes and that cellulose solubilisation is largely mediated by surface attached bacteria. However, little is known about the identity or the ecophysiology of cellulolytic microorganisms from landfills and anaerobic digesters. The aim of this study was to investigate an enriched cellulolytic microbial community from an anaerobic batch reactor. Chemical oxygen demand balancing was used to calculate the cellulose solubilisation rate and the degree of cellulose solubilisation. Fluorescence in situ hybridisation (FISH) was used to assess the relative abundance and physical location of three groups of bacteria belonging to the Clostridium lineage of the Firmicutes that have been implicated as the dominant cellulose degraders in this system. Quantitation of the relative abundance using FISH showed that there were changes in the microbial community structure throughout the digestion. However, comparison of these results to the process data reveals that these changes had no impact on the cellulose solubilisation in the reactor. The rate of cellulose solubilisation was approximately stable for much of the digestion despite changes in the cellulolytic population. The solubilisation rate appears to be most strongly affected by the rate of surface area colonisation and the biofilm architecture with the accepted model of first order kinetics due to surface area limitation applying only when the cellulose particles are fully covered with a thin layer of cells.     

Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: II. Quantification of inhibition and suitability of membrane reactors

Product inhibition of cellulolytic enzymes affects the efficiency of the biocatalytic conversion of lignocellulosic biomass to ethanol and other valuable products. New strategies that focus on reactor designs encompassing product removal, notably glucose removal, during enzymatic cellulose conversion are required for alleviation of glucose product inhibition. Supported by numerous calculations this review assesses the quantitative aspects of glucose product inhibition on enzyme-catalyzed cellulose degradation rates. The significance of glucose product inhibition on dimensioning of different ideal reactor types, i.e. batch, continuous stirred, and plug-flow, is illustrated quantitatively by modeling different extents of cellulose conversion at different reaction conditions. The main operational challenges of membrane reactors for lignocellulose conversion are highlighted. Key membrane reactor features, including system set-up, dilution rate, glucose output profile, and the problem of cellobiose are examined to illustrate the quantitative significance of the glucose product inhibition and the total glucose concentration on the cellulolytic conversion rate. Comprehensive overviews of the available literature data for glucose removal by membranes and for cellulose enzyme stability in membrane reactors are given. The treatise clearly shows that membrane reactors allowing continuous, complete, glucose removal during enzymatic cellulose hydrolysis, can provide for both higher cellulose hydrolysis rates and higher enzyme usage efficiency (kg_product/kg_enzyme). Current membrane reactor designs are however not feasible for large scale operations. The report emphasizes that the industrial realization of cellulosic ethanol requires more focus on the operational feasibility within the different hydrolysis reactor designs, notably for membrane reactors, to achieve efficient enzyme-catalyzed cellulose degradation.     

Liquefaction and gasification during anaerobic digestion of coffee waste by two-phase methane fermentation with slurry-state liquefaction

In order to improve the gas evolution rate during anaerobic digestion of coffee waste by two-phase methane fermentation with slurry-state liquefaction, the liquefaction and gasification processes were separately investigated. In the liquefaction process (including the acidification process), treatment at a pH above 6 had no major effects on the liquefaction and acidification rates. However, the VFA production rates were 880 and 320 mg/l·d during mesophilic (37°C) and thermophilic (53°C) liquefaction, respectively. Mesophilic conditions were superior to thermophilic conditions in the liquefaction. With respect to the gasification process, a high TOC volumetric loading rate of 21 g/l·d was achieved during thermophilic gasification. However, the mesophilic gasification did not yield stable data, even at a low TOC volumetric loading rate of 2 g/l·d. The gas yield was 1.7 l/g TOC consumed during thermophilic gasification. The thermophilic liquefaction and thermophilic gasification reactors were connected in series and a two-phase experiment was conducted with the reactors at various volumetric ratios. The maximum gas evolution rate of 1.43 l/l·d was achieved with a combination of a gasification reactor with a 0.45l working volume and liquefaction reactor with a 2l working volume. This rate was 1.7 times higher than the rate obtained in a previous study.     

Effect of organic loading rate on the stability, operational parameters and performance of a secondary upflow anaerobic sludge bed reactor treating piggery waste

A study of anaerobic digestion of piggery wastewater was carried out in a laboratory-scale sludge bed reactor as a secondary treatment. The effect of organic volumetric loading rates (BV) in the range of 1.0-8.1 g TCOD/ld on the process performance was evaluated. The best results were obtained at BV equal to or lower than 4 g TCOD/ld. At higher BV values, the removal efficiency of the process decreased suddenly. A linear relationship was found between the effluent SCOD and the TVFA/alkalinity ratio (P). A relationship was found among the different operational variables (BV , removal efficiency, effluent soluble COD, soluble COD removal rate (R), retention factor (phi), specific microbial growth rate (mu), methane production rate per volume of reactor and per volume of waste treated--QM and qM, respectively) and the corresponding regression equations were obtained. An increase of BV determined a decrease of removal efficiency, phi and qM and an increase of effluent soluble COD, mu, R and QM. The value of the maximum specific microbial growth rate (muM) determined through the equation that correlated BV and mu was found to be 0.19 d(-1). This value was of the same magnitude as those reported in other works of anaerobic digestion of piggery waste.     

Concurrent microscopic observations and activity measurements of cellulose hydrolyzing and methanogenic populations during the batch anaerobic digestion of crystalline cellulose

This study compares process data with microscopic observations from an anaerobic digestion of organic particles. As the first part of the study, this article presents detailed observations of microbial biofilm architecture and structure in a 1.25-L batch digester where all particles are of an equal age. Microcrystalline cellulose was used as the sole carbon and energy source. The digestions were inoculated with either leachate from a 220-L anaerobic municipal solid waste digester or strained rumen contents from a fistulated cow. The hydrolysis rate, when normalized by the amount of cellulose remaining in the reactor, was found to reach a constant value 1 day after inoculation with rumen fluid, and 3 days after inoculating with digester leachate. A constant value of a mass specific hydrolysis rate is argued to represent full colonization of the cellulose surface and first-order kinetics only apply after this point. Additionally, the first-order hydrolysis rate constant, once surfaces were saturated with biofilm, was found to be two times higher with a rumen inoculum, compared to a digester leachate inoculum. Images generated by fluorescence in situ hybridization (FISH) probing and confocal laser scanning microscopy show that the microbial communities involved in the anaerobic biodegradation process exist entirely within the biofilm. For the reactor conditions used in these experiments, the predominant methanogens exist in ball-shaped colonies within the biofilm.     

Trickle-bed root culture bioreactor design and scale-up: growth, fluid-dynamics, and oxygen mass transfer

Trickle-bed root culture reactors are shown to achieve tissue concentrations as high as 36 g DW/L (752 g FW/L) at a scale of 14 L. Root growth rate in a 1.6-L reactor configuration with improved operational conditions is shown to be indistinguishable from the laboratory-scale benchmark, the shaker flask (mu=0.33 day(-1)). These results demonstrate that trickle-bed reactor systems can sustain tissue concentrations, growth rates and volumetric biomass productivities substantially higher than other reported bioreactor configurations. Mass transfer and fluid dynamics are characterized in trickle-bed root reactors to identify appropriate operating conditions and scale-up criteria. Root tissue respiration goes through a minimum with increasing liquid flow, which is qualitatively consistent with traditional trickle-bed performance. However, liquid hold-up is much higher than traditional trickle-beds and alternative correlations based on liquid hold-up per unit tissue mass are required to account for large changes in biomass volume fraction. Bioreactor characterization is sufficient to carry out preliminary design calculations that indicate scale-up feasibility to at least 10,000 liters.     

Application of flowcell technology for monitoring biofilm development and cellulose degradation in leachate and rumen systems

In this study, a flat plate flowcell was modified to provide a reactor system that could maintain anaerobic, cellulolytic biofilms while providing the data needed to carry out a chemical oxygen demand mass balance to determine the cellulose digestion rates. The results showed that biofilms could be observed to grow and develop on cellulose particle surfaces from both anaerobic digester leachate and rumen fluid inocula. The observations suggest that the architecture of rumen and leachate derived biofilms may be significantly different with rumen derived organisms forming stable, dense biofilms while the leachate derived organisms formed less tenacious surface attachments. This experiment has indicated the utility of flowcells in the study of anaerobic biofilms.     

High-Rate two-phase process for the anaerobic degradation of cellulose, employing rumen microorganisms for an efficient acidogenesis

A novel two-stage anaerobic process for the microbial conversion of cellulose into biogas has been developed. In the first phase, a mixed population of rumen bacteria and ciliates was used in the hydrolysis and fermentation of cellulose. The volatile fatty acids (VFA) produced in this acidogenic reactor were subsequently converted into biogas in a UASB-type methanogenic reactor.A stepwise increase of the loading rate from 11.9 to 25.8 g volatile solids/L reactor volume/day (g VS/L/day) did not affect the degradation efficiency in the acidogenic reactor, whereas the methanogenic reactor appeared to be overloaded at the highest loading rate. Cellulose digestion was almost complete at all loading rates applied. The two-stage anaerobic process was also tested with a closed fluid circuit. In this instance total methane production was 0.438 L CH(4)g VS added, which is equivalent to 98% of the theoretical value. The application of rumen microorganisms in combination with a high-rate methane reactor is proposed as a means of efficient anaerobic degradation of cellulosic residues to methane. Because this newly developed two-phase system is based on processes and microorganisms from the ruminant, it will be referred to as "Rumen Derived Anaerobic Digestion" (RUDAD-) process.     

Carbon dioxide absorption characterization of a bioreactor for biomass production ofPhormidium bohneri: comparative study of three types of diffuser

Under both laboratory-controlled and outdoor conditions, mass culture ofPhormidium bohneri has shown low growth rates and filament aggregate or floc instability. In order to test for a possible carbonaceous supply limitation, a mass transfer characterization study of the reactor used was conducted. To examine different conditions of aeration, the overall volumetric mass transfer coefficient, K_La(C0₂), and the energy and physical transfer efficiency for CO₂ were determined for three air-flow rates and three types of diffuser. For the different aeration conditions studied, the reactor showed adequate mixing properties with respect to CO₂. However, under low air-flow rate conditions, even with the most efficient diffuser, the CO₂ transfer capacity appeared limiting. On the other hand, at high air-flow rates, floc breakage as a result of shear stress appeared to limit bioreactor efficiency.     

The effect of biomass density on cellulose solubilisation rates

The aim of this work was to compare the impact of inoculation density on the rate of cellulose hydrolysis by a rumen derived culture with that of a microbial enrichment from an organic waste anaerobic digester. The results showed a linear relationship between the mass of biomass at the start of the first order degradation phase (Xo) and the first order hydrolysis rate (r) for both rumen inoculated and leachate inoculated cellulose digestions and that the slopes of these relationships were not distinguishable. This suggested that differences in the microbial community, media and other environmental factors had a lesser impact on the hydrolysis rate compared to the effect of the number of cells in the system. This could be of great importance to industrial applications of anaerobic digestion technologies as it suggested that if cells densities in the waste treatment digesters could be boosted to match those seen in the rumen, then the rates of the cellulose hydrolysis would rise.     

Effect of wall growth on the kinetic modeling of nitrite oxidation in a CSTR

A simple kinetic model was developed for describing nitrite oxidation by autotrophic aerobic nitrifiers in a continuous stirred tank reactor (CSTR), in which mixed (suspended and attached) growth conditions prevail. The CSTR system was operated under conditions of constant nitrite feed concentration and varying volumetric flow rates. Experimental data from steady-state conditions in the CSTR system and from batch experiments were used for the determination of the model's kinetic parameters. Model predictions were verified against experimental data obtained under transient operating conditions, when volumetric flow rate and nitrite feed concentration disturbances were imposed on the CSTR. The presented kinetic modeling procedure is quite simple and general and therefore can also be applied to other mixed growth biological systems.     

In vitro protein digestion kinetics of protein sources for pigs

In current feed evaluation systems, the nutritional value of protein sources in diets for pigs is based on the ileal digestibility of protein and amino acids, which does not account for the kinetics of protein digestion along the gastrointestinal tract. The objective of the present study was to determine the in vitro protein digestion kinetics of different protein sources (soya bean meal (SBM), wheat gluten (WG), rapeseed meal (RSM), whey powder (WP), dried porcine plasma protein, yellow meal worm larvae and black soldier fly larvae (BSF)). Protein sources were incubated with pepsin at pH 3.5 for 0 to 90 min and subsequently with pancreatin at pH 6.8 for 0 to 210 min at 39°C. The in vitro protein digestion kinetics were described as the kinetics of nitrogen (N) solubilisation and the release of low molecular weight peptides (LMW) (<500 Da). The N solubilisation rate ranged from 0.025 min⁻¹ for BSF to 0.685 min⁻¹ for WP during the incubation with pepsin, and from 0.027 min⁻¹ for RSM to 0.343 min⁻¹ for WP during the incubation with pancreatin. The release rate of LMW peptides ranged from 0.027 min⁻¹ for WG to 0.093 min⁻¹ for WP during the incubation with pepsin, and from 0.029 min⁻¹ for SBM to 0.385 min⁻¹ for WP. Black soldier fly larvae showed a similar release rate of LMW peptides as WP during the incubation with pancreatin. At the end of the sequential incubation with pepsin (90 min) and pancreatin (210 min), WG and WP showed the highest percentage of N present in LMW peptides relative to total N (78% and 79%, respectively), whereas SBM showed the lowest (35%). In conclusion, protein sources for pig diets show substantial differences in in vitro protein digestion kinetics as measured by the kinetics of N solubilisation and the release of LMW peptides. The rate of release of LMW peptides was not correlated to the rate of N solubilisation for each of the protein sources evaluated.     

Carbon monoxide mass transfer for syngas fermentation in a stirred tank reactor with dual impeller configurations

This study compares the power demand and gas-liquid volumetric mass transfer coefficient, kLa, in a stirred tank reactor (STR) (T = 0.211 m) using different impeller designs and schemes in a carbon monoxide-water system, which is applicable to synthesis gas (syngas) fermentation. Eleven different impeller schemes were tested over a range of operating conditions typically associated with the "after large cavity" region (ALC) of a Rushton-type turbine (D/T = 0.35). It is found that the dual Rushton-type impeller scheme exhibits the highest volumetric mass transfer rates for all operating conditions; however, it also displays the lowest mass transfer performance (defined as the volumetric mass transfer coefficient per unit power input) for all conditions due to its high power consumption. Dual impeller schemes with an axial flow impeller as the top impeller show improved mass transfer rates without dramatic increases in power draw. At high gas flow rates, dual impeller schemes with a lower concave impeller have kLa values similar to those of the Rushton-type dual impeller schemes but show improved mass transfer performance. It is believed that the mass transfer performance can be further enhanced for the bottom concave impeller schemes by operating at conditions beyond the ALC region defined for Rushton-type impellers because the concave impeller can handle higher gas flow rates prior to flooding.     

A model for continuous enzymatic saccharification of cellulose with simultaneous removal of glucose syrup

Cellulase of Trichoderma viride was concentrated in various molecular cutoff membranes, and flux rates and retention of activity were studied under ultra-filtration conditions. Little or no Cellulase was discharged through the membranes tested. The concentrated (5–8-fold) enzymes were used to saccharify finely ground substrate (Solka Floe) in stirred tank (STR) and membrane reactors (MR). A pressure filtration vessel provided with a membrane for simultaneous removal of low molecular weight products (glucose) from the reacting system (Cellulose-Cellulase) is designated as a membrane reactor. Continuous digestion of dense cellulose suspension in the membrane reactor was achieved. Using PM-30 (Amicon) membrane reasonably high mass flux values (9.7–23.3 gals/ft²—day) were obtained in separating glucose from a digest of 30% cellulose suspension. Abcor membrane (HFA 300) was equally effective and necessitated less care in handling. Nearly 14% glucose concentration has been achieved in less than 50 hrs in STR by digesting a 30% cellulose suspension. Based on experimental data a model system is proposed for the continuous steady state Saccharification of ground substrate in which there is continuous removal of concentrated glucose syrup, and a feedback of enzyme.     

Process optimization of an auger pyrolyzer with heat carrier using response surface methodology

A 1 kg/h auger reactor utilizing mechanical mixing of steel shot heat carrier was used to pyrolyze red oak wood biomass. Response surface methodology was employed using a circumscribed central composite design of experiments to optimize the system. Factors investigated were: heat carrier inlet temperature and mass flow rate, rotational speed of screws in the reactor, and volumetric flow rate of sweep gas. Conditions for maximum bio-oil and minimum char yields were high flow rate of sweep gas (3.5 standard L/min), high heat carrier temperature (∼600 °C), high auger speeds (63 RPM) and high heat carrier mass flow rates (18 kg/h). Regression models for bio-oil and char yields are described including identification of a novel interaction effect between heat carrier mass flow rate and auger speed. Results suggest that auger reactors, which are rarely described in literature, are well suited for bio-oil production. The reactor achieved liquid yields greater than 73 wt.%.     

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.     

Influence of sparger on energy dissipation, shear rate, and mass transfer to sea water in a concentric-tube airlift bioreactor

Data on volumetric mass-transfer coefficient, K_La_L, in a 12 × 10⁻³ m³ airlift bioreactor are reported. Measurements were made in sea water. The superficial gas velocity ranged up to 0.21 m/s. Four cylindrical spargers (60–1000 μm pore size) were tested. In bubbly flow, the sparger pore size strongly influenced the K_La_L; the highest K_La_L values were obtained with the smallest pore size. In contrast, in the transition and heterogeneous flow regimes, the pore size had little influence on K_La_L. The best correlation of the mass transfer data was obtained when both gas holdup and liquid superficial velocity were taken as independent variables. Shear rates were estimated in the different zones of the reactor. The highest values were found in the bottom zone of the reactor and in the gas-liquid separator. The penetration and isotropic turbulence models were used to develop a semi-theoretical equation relating the volumetric mass-transfer coefficient to shear rate; hence providing a better understanding of how the operational variables may be manipulated to attain a moderate shear rate and an appropriate level of mass transfer, two extremely important parameters for the growth of sensible microorganisms as those used in marine biotechnology.     

Dextranase production by recombinant Pichia pastoris under operational volumetric mass transfer coefficient (kLa) and volumetric gassed power input (Pg/V) attainable at commercial large scale

Abstract

Most of the reported bioprocesses carried out by the methylotrophic yeast Pichia pastoris have been performed at laboratory scale using high power inputs and pure oxygen, such conditions are not feasible for industrial large-scale processes. In this study, volumetric mass transfer (k_La) and volumetric gassed power input (Pg/V) were evaluated within values attainable in large-scale production as scale-up criteria for recombinant dextranase production by Mut^S P. pastoris strain. Cultures were oxygen limited when the volumetric gassed power supply was limited to 2?kW m^?3. Specific growth rate, and then dextranase production, increased as k_La and Pg/V did. Meanwhile, specific production and methanol consumption rates were constant, due to the limited methanol condition also achieved at 2?L bioprocesses. The specific dextranase production rate was two times higher than the values previously reported for a Mut⁺ strain. After a scale-up process, at constant k_La, the specific growth rate was kept at 30?L bioprocess, whereas dextranase production decreased, due to the effect of methanol accumulation. Results obtained at 30?L bioprocesses suggest that even under oxygen-limited conditions, methanol saturated conditions are not adequate to express dextranase with the promoter alcohol oxidase. Bioprocesses developed within feasible and scalable operational conditions are of high interest for the commercial production of recombinant proteins from Pichia pastoris.     

Kinetics of Cellulose Digestion by Fibrobacter succinogenes S85

Growing cultures of Fibrobacter succinogenes S85 digested cellulose at a rapid rate, but nongrowing cells and cell extracts did not have detectable crystalline cellulase activity. Cells that had been growing exponentially on cellobiose initiated cellulose digestion and succinate production immediately, and cellulose-dependent succinate production could be used as an index of enzyme activity against crystalline cellulose. Cells incubated with cellulose never produced detectable cellobiose, and cells that were preincubated for a short time with thiocellobiose lost their ability to digest cellulose (competitive inhibition [K(infi)] of only 0.2 mg/ml or 0.56 mM). Based on these results, the crystalline cellulases of F. succinogenes were very sensitive to feedback inhibition. Different cellulose sources bound different amounts of Congo red, and the binding capacity was HCl-regenerated cellulose > ball-milled cellulose > Sigmacel > Avicel > filter paper. Congo red binding capacity was highly correlated with the maximum rates of metabolism of cellulose digestion and inversely related to K(infm). Congo red (250 (mu)g/ml) did not inhibit the growth of F. succinogenes S85 on cellobiose, but this concentration of Congo red inhibited the rate of ball-milled cellulose digestion. A Lineweaver-Burk plot of ball-milled cellulose digestion rate versus the amount of cellulose indicated that Congo red was a competitive inhibitor of cellulose digestion (K(infi) was 250 (mu)g/ml).     

Comparative evaluation of biogas production from dairy manure and co‐digestion with maize silage by CSTR and new anaerobic hybrid reactor

This study aimed to investigate potential methane production through anaerobic digestion of dairy manure and co‐digestion with maize silage. Two different anaerobic reactor configurations (single‐stage continuously stirred tank reactor [CSTR] and hybrid anaerobic digester) were used and biogas production performances for each reactor were compared. The HR was planned to enable phase separation in order to improve process stability and biogas production under higher total solids loadings (≥4%). The systems were tested under six different organic loading rates increased steadily from 1.1 to 5.4 g VS/L.d. The CSTR exhibited lower system stability and biomass conversion efficiency than the HR. The specific biogas production of the hybrid system was between 440 and 320 mL/gVS with 81–65% volatile solids (VS) destruction. The hybrid system provided 116% increase in specific biogas production and VS destruction improved by more than 14%. When MS was co‐digested together with dairy manure, specific biogas production rates increased about 1.2‐fold. Co‐digestion was more beneficial than mono‐material digestion. The hybrid system allowed for generating methane enriched biogas (>75% methane) by enabling phase separation in the reactor. It was observed that acidogenic conditions prevailed in the first two compartments and the following two segments as methanogenic conditions were observed. The pH of the acidogenic part ranged between 4.7 and 5.5 and the methanogenic part was between 6.8 and 7.2.