Bioreactor Design Research Articles

Lab-Scale Cultivation of Cupriavidus necator on Explosive Gas Mixtures: Carbon Dioxide Fixation into Polyhydroxybutyrate.

Aerobic, hydrogen oxidizing bacteria are capable of efficient, non-phototrophic CO2 assimilation, using H2 as a reducing agent. The presence of explosive gas mixtures requires strict safety measures for bioreactor and process design. Here, we report a simplified, reproducible, and safe cultivation method to produce Cupriavidus necator H16 on a gram scale. Conditions for long-term strain maintenance and mineral media composition were optimized. Cultivations on the gaseous substrates H2, O2, and CO2 were accomplished in an explosion-proof bioreactor situated in a strong, grounded fume hood. Cells grew under O2 control and H2 and CO2 excess. The starting gas mixture was H2:CO2:O2 in a ratio of 85:10:2 (partial pressure of O2 0.02 atm). Dissolved oxygen was measured online and was kept below 1.6 mg/L by a stepwise increase of the O2 supply. Use of gas compositions within the explosion limits of oxyhydrogen facilitated production of 13.1 ± 0.4 g/L total biomass (gram cell dry mass) with a content of 79 ± 2% poly-(R)-3-hydroxybutyrate in a simple cultivation set-up with dissolved oxygen as the single controlled parameter. Approximately 98% of the obtained PHB was formed from CO2.

Emerging Paradigms in Bioengineering the Lungs.

In end-stage lung diseases, the shortage of donor lungs for transplantation and long waiting lists are the main culprits in the significantly increasing number of patient deaths. New strategies to curb this issue are being developed with the help of recent advancements in bioengineering technology, with the generation of lung scaffolds as a steppingstone. There are various types of lung scaffolds, namely, acellular scaffolds that are developed via decellularization and recellularization techniques, artificial scaffolds that are synthesized using synthetic, biodegradable, and low immunogenic materials, and hybrid scaffolds which combine the advantageous properties of materials in the development of a desirable lung scaffold. There have also been advances in the design of bioreactors in terms of providing an optimal regenerative environment for the maturation of functional lung tissue over time. In this review, the emerging paradigms in the field of lung tissue bioengineering will be discussed.

Towards the biomimetic design of hollow fiber membrane bioreactors for bioartificial organs and tissue engineering: A micro-computed tomography (μCT) approach

Hollow fiber membrane bioreactors (HFMBs) with cells cultured in the extracapillary space (ECS) have been proposed for bioartificial organs, to assist patients with failing organs, or to produce in vitro engineered biological substitutes of tissues and organs. They have not gained clinical acceptance yet. One factor limiting therapeutic application is the irregular membrane distribution in the HFMB shell, often considered a typical feature of clinical-scale HFMBs. Such distribution does not permit good control of shell spaces, prevents from offering cells a template structure mimicking the tissue-specific extracellular matrix (ECM) and an adequate supply of oxygen and nutrients, and limits control over cell migration, organization, and differentiation in the ECS. In this study, micro-computed tomography and image analysis techniques were used to characterize the space distribution in the shell of HFMBs varying for membrane packing density and bundling technique, and to investigate whether and how it is possible to manufacture HFMBs in which the distribution of intermembrane spaces in the ECS is uniform and biomimetic. Results suggest that the arrangement in HFMBs of hollow fiber membranes bundled in rolled cross-woven mats at high packing density permits to obtain a uniform shell-side membrane distribution with pore size distribution favoring cells migration around the membranes, and mimicking the ECM structure of bone tissue. • HFMBs were equipped with membranes in loose bundles or in cross-woven mats at varying packing density in the ECS. • Voids distribution in ECS was characterized by μCT and image analysis and was compared to natural bone and a synthetic bone substitute. • Results show that in HFMBs with densely packed membranes in cross-woven mats voids distribution is uniform and mimics bone ECM.

Review of the structures and functions of algal photoreceptors to optimize bioproduct production with novel bioreactor designs for strain improvement.

Microalgae are important renewable feedstock to produce biodiesel and high-value chemicals. Different wavelengths of light influence the growth and metabolic activities of algae. Recent research has identified the light-sensing proteins called photoreceptors that respond to blue or red light. Structural elucidations of algal photoreceptors have gained momentum over recent years. These include channelrhodopsins, PHOT proteins, animal-like cryptochromes, and blue-light sensors utilizing flavin-adenine dinucleotide proteins. Pulsing light has also been investigated as a means to optimize energy inputs into bioreactors. This study summarizes the current structural and functional basis of photoreceptor modulation to optimize the growth, production of carotenoids and other high-value metabolites from microalgae. The review also encompasses novel photobioreactor designs that implement different light regimes including light wavelengths and time to optimize algal growth and desired metabolite profiles for high-value products.

A Mathematical Model of a Valve-Controlled Bioreactor for Platelet Production

As blood donor numbers decrease, while demand for platelets increases, hospitals worldwide are becoming increasingly vulnerable to critical platelet shortages. Alternative methods of supplying platelets are therefore required. One approach is to engineer platelets in vitro in a bioreactor. To characterise such a system, we develop a mathematical model of a novel platelet bioreactor described in Shepherd et al. (Biomaterials, 2018, 182, 135–144). The bioreactor consists of upper and lower tube systems, with a cell-seeded porous collagen scaffold situated between them. Flow through the system is driven by gravity, and controlled by valves on each of the inlets and outlets. The bioreactor is long relative to its width, a feature which we exploit to derive a lubrication reduction of the Navier-Stokes equations for flow in the tube systems, coupled to Darcy flow through the porous scaffold. Flow in the tube systems and scaffold are coupled to form a network model for the bioreactor flow. We characterise the effect of geometrical parameters and valve configuration and synchronisation, on the fluxes through the bioreactor and shear stress experienced by cells in the scaffold. The simplicity of the model means that parameter sweeps take only seconds or minutes to perform, making the model a convenient tool for future bioreactor design optimisation.

Simulation of two‐phase air–liquid flows in a closed bioreactor loop: Numerical modeling, experiments, and verification

Improvements in technology for the production of microbial biomass from natural gas, which have been gaining importance in recent years, require the development and optimization of energy‐efficient bioreactor designs. The most advanced methods for developing and scaling up industrial biotechnological processes rely on detailed hydrodynamic modeling, in addition to classical approaches, as the main tool for analysis and large‐scale transfer of experimental information. This paper presents the results of a simulation of flows in a bioreactor circuit using the latest mathematical methods and numerical flow analysis tools for two‐phase bubble flows based on experimental data.

Design, fabrication, and characterization of a multimodal reconfigurable bioreactor for bone tissue engineering.

In the past decades, bone tissue engineering developed and exploited many typologies of bioreactors, which, besides providing proper culture conditions, aimed at integrating those bio‐physical stimulations that cells experience in vivo, to promote osteogenic differentiation. Nevertheless, the highly challenging combination and deployment of many stimulation systems into a single bioreactor led to the generation of several unimodal bioreactors, investigating one or at mostly two of the required biophysical stimuli. These systems miss the physiological mimicry of bone cells environment, and often produced contrasting results, thus making the knowledge of bone mechanotransduction fragmented and often inconsistent. To overcome this issue, in this study we developed a perfusion and electroactive‐vibrational reconfigurable stimulation bioreactor to investigate the differentiation of SaOS‐2 bone‐derived cells, hosting a piezoelectric nanocomposite membrane as cell culture substrate. This multimodal perfusion bioreactor is designed based on a numerical (finite element) model aimed at assessing the possibility to induce membrane nano‐scaled vibrations (with ~12 nm amplitude at a frequency of 939 kHz) during perfusion (featuring 1.46 dyn cm−2 wall shear stress), large enough for inducing a physiologically‐relevant electric output (in the order of 10 mV on average) on the membrane surface. This study explored the effects of different stimuli individually, enabling to switch on one stimulation at a time, and then to combine them to induce a faster bone matrix deposition rate. Biological results demonstrate that the multimodal configuration is the most effective in inducing SaOS‐2 cell differentiation, leading to 20‐fold higher collagen deposition compared to static cultures, and to 1.6‐ and 1.2‐fold higher deposition than the perfused‐ or vibrated‐only cultures. These promising results can provide tissue engineering scientists with a comprehensive and biomimetic stimulation platform for a better understanding of mechanotransduction phenomena beyond cells differentiation.

Trickle-Bed Bioreactors for Acetogenic H2/CO2 Conversion

Acetic acid is an essential industrial building block and can be produced by acetogenic bacteria from molecular hydrogen (H2) and carbon dioxide (CO2). When gasses are supplied as substrates, bioreactor design plays an important role for their availability. Trickle-bed bioreactors (TBs) have an enhanced gas-to-liquid mass transfer and cells remain in the system by forming a biofilm on the carriers. So far, TBs have been investigated extensively for bio-methanation processes, whereas studies for their use in acetic acid production are rare. In this study, we evaluated the reproducibility of two parallel TBs for acetic acid production from H2:CO2(= 70:30) by a mixed culture with a gas flow rate of 3.8 mL min−1and a medium flow rate of 10 mL min−1. Additionally, the effect of glucose addition during the starting phase on the resulting products and microbial composition was investigated by setting up a third TB2. Partial medium exchanges to decrease the internal acetic acid concentration (AAC) combined with recycling of withdrawn cells had a positive impact on acetic acid production rates with maxima of around 1 g L−1d−1even at high AACs of 19–25 g L−1. Initial glucose addition resulted in the accumulation of unwanted butyric acid up to concentrations of 2.60 ± 0.64 g L−1. The maximum AAC of 40.84 g L−1was obtained without initial glucose addition. The main families identified in the acetogenic TBs were Peptococcaceae, Ruminococcaceae, Planococcaceae, Enterobacteriaceae, Clostridiaceae, Lachnospiraceae, Dysgonomonadaceae and Tannerellaceae. We conclude that a TB is a viable solution for conversion of H2/CO2to acetate using an anaerobic enrichment culture.

Large-scale spatial characterization and liquefaction resistance of sand by hybrid bacteria induced biocementation

Microbially induced calcite precipitation (MICP) has received greater attention of geotechnical engineers recently as a potentially effective and sustainable approach for biocementation of sands. Present study investigates spatial variability and synergistic effects of bacterial hybrids of Sporosarcina (S.) pasteurii and Bacillus (B.) sphaericus for sand biocementation which is important to explore to stimulate real field conditions where multiple urease producing can be present in soil environment. Large-scale testing was conducted in rectangular tank (135 cm × 113 cm × 65 cm) filled with Narmada sand (1.21 Ton), treated using bio-chem distribution system consisting of bioreactor, chemical mixer, circulation tank, injection pipes, treatment tank, and drainage outlets. The design of bioreactor and slotted injection pipes are the major novel aspect of this study for on-site bacterial cultivation and uniform calcite precipitation throughout the treatment depth to increase liquefaction resistance of sand which was not investigated in previous large-scale testing studies. Bacterial hybrid was first cultivated, then inoculum of 10 L was prepared, and finally, 200 L of bacterial solution was cultivated in bioreactor. Sand was bioaugmented with hybrid bacteria in tank, subsequently injected 0.50 M urea and cementation solution periodically up to 8 days. Biosparging was also implemented for 10–12 h daily using air pumps to ensure aerobic conditions. During the treatment, pH, electrical conductivity (EC), and total dissolved solids (TDS) were measured periodically as indicators of biogeochemical reactions occurring during biotreatment. Dynamic cone penetration tests (DCPTs) were performed and DCPT-blows up to 30 cm depth were measured at 20 locations. Contours of dynamic cone penetration index (DCPI) and calcite precipitation were used to assess spatial variability of biocementation. Average DCPT blow count for untreated sand was 5, but it increased up to 50 with 10.6% calcite content, for 30 cm penetration. The DCPT results showed significant improvement in liquefaction resistance. Biocemented samples were collected from various locations to measure calcite content and conduct SEM. Overall, the large-scale testing showed effective and substantial biocementation of sand using the cultivated hybrid bacteria and bio-chem distribution/air sparging system developed in this study. The spacing of injection points and the amounts of injection biotreatment solutions can be optimized to achieve uniform, adequate, and cost-effective biotreatment under actual field applications.

Modular Bioreactor Design for Directed Tendon/Ligament Tissue Engineering.

Functional tissue-engineered tendons and ligaments remain to be prepared in a reproducible and scalable manner. This study evaluates an acellular 3D extracellular matrix (ECM) scaffold for tendon/ligament tissue engineering and their ability to support strain-induced gene regulation associated with the tenogenesis of cultured mesenchymal stromal cells. Preliminary data demonstrate unique gene regulation patterns compared to other scaffold forms, in particular in Wnt signaling. However, the need for a robust bioreactor system that minimizes process variation was also evident. A design control process was used to design and verify the functionality of a novel bioreactor. The system accommodates 3D scaffolds with clinically-relevant sizes, is capable of long-term culture with customizable mechanical strain regimens, incorporates in-line load measurement for continuous monitoring and feedback control, and allows a variety of scaffold configurations through a unique modular grip system. All critical functional specifications were met, including verification of physiological strain levels from 1–10%, frequency levels from 0.2–0.5 Hz, and accurate load measurement up to 50 N, which can be expanded on the basis of load cell capability. The design process serves as a model for establishing statistical functionality and reliability of investigative systems. This work sets the stage for detailed analyses of ECM scaffolds to identify critical differentiation signaling responses and essential matrix composition and cell–matrix interactions.

A Novel Bioreactor for Reconstitution of the Epithelium and Submucosal Glands in Decellularized Ferret Tracheas.

Tracheal grafts introduce the possibility to treat airway pathologies that require resection. While there has been success with engraftment of the surface airway epithelium (SAE) onto decellularized tracheas, there has been minimal advancement in regenerating the submucosal glands (SMGs). We designed a cost-effective open-system perfusion bioreactor to investigate the engraftment potential of ferret SAEs and murine myoepithelial cells (MECs) on a partly decellularized ferret trachea with the goal of creating a fully functional tracheal replacement. An air–liquid interface was also arranged by perfusing humidified air through the lumen of a recellularized conduit to induce differentiation. Our versatile bioreactor design was shown to support the successful partial decellularization and recellularization of ferret tracheas. The decellularized grafts maintained biomechanical integrity and chondrocyte viability, consistent with other publications. The scaffolds supported SAE basal cell engraftment, and early differentiation was observed once an air–liquid interface had been established. Lastly, MEC engraftment was sustained, with evidence of diffuse SMG reconstitution. This model will help shed light on SMG regeneration and basal cell differentiation in vitro for the development of fully functional tracheal grafts before transplantation.

Removal effect and mechanism of typical pharmaceuticals and personal care products by AAO-MBR and UV/Chlorine in black water

Pharmaceuticals and personal care products (PPCPs) are emerging pollutants that have gradually attracted the attention of researchers. PPCPs in urban sewage treatment plants mainly originate from human excreta, while water that is mainly composed of human excreta and flushing water is referred to as black water. Based on a traditional anaerobic-anoxic-oxic-membrane bioreactor (AAO-MBR) design, we optimized the strategy of prolonging the anaerobic-anoxic section length and tested its effect on the bioreactor's efficiency in removing conventional pollutants and PPCPs from blackwater. We assessed the PPCP removal mechanism and efficiency of the AAO-MBR system and a combined advanced ultraviolet (UV)/chlorine treatment system. Under the condition with a hydraulic retention time of 40 h, reflux ratio of the mixed liquid of 400%, and carbon source addition to the blackwater (chemical oxygen demand = 780 mg/L), the removal of ibuprofen, carbamazepine, and bezafibrate by the improved AAO-MBR process exceeded 90%. The PPCP removal mechanisms in the AAO-MBR system were mainly biodegradation, sludge adsorption, and effluent residue. More than 95% of ibuprofen and bezafibrate were biodegraded, and the remaining portions were adsorbed on sludge or remained in the effluent, whereas only approximately 50% of carbamazepine was biodegraded, and the rest was adsorbed on sludge or remained in the effluent. When the UV intensity was 8.0 W/m2, the pH was 6, and the free chlorine concentration was 18 mg/L in the NaClO system, the target PPCPs were completely removed after 23 min. The effective removal of PPCPs from blackwater is essential for alleviating potential environmental risks. Our research provides technical support for the discharge of conventional pollutants and the coordinated removal of PPCPs using a UV/chlorine treatment process.

Modeling fixed bed bioreactors for isopropanol and butanol production using Clostridium beijerinckii DSM 6423 immobilized on polyurethane foams

Isopropanol-butanol fermentation carried out by C. beijerinckii can be improved by immobilizing cells in a fixed bed bioreactor. In this study, a fixed bed bioreactor was developed using polyurethane foams as a solid support for biofilm growth. The dilution rate was increased over time to follow biofilm development while avoiding solvent accumulation within the bioreactor. A phenomenological model was designed and successfully described batch and continuous fixed bed bioreactor dynamics. The parameters regarding biofilm detachment and attachment were estimated using a bioreactor design allowing biofilm sampling during a continuous fermentation. This last experiment was operated during 550 h and reached a maximum productivity of 1.5 g.L −1.h −1. The model prediction capacity was tested by describing a continuous fermentation in a fixed bed bioreactor column over 800 h. This latter bioreactor enabled a maximum productivity of 2.5 g.L −1 h −1 in 650 h of continuous fermentation. A high solvent concentration (13.5 g.L −1) with a butanol concentration close to the toxicity threshold (7.5 g.L −1) was obtained during the fermentation process. The process enhanced the isopropanol butanol fermentation productivity. The model properly described the batch fermentation and the immobilized cells fermentation. However, a 20% difference between experimental and modeled data was observed. Therefore, the model should be improved to better described such fermentation systems.

NITRATE REMOVAL IN WOODCHIP DENITRIFICATION BIOREACTOR – AN APPROACH COMBINING MATHEMATICAL MODELLING AND PI CONTROL

A mathematical model of nitrate removal in woodchip denitrification bioreactor based on field experiment measurements was developed in this study. The approach of solving inverse problem for nonlinear system of differential convection-reaction equations was applied to optimize the efficiency of nitrate removal depending on bioreactor’s length and flow rate. The approach was realized through the developed algorithm containing a nonlocal condition with an incorporated PI controller. This allowed to adjust flow rate for varying inflow nitrate concentrations by using PI controller. The proposed model can serve as a useful tool for bioreactor design. The main outcome of the model is a mathematical relationship intended for bioreactor length selection when nitrate concentration at the inlet and the flow rate are known. Custom software was developed to solve the system of differential equations aiming to ensure the required nitrate removal efficiency.

Kinetic modeling of fungal biomass growth and lactic acid production in Rhizopus oryzae fermentation by using grape stalk as a solid substrate.

This work proposes the use of grape stalk, a residue from the wine industry, as a solid substrate for R. oryzae NCIM 1299 fermentation to obtain lactic acid. The use of filamentous fungi on grape stalk as a fermentative solid substrate has not been studied yet making this study an innovative research work. Grape Stalk was chemically characterized, and according to its properties, it was deemed as an innovative material with great potential to be used as a fermentation substrate. R. oryzae growth kinetic determination needs the measurement of the biomass dry weight of fungus versus time, but the filamentous fungi penetration into the solid substrate makes the quantitative biomass removal from the substrate practically impossible. Therefore, fungal growth on membrane experiment was performed making gravimetric quantification of fungal biomass feasible. Two mediums were used comparatively, potato dextrose agar and a medium designed with agar and grape stalk for 5 days at 30 °C and 50%RH. Logistic, First Order and First Order Plus Dead Time models were used to model biomass growth, where these last two models have not been previously used for microbial kinetics studies. Growth kinetic parameters ( X max , μ max , T p and T 0 ) were found for all models and the First Order model showed the best fit with an R 2 equal to 91.84%. The Luedeking-Piret model was used to model lactic acid production and provided the parameters Y p/x and m p , with an R 2 equal to 90.05%. The kinetic parameters found here are also of great interest for further bioreactor designs. • Grape stalk was characterized as a fungal fermentation substrate. • Grape stalk, raw material in fungal fermentation trials for Lactic acid production. • Cell biomass and lactic acid production kinetic modeling by membrane experiments. • First Order model for fungal biomass growth showed best data fitting. • The Luedeking Piret model parameters were obtained for Lactic Acid production.

Optimizing in-situ product removal operation in a bioreactor for different production scenarios

Mathematical models and optimization problems can be a valuable tool to optimize the operational design of bioreactors. In the present contribution a bioreactor model, implementing in-situ product removal (ISPR) is presented and used to demonstrate potential real world operational strategies that can be applied to the system. Different optimization objectives are formulated in order to find strategies (i.e. operational designs) that maximize the yield and/or the productivity. The decision variables reflect when feeding pulses should be introduced, how much feeding should be added and when the extraction cycles should take place. The first optimization problem focuses on maximizing the yield of the system by means of a single objective optimization. This solution is the most robust and easiest to implement with no requirements for online measurements or control systems. The optimization problem focusing on maximizing the productivity requires solving a stochastic optimization problem to ensure the robustness of the solution, as trying to maximize the productivity was seen to be very sensitive to model uncertainty. Despite the robustness of the proposed strategy online measurements, monitoring propionate, is advised. Because both yield and productivity are important performance indexes, a multi-objective optimization can be used to consider an acceptable performance of both objectives at the same time. This strategy results in a set of solutions representing the potential compromises among the objectives. However, these solutions certainly require online measurements and control systems to implement them correctly.

In situ resource utilisation: The potential for space biomining

The world is entering a new era of exploring and exploiting outer space. The revolution in small, low-cost satellites, the recent initiatives from some countries to establish a legal framework, the increasing demand for technology metals and advances in space additive manufacturing have renewed the interest in space mining. Biomining, the use of microorganisms to extract and recover valuable metals from minerals and wastes, could be used as alternative ISRU technology for harnessing space resources. This paper reviews in situ resources available on the Moon, Mars, and Near-Earth Asteroids (NEAs) for implementing biomining processes in space, the effects of the space environment on biomining microbes, and space-based bioreactor designs that will enable leaching of metals from regoliths. A comparison between terrestrial and space biomining will also be presented, focusing on the differences in the composition of minerals on Earth and space, the types of microorganisms used for leaching, and the parameters that need to be optimised in the space biomining processes. Next steps to mature biomining approaches by combining knowledge from synthetic biology, systems biology, geomicrobiology and process engineering for space applications will also be explored. Through an integrative effort of these fields, biomining processes commonly employed on Earth can be harnessed for sustainable space exploration.

Chaotic printing of hydrogel carriers for human mesenchymal stem cell expansion

Increasing expansion rate in cell manufacturing systems would allow faster delivery of life-saving treatments to a greater number of patients. Chaotic printing is a novel biofabrication technology that could improve cell expansion capabilities by producing hydrogel filaments layered with open channels, resulting in significantly higher surface area-per-unit-volume of interface between cells and nutrient media compared to existing systems. In this study, we chaotically printed hydrogel filaments laden with clinically-relevant, bone marrow-derived human mesenchymal stem cells. After the first week of a 21-day expansion period, cell-laden hydrogel filaments containing open channels had an optimal expansion rate and 2.1x as many cells as filaments without channels. We then validated a novel bioreactor design for expanding cells under flowing nutrient media conditions.

Biogas Potential Assessment of the Composite Mixture from Duckweed Biomass

The article presents the research results of anaerobic digestion processes in bioreactors of composite mixtures based on initial and residual biomass of Lemna minor duckweed and additives: inoculum (manure), food waste, and spent sorbents to determine biogas potential (biogas volume, methane content). Duckweed Lemna minor, which is widespread in freshwater reservoirs, is one of the promising aquatic vegetation species for energy use. Residual biomass is obtained by chemically extracting valuable components from the primary product. The purpose of the research was to evaluate the possibility of the energy potential of residual biomass of Lemna minor to reduce the consumption of fossil fuels and reduce greenhouse gas emissions. This is in line with the International Energy Agency (IEA) scenarios for the reduction of environmental impact. The obtained results confirm the feasibility of using this type of waste for biogas/biomethane production. The recommendations on the optimal composition of the mixture based on the residual biomass of Lemna minor, which will allow for an increase in biogas production, are given. The obtained data can be used in the design of bioreactors.

Anaerobic Treatment for Palm Oil Mill Effluent Using Covered In-the Ground Anaerobic Reactor (CIGAR)

Wastewater from crude palm oil mills contains high organic matter, which potentially produces biogas through anaerobic digestion processes. The design and operation of an anaerobic bioreactor require a good understanding of the reaction kinetic in the bioreactor. This study aimed to evaluate the biogas production from POME and to determine the kinetic parameters of microbial growth and the substrate utilization rates in a CIGAR. An experiment was conducted using a 5-m3 bioreactor with a working volume of 4.4 m3. Wastewater from the Bekri palm oil mill was stored in a 5-m3 tank. After stabilization, the wastewater was loaded into the reactor at a rate of 100 to 250 L/d, corresponding to a COD loading rate of 1.373-3.097 kg·m-3.d-1, and an HRT of 18-44 days. Monod, Contois, Moser, and Shuler kinetic models were evaluated. The results showed that the Shuler model performed best for microbial activities, while the first order reaction model performed best for the substrate utilization kinetic. The maximum specific growth rate (μmax) for the Shuler model was 0.052 d-1 and the saturation constant (Kso) was 0.119. The maximum substrate utilization rate constant (ks) was 2.183 d-1 and biomass yield (Yx/s) 0.024 kg/kg. The maximum average efficiency of anaerobic degradation (34.4%) occurred at a feeding rate of 100 L/d with methane yield of 0.120 Nm3/kg of removed COD. This value is relatively low compared to the maximum potential of 0.350 Nm3/kg CODr.