Bioreactor Volume Research Articles

Evaluating the potential role of denitrifying bioreactors in reducing watershed-scale nitrate loads: A case study comparing three Midwestern (USA) watersheds

The transport of nitrate (NO3-N) from agricultural lands to surface waters is a complex and recalcitrant problem. Subsurface drainage systems that are especially prevalent in the corn-growing regions of the Midwestern USA facilitate NO3-N transport. Several conservation practices, including fertilizer and manure management, cover crops, natural and installed wetlands, and wood-chip denitrification bioreactors are options that can mitigate NO3-N losses from agricultural lands. Using simple methods of estimation we examine the cumulative volume of denitrification bioreactors required to treat various amounts of NO3-N in base flow, a proxy for tile drainage, at the watershed scale. The use of load duration curves from three different watersheds shows that NO3-N transport is disproportionately skewed toward larger daily base flows. Approximately 50% of the annual NO3-N is transported in largest 30% of daily base flows. Using previous estimates of NO3-N removal by wood-chip bioreactors, we calculated cumulative bioreactor volumes needed to achieve a range of hydraulic residence times (HRT) given rates of base flow observed in three agricultural watersheds. These analyses suggest that cumulative watershed bioreactor volumes sufficient to achieve an HRT of 0.5 days will reduce at least 20% of the total annual NO3-N loss in one watershed and 30% in the other two watersheds. The area required for wood-chip bioreactors would be at most 0.27% of the watershed area.

In situ UV disinfection of a waveguide-based photobioreactor.

Compact waveguide-based photobioreactors with high surface area-to-volume ratios and optimum light-management strategies have been developed to achieve high volumetric productivities within algal cultures. The light-managing strategies have focused on optimizing sunlight collection, sunlight filtration, and light delivery throughout the entire bioreactor volume by using light-directing waveguides. In addition to delivering broad-spectrum or monochromatic light for algal growth, these systems present an opportunity for advances in photobioreactor disinfection by using germicidal ultraviolet (UV) light. Here, we investigated the efficacy of in situ, nonchemical UV treatment to disinfect a heterotrophic contaminant in a compact photobioreactor. We maintained a >99% pure culture of Synechocystis sp. PCC 6803 for an operating period exceeding 3 weeks following UV treatment of an intentionally contaminated waveguide photobioreactor. Without UV treatment, the culture became contaminated within only a few days (control). We developed a theoretical model to predict disinfection efficiency based on operational parameters and bioreactor geometry, and we verified it with experimental results to predict the disinfection efficiency of a Bacillus subtilis spore culture.

Simultaneous Production of Hydrogen and Methane from Cassava Wastewater Using a Two Stage Upflow Anaerobic Sludge Blanket System Under Thermophilic Operation

The objective of this study was to investigate the hydrogen and methane production from cassava wastewater by using a two stage upflow anaerobic sludge blanket (UASB) system under thermophillic operation (55 °C). The recycle ratio of the effluent from the methane bioreactor-to-the feed flow rate was fixed at 1:1 and the solution pH in the hydrogen bioreactor was maintained at 5.5 by a pH controller while the solution pH of the methane bioreactor was not controlled. The liquid working volumes of the hydrogen and methane bioreactors were 4 and 24 L, respectively. The two stage UASB system was operated at different COD loading rates (30, 60, 90, 120, and 150 kg/m 3 d based on the hydrogen bioreactor volume). Under the optimum COD loading rate of 90 kg/m 3 d based on the hydrogen bioreactor volume corresponding to 15 kg/m 3 d based on the methane bioreactor volume for the hydrogen bioreactor, the produced gas contained 40 % H2, 52 % CO2 and 8 % CH4 and the system provided a maximum hydrogen yield and specific hydrogen production rate of 18 mL/g COD removed and 520 mL/L d, respectively. Under this optimum COD loading rate, the produced gas of the methane bioreactor contained 65 % CH4 and 35 % CO2 without hydrogen and the system provided a maximum methane yield and specific methane production rate of 115 mL/g COD removed and 650 mL/L d, respectively. The operation of recycling from methane bioreactor to hydrogen bioreactor optimized the use of NaOH for pH control in the hydrogen production step.

Microbial lipid production: screening with yeasts grown on Brazilian molasses

Rhodotorula glutinis CCT 2182, Rhodosporidium toruloides CCT 0783, Rhodotorula minuta CCT 1751 and Lipomyces starkeyi DSM 70296 were evaluated for the conversion of sugars from Brazilian molasses into single-cell oil (SCO) feedstock for biodiesel. Pulsed fed-batch fermentations were performed in 1.65 l working volume bioreactors. The maximum specific growth rate (µmax), lipid productivity (Pr) and cellular lipid content were, respectively, 0.23 h(-1), 0.41 g l(-1) h(-1), and 41% for Rsp. toruloides; 0.20 h(-1), 0.27 g l(-1) h(-1), and 36% for Rta. glutinis; 0.115 h(-1), 0.135 g l(-1) h(-1), and 27 % for Rta. minuta; and 0.11 h(-1), 0.13 g l(-1) h(-1), and 32% for L. starkeyi. Based on their microbial lipid productivity, content, and profile, Rsp. toruloides and Rta. glutinis are promising candidates for biodiesel production from Brazilian molasses. All the oils from the yeasts were similar to the composition of plant oils (rapeseed and soybean) and could be used as raw material for biofuels, as well as in food and nutraceutical products.

Increased nitrogen removal in existing volumes at Sundet wastewater treatment plant, Växjö

Many wastewater treatment plants need to improve their nitrogen removal due to stricter requirements and increasing loads. This often means larger bioreactor volumes, which can be very expensive and is sometimes impossible if space is limited. Therefore, there is a need for compact hybrid solutions that can increase capacity within existing volumes. Two full-scale demonstration projects using moving bed biofilm reactor (MBBR) technology has proven to be an efficient way to treat nitrogen in existing volumes at Sundet wastewater treatment plant in Växjö. Increased nitrification and denitrification capacity in parts of the main stream were demonstrated through the Hybas™ process, a combination of MBBR and activated sludge using the integrated fixed-film activated sludge technology. The ANITA™ Mox process, using autotrophic N-removal through anaerobic ammonium oxidation (anammox), provided high nitrogen removal for the sludge liquor. Data collected on-site for over a year are analyzed and compared with the performance of conventional treatment systems. These two full-scale demonstration projects have been a successful learning experience in identifying and correcting both process and operational issues, which may not have arisen at pilot scale. The set objectives in terms of nitrogen removal were met for both processes and design modifications have been identified that will improve future operation at Sundet WWTP.

Rapid high-throughput characterisation, classification and selection of recombinant mammalian cell line phenotypes using intact cell MALDI-ToF mass spectrometry fingerprinting and PLS-DA modelling

Despite many advances in the generation of high producing recombinant mammalian cell lines over the last few decades, cell line selection and development is often slowed by the inability to predict a cell line's phenotypic characteristics (e.g. growth or recombinant protein productivity) at larger scale (large volume bioreactors) using data from early cell line construction at small culture scale. Here we describe the development of an intact cell MALDI-ToF mass spectrometry fingerprinting method for mammalian cells early in the cell line construction process whereby the resulting mass spectrometry data are used to predict the phenotype of mammalian cell lines at larger culture scale using a Partial Least Squares Discriminant Analysis (PLS-DA) model. Using MALDI-ToF mass spectrometry, a library of mass spectrometry fingerprints was generated for individual cell lines at the 96 deep well plate stage of cell line development. The growth and productivity of these cell lines were evaluated in a 10L bioreactor model of Lonza's large-scale (up to 20,000L) fed-batch cell culture processes. Using the mass spectrometry information at the 96 deep well plate stage and phenotype information at the 10L bioreactor scale a PLS-DA model was developed to predict the productivity of unknown cell lines at the 10L scale based upon their MALDI-ToF fingerprint at the 96 deep well plate scale. This approach provides the basis for the very early prediction of cell lines’ performance in cGMP manufacturing-scale bioreactors and the foundation for methods and models for predicting other mammalian cell phenotypes from rapid, intact-cell mass spectrometry based measurements.

Effect of Parameters on Treatment of Industrial Effluents Using Inverse Fluidized Bed Bioreactor: Statistical Analysis

Phenolic wastewater (effluents from steel plant) was treated in an inverse fluidized bed (IFB) bioreactor. Effects of different system parameters (viz. residence time (t), settled bed to bioreactor volume ratio (Vb/Vr), water flow rate, static bed height, and gas flow rate) on removal of chemical oxygen demand (COD) were studied to determine the optimum conditions. Optimal conditions corresponding to the largest COD removal were obtained at Vb/Vr ratio of 0.55 and gas flow of 40 LPH with time period greater than 62 h. COD removal was also measured with the addition of mineral salts in the wastewater. The results thus obtained with and without the addition of mineral salt to the wastewater were compared. Addition of nutrient salts to the wastewater yielded better percentage of COD removal. The effect of different types of salts on COD removal was also studied with respect to the biomass growth. Experiments were also carried out by continuous culturing of biomass samples. Conversions greater than 68% and 84% were achieved with batch and continuous mode operation respectively, implying that IFB bioreactor can be used successfully for the treatment of industrial effluents.

Stability of the process of simultaneous saccharification and fermentation of corn flour. The effect of structural changes of starch by stillage recycling and scaling up of the process

Intensive development of the transport sector and a rise in the prices of fossil fuels boost the demand for fuels from alternative sources of energy, including biofuels. New energy-efficient technologies of fuel production from renewable resources are developed. The aim of the present study was to examine the factors influencing the effectiveness of the process of simultaneous saccharification and fermentation of corn flour with full stillage recycling. The effect of structural changes of starch granules during the long-term repeated SSF process as well as the scale of the process were investigated. Commercially available STARGEN 001 enzymatic preparation and Saccharomyces cerevisiae strain Red Star Ethanol Red were used in the experiment. The results proved that raw material quality is of the utmost importance for the effectiveness of this processes. Bacterial contamination of the raw material caused decreased ethanol productivity despite similar substrate utilization. Process scale turned out to be a second significant factor influencing the SSF outcome. Increase of bioreactor volume resulted in decreased productivity. Repeated stillage recycling and the resulting concentration of broth ingredients has a lesser impact on the process. Ethanol content and the amount of residual starch was independent of the number of operation cycles. Formation of porous granules is predominant as starch undergoes hydrolysis. The affinity of the amylolytic enzyme used towards crystalline and amorphous regions is equal.

Biomass Production and Inoculation of Industrial Bioleaching Processes

Microbial activity inleaching processes accounts for 4% of today’s copper produced in the world. Factorsrelated with lesser overall metal recoveries, no recovery of precious metalsand molybdenum in comparison with conventional concentration/smelting &refining technologies and the high prices of metals inhibit the use of bioleachingat a larger scale. In order to increase bioleaching rates and overall metal recoveries,continuous inoculation of the ore with a leaching solution containing specific adaptedconsortium of microorganisms, allows an early expression of microbial activity,reducing 2-3 fold the time required by ore native bearing microflora to grow.This leaching solution concentrated in microorganisms can be produced by meansof bioreactors operated in continuous regime. Unfortunately biomining microorganisms have a very low duplication timewhen comparing to common microbes like E.colior B. subtilis, that forces the useof huge volume bioreactors in the case of conventional bioreactors, to ensurethe growth of microorganisms have sufficient residence time. To overcome thisproblem, we have designed a very efficient air-lift bioreactor (Patent Registration No. CL 48319), that can be used at industrial operations for the production ofsolutions with a high concentration of biomining microorganisms, for theinoculation of bioleaching heaps, with lesser residence time in comparison toconventional bioreactors. Ourbioreactor has an internal recirculation for producing sulfide-ore bioleachingsolutions, with a phase-separating and solids-recirculation system, without theneed to impel the suspension containing the solids towards the bioreactor withpumps, using diatomaceous earth, ferric precipitates and/or elemental sulfur asa solid support to immobilize iron and/or sulfur-oxidizing microorganisms. Dependingon the source of energy supplied for the growth of the microorganisms, thebioreactor can produce either a solution concentrated in ferric ions andiron-oxidizing bacteria or sulfur oxidizing bacteria. In order to validate ourbioreactor design at industrial scale, a trial was carried out in an air-liftbioreactor of 35 m3 nominal capacity, which is part of a biomassplant located in Radomiro Tomic Division of CODELCO. In this article, theresults of the test proving the advantages and satisfactory design of ourbioreactor for producing continuously iron-oxidizing bacteria and sulfuroxidizing bacteria for inoculation and irrigation of heaps and dumps are shown.

On Geometry and Scale of a Stochastic Chemostat

A chemostat is a fixed volume bioreactor in which micro–organisms are grown in a continuously renewed liquid medium. We propose a stochastic model for the evolution of the concentrations in the single species and single substrate case. It is obtained as a diffusion approximation of a pure jump Markov process, whose increments are comparable in mean with the deterministic model. A specific time scale, related to the noise intensity, is considered for each source of variation. The geometric structure of the problem, usable by identification procedures, is preserved both in the drift and diffusion term. We study the properties of this model by numerical experiments.

Process cost and facility considerations in the selection of primary cell culture clarification technology

The bioreactor volume delineating the selection of primary clarification technology is not always easily defined. Development of a commercial scale process for the manufacture of therapeutic proteins requires scale-up from a few liters to thousands of liters. While the separation techniques used for protein purification are largely conserved across scales, the separation techniques for primary cell culture clarification vary with scale. Process models were developed to compare monoclonal antibody production costs using two cell culture clarification technologies. One process model was created for cell culture clarification by disc stack centrifugation with depth filtration. A second process model was created for clarification by multi-stage depth filtration. Analyses were performed to examine the influence of bioreactor volume, product titer, depth filter capacity, and facility utilization on overall operating costs. At bioreactor volumes <1,000 L, clarification using multi-stage depth filtration offers cost savings compared to clarification using centrifugation. For bioreactor volumes >5,000 L, clarification using centrifugation followed by depth filtration offers significant cost savings. For bioreactor volumes of ∼ 2,000 L, clarification costs are similar between depth filtration and centrifugation. At this scale, factors including facility utilization, available capital, ease of process development, implementation timelines, and process performance characterization play an important role in clarification technology selection. In the case study presented, a multi-product facility selected multi-stage depth filtration for cell culture clarification at the 500 and 2,000 L scales of operation. Facility implementation timelines, process development activities, equipment commissioning and validation, scale-up effects, and process robustness are examined.

Modelling of a Fed-Batch Culture Applying Simulated Annealing

In this paper the metaheuristic Simulated Annealing (SA) is applied for parameter identification of non-linear model of cultivation process. SA algorithm is a stochastic relaxation technique, using the Metropolis algorithm based on the Boltzmann distribution in statistical mechanics, for solving nonconvex optimization problems. A real E. coli MC4110 fed-batch cultivation process is considered. The mathematical model is presented by a system of five ordinary differential equations, describing the regarded cultivation process variables - biomass, substrate, acetate, dissolved oxygen and bioreactor volume increasing. The obtained criteria values show that the developed model is adequate and has a high degree of accuracy. The presented results are a confirmation of successful application of the SA algorithm and of the choice of SA algorithm parameters.

Interrelationships between bioreactor volume, effluent recycle rate, temperature, pH, %H2, hydrogen productivity and hydrogen yield with undefined bacterial cultures

Simultaneous achievement of high volumetric productivities (HP=231.3mmolH2/L/h) and high hydrogen yields (HY=3.55molH2/mol glucose) was obtained by increasing the temperature to 70°C and by reducing the total bioreactor system volume (V) to 5.74L and increasing the degassed effluent recycle rate (Fer) to 3.2L/min, giving a V/Fer value of 1.8min. The bioprocess involved the recycling of degassed effluent at a high flow rate through a quasi-stationary fluidized granular bed. In this process the rate of physical removal of H2 trapped in the bulk liquid phase surrounding the fluidized granules reduced the thermodynamic constraints preventing the simultaneous achievement of high HPs and high HYs in the anaerobic fluidized granular bed bioreactor. Energy balance analysis showed that with heat recycling the bioreactor system could achieve a net positive volumetric energy output of 11.76W/L at an energy efficiency of 49.3%.

Performance Evaluation of Four Field-Scale Agricultural Drainage Denitrification Bioreactors in Iowa

Recently, interest in denitrification bioreactors to reduce the amount of nitrate in agricultural drainage has led to increased installations across the U.S. Midwest. Despite this recent attention, there are few peer-reviewed, field-scale comparative performance studies investigating the effectiveness of these denitrification bioreactors. The object of this work was to analyze nitrate removal performance from four existing bioreactors in Iowa, paying particular attention to potential performance-affecting factors including retention time, influent nitrate concentration, temperature, flow rate, age, length-to-width ratio, and cross-sectional shape. Based on a minimum of two years of water quality data from each of the four bioreactors, annual removal rates ranged from 0.38 to 7.76 g N m-3 bioreactor volume d-1. Bioreactor and total (including bypass flow) nitrate-nitrogen load reductions ranged from 12% to 76% (mean 45%) and from 12% to 57% (mean 32%), respectively, removing from 0.5 to 15.5 kg N ha-1 drainage area. Multiple regression analyses showed that temperature and influent nitrate concentration were the most important factors affecting percent bioreactor nitrate load reduction and nitrate removal rate, respectively. This analysis also indicated that load reductions within the bioreactor were significantly impacted by retention time at three of the four reactors. More field-scale performance data from bioreactors of different designs and from multiple locations around the Midwest are necessary to further enhance understanding of nitrate removal in these systems and their potential to positively impact water quality.

Moving bed technology as an alternative solution for reducing bioreactor volume

Moving bed technology as an alternative solution for reducing bioreactor volume

Continuous perfusion culture of encapsulated hybridoma cells

AbstractBACKGROUND: The production of Monoclonal antibodies (mAbs) is often performed in batch or fed‐batch operations where low cell densities and low volumetric productivities are achieved. The main bottleneck of both processes is the short operating time with productive cells at maximum cell concentration.RESULTS: The process studied in this work is based on a fluidized‐bed bioreactor culture of encapsulated KB26.5 cells in a liquid core of calcium alginate microcapsules as a culture strategy to produce IgG3. First, DMEM medium was modified in order to protect the microcapsules from degradation, and later, the optimal operating conditions were set. Under these conditions encapsulated KB26.5 cells reached cell densities of 1.05 × 108cells mL−1 or 9.8 × 106 cells mL−1 (referred to the inner capsule volume or total bioreactor volume, respectively), and a mAb volumetric productivity of 2.75 µg mL−1 h−1.CONCLUSIONS: The productivity of encapsulated KB26.5 cells in perfusion culture was enhanced significantly in comparison with batch and fed‐batch processes. Continuous operation of the perfusion culture for periods longer than 35 days, represented a volumetric productivity about five‐fold higher than conventional operations. However, the fluidized‐bed also showed limitations such as low cell viability at high cell densities due to the mass transfer limitations of large molecules inside the microcapsules. Copyright © 2011 Society of Chemical Industry

Design of a stirred multiwell bioreactor for expansion of CD34+ umbilical cord blood cells in hypoxic conditions

Besides having a metabolic role, oxygen is recognized as an important signaling stimulus for stem cells. In hematopoiesis, hypoxia seems to favor stem cell self-renewal. In fact, long-term repopulating hematopoietic stem cells reside in bone marrow at concentrations as low as 1% oxygen. However, O2 concentration is difficult to control in vitro. Thermodynamically, we found significant differences between O2 solubility in different media, and in presence of serum. Furthermore, we verified that medium equilibration with a hypoxic atmosphere requires several hours. Thus, in a static culture, the effective O2 concentration in the cell immediate microenvironment is difficult to control and subject to concentration gradients. Stirred systems improve homogeneity within the culture volume. In this work, we developed a stirred bioreactor to investigate hypoxia effect on the expression of stem cell markers in CD34+ cells from umbilical cord blood. The stirring system was designed on top of a standard six-well plate to favor continuity with conventional static conditions and transfer of culture protocols. The bioreactor volume (10 mL/well) is suitable for cell expansion and multiparametric flow cytometry analyses. First, it was tested at 21% O2 for biocompatibility and other possible effects on the cells compared to static conditions. Then, it was used to study c-kit expression of CD34+ cells at 5% O2, using 21%-O2 cultures as a control. In hypoxia we found that CD34+ cells maintained a higher expression of c-kit. Further investigation is needed to explore the dynamics of interaction between oxygen- and c-kit-dependent pathways at the molecular level.

MBBR evaluation for oil refinery wastewater treatment, with post-ozonation and BAC, for wastewater reuse

This work evaluated the performance of a Moving Bed Biofilm Reactor (MBBR) in the treatment of an oil refinery wastewater. Also, it investigated the possibility of reuse of the MBBR effluent, after ozonation in series with a biological activated carbon (BAC) column. The best performance of the MBBR was achieved with a hydraulic retention time (HRT) of 6 hours, employing a bed to bioreactor volume ratio (V(B)/V(R)) of 0.6. COD and N-NH₄(+) MBBR effluent concentrations ranged from 40 to 75 mg L⁻¹ (removal efficiency of 69-89%) and 2 to 6 mg L⁻¹ (removal efficiency of 45-86%), respectively. Ozonation carried out for 15 min with an ozone concentration of 5 mg L⁻¹ was able to improve the treated wastewater biodegradability. The treatment performance of the BAC columns was practically the same for ozonated and non ozonated MBBR effluents. The dissolved organic carbon (DOC) content of the columns of the activated carbon columns (CAG) was in the range of 2.1-3.8 mg L⁻¹, and the corresponding DOC removal efficiencies were comprised between 52 and 75%. The effluent obtained at the end of the proposed treatment presented a quality, which meet the requirements for water reuse in the oil refinery.

Production, purification and tumor necrosis factor-α (TNF-α) release capability of exopolysaccharide from Laetiporus sulphureus (Bulliard: Fries) Bondartsev & Singer in submerged cultures

The optimal culture conditions of exopolysaccharide (EPS) productions in submerged culture by Laetiporus sulphureus were determined in flask cultures. Optimal medium composition was determined as follows (g/l): glucose 10, yeast extract 5, KH2PO4 3. The optimal temperature and initial pH for EPS production were 31°C and initial pH 4.0, respectively. The optimal culture medium was then cultivated in a 5-l and a 20-l stirred tank fermenter at 1vvm (volume of aeration per volume of bioreactor per min) aeration rate, 150rpm agitation speed, controlled pH 4.0 and 31°C. In the optimal culture medium, the maximum EPS production in a 5-l and a 20-l stirred tank fermenter was 367 and 356mg/l, respectively, resulting in a 57% and 51% enhancement of EPS fermentation production compared with those from the basal medium cultures. Two main fractions, Fr-I and Fr-II, of crude EPS extracted from optimal L. sulphureus culture were obtained by a Sepharose CL-4B column chromatography. The high-performance gel-permeation chromatography (HPGPC) analysis exhibited that the number average molecular weight (Mn) of two polysaccharides were 4290 and 1120kDa, respectively. The protein contents of two fractions were 36.98% and 12.31% (w/w), respectively. All polysaccharide fractions showed significantly higher tumour necrosis factor-α (TNF-α) release capability on macrophage cells. The results obtained are useful in optimization of L. sulphureus culture for efficient production of a potent immunostimulating EPS in the submerged culture.

Biological hydrogen production by the algal biomass Chlorella vulgaris MSU 01 strain isolated from pond sediment

Chlorella vulgaris MSU 01 strain isolated from the sediment of the pond is able to produce molecular hydrogen in a clean way. To relate the dynamic coupling between the cultural conditions and biological responses, an original lab scale set up has been developed for hydrogen production. Different sources like mannitol, glucose, alanine, citric acid, aspartic acid, l-alanine, l-cysteine, sodium succinate and sodium pyruvate were used for algal media optimization. Corn stalk, from 1 to 5 g/L was tested for the effective algal growth and hydrogen production. The cell concentration of 1.6-19 g/L dry cell weight (DCW) was found at the 10th day. The kinetic parameters involved in the hydrogen production at 4 g/L corn stalk using the algal inoculum (50 mL) in the bioreactor volume (500 mL) was found to be with the hydrogen production potential (P(s)) of 7.784 mL and production yield of (P(r)) 5.534 mL respectively. The growth profile of the algal biomass at the above mentioned condition expressed the logistic model with R(2) 0.9988. The final pH of the broth was increased from 7.0 to 8.5-8.7. The anaerobic fermentation by C. vulgaris MSU 01 strain involved in the conversion process of complex carbon source has increased the H(2) evolution rate and higher butyrate concentration in the fermentate.