Operation strategies for biohydrogen production with a high-rate anaerobic granular sludge bed bioreactor

Long-term operation for biohydrogen production with an efficient carrier-induced granular sludge bed (CIGSB) bioreactor had encountered problems with poor biomass retention at a low hydraulic retention (HRT) as well as poor mass-transfer efficiency at a high HRT or under a prolonged operation period. This work was undertaken to develop strategies enabling better biomass retention and mass-transfer efficiency of the CIGSB reactors. Supplementation of calcium ion was found to enhance mechanical strength of the granular sludge. Addition of 5.4–27.2 mg/l of Ca²⁺ also led to an over three-fold increase in biomass concentration and a nearly five-fold increase in the H₂ production rate (up to 5.1 l H₂/h/l). Two reflux strategies were utilized to enhance the mass-transfer efficiency of the CIGSB system. The liquid reflux (LR) strategy enhanced the H₂ production rate by 2.2-fold at an optimal liquid upflow velocity of 1.09 m/h, which also gave a maximal biomass concentration of ca. 22 g VSS/l. Similar optimal H₂ production rate was also obtained with the gas reflux (GR) strategy at a rate of 1.0–1.49 m/h, whereas the biomass concentration decreased to 2–7 g VSS/l and thereby the specific H₂ production rate was higher than that with LR. The operation strategies applied in this work were effective to allow stable and efficient H₂ production for nearly 100 days.     

Optimization of fermentative biohydrogen production by response surface methodology using fresh leachate as nutrient supplement

Fresh compost leachate was used as a nutrients source to facilitate anaerobic fermentative hydrogen production from glucose inoculated with mixed culture. The optimum condition for hydrogen production was predicted by response surface methodology (RSM). The model showed the maximum cumulative hydrogen volume (469.74 mL) and molar hydrogen yield (1.60 mol H₂/mol glucose) could be achieved at 6174.93 mg/L glucose and 3383.20 mg COD/L leachate. According to the predicted optimal condition, four tests were carried out to validate the predicted values and evaluate the leachate’s effect on co-fermentation with juice wastewater. A maximum cumulative hydrogen volume of 587.05 ± 15.08 mL was obtained in co-fermentation test, and the molar hydrogen yield reached 2.06 ± 0.06 mol H₂/mol glucose. The co-fermentation of fresh leachate and glucose/juice wastewater was a combination of acetic acid and butyric acid type-fermentation. The results demonstrated that leachate can serve as a nutrients source for biohydrogen production.     

Using a starch-rich mutant of Arabidopsis thaliana as feedstock for fermentative hydrogen production

A mutant plant (Arabidopsis thaliana), sex1-1 (starch excess 1-1), accumulating high starch content in leaves was created to serve as better biomass feedstock for a H₂-producing strain Clostridium butyricum CGS2, which efficiently utilizes starch for H₂ production but cannot assimilate cellulosic materials. The starch content of the mutant plant increased to 10.67 mg/fresh weight, which is four times higher than that of wild type plant. Using sex1-1 mutant plant as feedstock, C. butyricum CGS2 could produce 490.4 ml/l of H₂ with a H₂ production rate of 32.9 ml/h/l. The H₂ production performance appeared to increase with the increase in the concentration of mutant plant from 2.5 to 10 g/l. The highest H₂ to plant biomass yield was nearly 49 ml/g for the mutant plant. This study successfully demonstrated the feasibility of using a starch-rich mutant plant for more effective bioH₂ production with C. butyricum CGS2.     

On-line monitoring of oxygen in Tubespin, a novel, small-scale disposable bioreactor

A novel, optical sensor was fixed in a new type of disposable bioreactor, Tubespin, for the on-line (real-time) monitoring of dissolved oxygen concentrations during cell culture. The cell density, viability and volumetric mass transfer coefficient were also determined to further characterize the bioreactors. The k_La value of the Tubespin at standard conditions was 24.3 h⁻¹, while that of a spinner flask was only 2.7 h⁻¹. The maximum cell density in the Tubespin bioreactor reached 6 × 10⁶ cells mL⁻¹, which was two times higher than the cell density in a spinner flask. Furthermore, the dynamic dissolved oxygen level was maintained above 90% air-saturation in the Tubespin, while the value was only 1.9% in a spinner flask. These results demonstrate the competitive advantage of using the Tubespin system over spinner flasks for process optimization and scale-down studies of oxygen transfer and cell growth.     

Effects of oxygen volumetric mass transfer coefficient and pH on lipase production by Staphylococcus warneri EX17

The principal objectives of this study were to evaluate the kinetics of lipase production by Staphylococcus warneri EX17 under different oxygen volumetric mass transfer coefficients (k_La) and pH conditions in submerged bioreactors, using glycerol (a biodiesel by-product) as a carbon source. Cultivations were conducted at different k_La (26, 38, 50, and 83 h⁻¹) and pH values (6.0, 7.0, and 8.0). The optimal k_La and pH were 38 h⁻¹ and 7.0, respectively. Under these conditions, the maximal cell production obtained was 8.0 g/L, and the volumetric and specific lipase production reached high levels of activity, approximately 800 U/L and 150 U/g cell, respectively, after 12 h of cultivation. This result was approximately five times higher than that obtained in the shake flask cultures. The relationship between cell growth and lipase production was found to be associated with growth by the Luedeking-Piret model.     

Bioreactor design for continuous dark fermentative hydrogen production

Dark fermentative H₂ production (DFHP) has received increasing attention in recent years due to its high H₂ production rate (HPR) as well as the versatility of the substrates used in the process. For most studies in this field, batch reactors have been applied due to their simple operation and efficient control; however, continuous DFHP operation is necessary from economical and practical points of view. Continuous systems can be classified into two categories, suspended and immobilized bioreactors, according to the life forms of H₂ producing bacteria (HPB) used in the reactor. This paper reviews operational parameters for bioreactor design including pH, temperature, hydraulic retention time (HRT), and H₂ partial pressure. Also, in this review, various bioreactor configurations and performance parameters including H₂ yield (HY), HPR, and specific H₂ production rate (SHPR) are evaluated and presented.     

Citric acid production and morphology of Aspergillus niger as functions of the mixing intensity in a stirred tank and a tubular loop bioreactor

The relationship between Aspergillus niger morphology and citric acid production was investigated in two reactor systems with different configurations, a tubular loop and a stirred tank bioreactor, with operating volumes of 6 and 8 dm³, respectively. Morphology was quantified by image analysis. In each system, morphology, characterized by the parameter P (mean convex perimeter of clumps), and citric acid production, were agitation-dependent and closely linked. Increased agitation caused a reduction of clump sizes and results when both reactors demonstrate that the parameter P should not exceed a threshold value in order to achieve increased productivities. The results obtained from the two reactors were in agreement, both qualitatively and quantitatively. Reducing the fundamentally different mixing conditions of the two bioreactors to the order of the dimensionless mixing parameter relative mixing time (τ_m), results showed that the loop simulated the stirred tank. Also, relationships valid for one system accurately described the results obtained from the other system, demonstrating the validity of the relationship between morphology and productivity for the particular fermentation, regardless of the reactor type. Previous attempts to evaluate the use of loop configurations as scale-up tools and their performance as bioreactors, neglected the morphology of the producer micro-organisms. This study demonstrated the close link between morphology and productivity for citric acid production by A. niger, and identified a morphology parameter that was used successfully to characterize the process performance.     

Performance evaluation and phylogenetic characterization of anaerobic fluidized bed reactors using ground tire and pet as support materials for biohydrogen production

This study evaluated two different support materials (ground tire and polyethylene terephthalate [PET]) for biohydrogen production in an anaerobic fluidized bed reactor (AFBR) treating synthetic wastewater containing glucose (4000 mg L⁻¹). The AFBR, which contained either ground tire (R1) or PET (R2) as support materials, were inoculated with thermally pretreated anaerobic sludge and operated at a temperature of 30 °C. The AFBR were operated with a range of hydraulic retention times (HRT) between 1 and 8 h. The reactor R1 operating with a HRT of 2 h showed better performance than reactor R2, reaching a maximum hydrogen yield of 2.25 mol H₂ mol⁻¹ glucose with 1.3 mg of biomass (as the total volatile solids) attached to each gram of ground tire. Subsequent 16S rRNA gene sequencing and phylogenetic analysis of particle samples revealed that reactor R1 favored the presence of hydrogen-producing bacteria such as Clostridium, Bacillus, and Enterobacter.     

Significance of oxygen supply in production of a novel antibiotic by Pseudomonas sp. SJT25

A new agricultural antibiotic, 2-heptyl-5-hexylfuran-3-carboxylic acid (HHCA), was recently discovered, but its further application was limited due to its low production titer. In shake flask fermentation, the effect of initial K(L)a within the range of 2.12-18.87 h?1 on HHCA production was investigated. The cell growth and glycerol consumption were faster at a higher initial K(L)a value, but the maximum production (14.43 mg/L) was attained at an initial K(L)a value of 12.46 h?1. The oxygen supply information was further applied to a 2-L bubble column bioreactor (BCB) by varying initial K(L)a from 1.45 to 30.18 h?1, and the hyperproduction of HHCA was achieved at a relatively low initial K(L)a around 5-10 h?1. The control of oxygen supply is considered to be an important strategy to enhance HHCA production, and the information obtained will be useful to production of this powerful new antibiotic on a large scale.     

Effects of key operational parameters on biohydrogen production via anaerobic fermentation in a sequencing batch reactor

In this study, a series of tests were conducted in a 6 L anaerobic sequencing batch reactor (ASBR) to investigate the effect of pH, hydraulic retention time (HRT) and organic loading rate on biohydrogen production at 28 °C. Sucrose was used as the main substrate to mimic carbohydrate-rich wastewater and inoculum was prepared from anaerobic digested sludge without pretreatment. The reactor was operated initially with nitrogen sparging to form anaerobic condition. Results showed that methanogens were effectively suppressed. The optimum pH value would vary depending on the HRT. Maximum hydrogen production rate and yield of 3.04 L H₂/L reactor d and 2.16 mol H₂/mol hexose respectively were achieved at pH 4.5, HRT 30 h, and OLR 11.0 kg/m³ d. Two relationships involving the propionic acid/acetic acid ratio and ethanol/acetic acid ratio were derived from the analysis of the metabolites of fermentation. Ethanol/acetic acid ratio of 1.25 was found to be a threshold value for higher hydrogen production.     

Study of the oxygen transfer in a disposable flexible bioreactor with surface aeration in vibrated medium

The oxygen mass transfer is a critical design parameter for most bioreactors. It can be described and analyzed by means of the volumetric mass transfer coefficient K _L a. This coefficient is affected by many factors such as geometrical and operational characteristics of the vessels, type, media composition, rheology and microorganism’s morphology and concentration. In this study, we aim to develop and characterize a new culture system based on the surface aeration of a flexible, single-used bioreactor fixed on a vibrating table. In this context, the K _L a was evaluated using a large domain of operating variables such as vibration frequency of the table, overpressure inside the pouch and viscosity of the liquid. A novel method for K _L a determination based on the equilibrium state between oxygen uptake rate and oxygen transfer rate of the system at given conditions was also developed using resting cells of baker’s fresh yeast with a measured oxygen uptake rate of 21 mg g⁻¹ h⁻¹ (at 30°C). The effect of the vibration frequency on the oxygen transfer performance was studied for frequencies ranging from 15 to 30 Hz, and a maximal K _L a of 80 h⁻¹ was recorded at 30 Hz. A rheological study of the medium added with carboxymethylcellulose at different concentrations and the effect of the liquid viscosity on K _L a were determined. Finally, the mixing time of the system was also measured using the pH method.     

Fluid mixing and oxygen transfer in cell suspensions ofTaxus chinensis in a novel stirred bioreactor

In high-density plant cell cultures, mixing and mass transfer are two key issues, which should be emphasized for process optimization. In this work, both mixing and oxygen transfer characteristics of cell suspensions ofTaxus chinensis were studied in a new centrifugal impeller bioreactor with a working volume of 1.2 L. The mixing time (t _M) and the volumetric oxygen transfer coefficient (K _L a) under different operational conditions were determined in both tap water and cell suspensions of 100–400 g fresh weight/L (i.e., 5.65–23.1 g DW/L). At an aeration rate of 0.1 L/min,t _M decreased from 10.6s at 30 rpm to 2.89 s at 200 rpm under 100 g FW/L, and from 9.63 s (120 rpm) to 4.05 s (300 rpm) under 400 g FW/L. Compared with the effect of agitation, aeration was less significant to the suspension mixing. At a relatively high agitation speed (e.g., 200 rpm),t _M remained almost the same even though aeration rate was changed from 0.1 to 0.4 L/min. Thet _M value increased slowly from 3.98 to 5.26 s at 120 rpm when the cell density was raised from 100 to 250 g FW/L. A rapid increase of botht _M and the suspension viscosity was observed at a cell density above 300 g FW/L. As expected, theK _L a value increased with an increase of aeration rate and agitation speed, but decreased with an increase of cell density. The quantitative data obtained here are useful to investigate the effect of mixing stress on the cell physiology and metabolism ofTaxus chinensis in the bioreactor. This paper is dedicated by JJZ to his colleague Prof. Jun-Tang Yu on the occasion of his 70 birthday.     

kLa of stirred tank bioreactors revisited

By means of improved feedback control k_La measurements become possible at a precision and reproducibility that now allow a closer look at the influences of power input and aeration rate on the oxygen mass transfer. These measurements are performed online during running fermentations without a notable impact on the biochemical conversion processes. A closer inspection of the mass transfer during cultivations showed that at least the number of impellers influences mass transfer and mixing: On the laboratory scale, two hollow blade impellers clearly showed a larger k_La than the usually employed three impeller versions when operated at the same agitation power and aeration rate. Hollow blade impellers are preferable under most operational conditions because of their perfect gas handling capacity. Mixing time studies showed that these two impeller systems are also preferable with respect to mixing. Furthermore the widths of the baffle bars depict a significant influence on the k_La. All this clearly supports the fact that it is not only the integral power density that finally determines k_La.     

Use of computational fluid dynamics as a tool for establishing process design space for mixing in a bioreactor

The concept of "design space" plays an integral part in implementation of quality by design for pharmaceutical products. ICH Q8 defines design space as "the multidimensional combination and interaction of input variables (e.g., material attributes) and process parameters that have been demonstrated to provide assurance of quality. Working within the design space is not considered as a change. Movement out of the design space is considered to be a change and would normally initiate a regulatory post-approval change process. Design space is proposed by the applicant and is subject to regulatory assessment and approval." Computational fluid dynamics (CFD) is increasingly being used as a tool for modeling of hydrodynamics and mass transfer. In this study, a laboratory-scale aerated bioreactor is modeled using CFD. Eulerian-Eulerian multiphase model is used along with dispersed k-ε turbulent model. Population balance model is incorporated to account for bubble breakage and coalescence. Multiple reference frame model is used for the rotating region. We demonstrate the usefulness of CFD modeling for evaluating the effects of typical process parameters like impeller speed, gas flow rate, and liquid height on the mass transfer coefficient (k(L)a). Design of experiments is utilized to establish a design space for the above mentioned parameters for a given permissible range of k(L)a.     

Influence of agitation and aeration on growth and antibiotic production by <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis>

The effect of agitation and aeration on the growth and antibiotic production by Xenorhabdus nematophila YL001 grown in batch cultures were investigated. Efficiency of aeration and agitation was evaluated through the oxygen mass transfer coefficient (K _L a). With increase in K _L a, the biomass and antibiotic activity increased. Activity units of antibiotic and dry cell weight were increased to 232 U ml⁻¹ and 19.58 g l⁻¹, respectively, productivity in cell and antibiotic was up more than 30% when K _L a increased from 115.9 h⁻¹ to 185.7 h⁻¹. During the exponential growth phase, DO concentration was zero, the oxygen supply was not sufficient. So, based on process analysis, a three-stage oxygen supply control strategy was used to improved the DO concentration above 30% by controlling the agitation speed and aeration rate. The dry cell weight and activity units of antibiotic were further increased to 24.22 g l⁻¹ and 249 U ml⁻¹, and were improved by 24.0% and 7.0%, compared with fermentation at a constant agitation speed and a constant aeration rate (300 rev min⁻¹, 2.5 l min⁻¹).     

Process development and intensification for enhanced production of Bacillus lipopeptides

The growing interest in Bacillus lipopeptides for high-value applications has driven process design, development and optimization for enhanced lipopeptide production. Traditional optimization approaches have been directed towards improving the overall titres by modification of media components and environmental parameters, almost exclusively in submerged cultures. Carbon and nitrogen sources, trace elements and oxygen availability have all been demonstrated to exhibit significant influences on lipopeptide yield, productivity and selectivity. This insight into process-linked kinetics, especially selectivity, has led to the introduction of novel process intensification and integration strategies which further promote process efficiency, and which include foam fractionation, inverse fluidization, rotating disc contacting and microfiltration with recycle. These strategies have not only transformed the production capabilities, but have also successfully integrated upstream production with downstream purification through cell retention and in situ product removal. This review analyses and critically discusses the impact of process conditions and process optimization strategies for improving lipopeptide production kinetics, specifically highlighting the emerging trend of process intensification and integration strategies and further, proposes a heuristic route to enhance lipopeptide production.     

Using CFD simulations and statistical analysis to correlate oxygen mass transfer coefficient to both geometrical parameters and operating conditions in a stirred-tank bioreactor

Optimization of a bioreactor design can be an especially challenging process. For instance, testing different bioreactor vessel geometries and different impeller and sparger types, locations, and dimensions can lead to an exceedingly large number of configurations and necessary experiments. Computational fluid dynamics (CFD), therefore, has been widely used to model multiphase flow in stirred-tank bioreactors to minimize the number of optimization experiments. In this study, a multiphase CFD model with population balance equations are used to model gas–liquid mixing, as well as gas bubble distribution, in a 50 L single-use bioreactor vessel. The vessel is the larger chamber in an early prototype of a multichamber bioreactor for mammalian cell culture. The model results are validated with oxygen mass transfer coefficient (k_La) measurements within the prototype. The validated model is projected to predict the effect of using ring or pipe spargers of different sizes and the effect of varying the impeller diameter on k_La. The simulations show that ring spargers result in a superior k_La compared to pipe spargers, with an optimum sparger-to-impeller diameter ratio of 0.8. In addition, larger impellers are shown to improve k_La. A correlation of k_La is presented as a function of both the reactor geometry (i.e., sparger-to-impeller diameter ratio and impeller-to-vessel diameter ratio) and operating conditions (i.e., Reynolds number and gas flow rate). The resulting correlation can be used to predict k_La in a bioreactor and to optimize its design, geometry, and operating conditions.     

Shake-flask and bench-scale stirred tank bioreactor production optimization of a thermoalkaline protease from Bacillus cereus SIU1 using one-factor-at-a-time and response surface (statistical) methodologies

Abstract

We report the optimization of production of a halotolerant, thermoalkaline protease by Bacillus cereus SIU1, at shake-flask and bench-scale bioreactor level, using conventional and response surface methods. The basal medium supplemented with optimized (w/v) 0.8% glucose, 1.5% peptone, and 0.4% yeast extract produced 224 Uml^{? 1} alkaline protease after 20 h incubation. Enzyme yield was further increased to 491 Uml^{? 1} when the fermentation broth was supplemented with 0.02% (w/v) Ca²⁺. Optimization of physical factors resulted in still higher protease level of 651 Uml^{? 1} within 18 h fermentation at initial pH 9.0, 50°C, and 150 rpm agitation. Statistically designed experiments revealed significant effects of peptone and CaCl₂ on protease production. A maximum of 749 protease Uml^{? 1} was produced at optimum factor levels (w/v) of peptone 1.75%, yeast extract 0.4%, CaCl₂ 0.025%, and pH 9.0 after 18 h incubation. Optimization of agitation and aeration rates in bench-scale bioreactors further enhanced the enzyme yield to 941 protease Uml^{? 1} at 125 rpm and 2.0 vvm aeration. Optimization of protease production by conventional and statistical approaches resulted in a ～10.7-fold increase (941 Uml^{? 1}) compared to un-optimized conditions (88 Uml^{? 1}).     

From field sampling to pneumatic bioreactor mycelia production of the ectomycorrhizal mushroom Laccaria trichodermophora

In order to increase survival rates of greenhouse seedlings destined for restoration and conservation programs, successful mycorrhization of the seedlings is necessary. To reforest forest ecosystems, host trees must be inoculated with ectomycorrhizal fungi and, in order to guarantee a sufficient supply of ectomycorrhizal inoculum, it is necessary to develop technologies for the mass production of ectomycorrhizal fungi mycelia. We selected the ectomycorrhizal fungus Laccaria trichodermophora, due to its ecological traits and feasible mycelia production in asymbiotic conditions. Here, we report the field sampling of genetic resources, as well as the highly productive nutritional media and cultivation parameters in solid cultures. Furthermore, in order to achieve high mycelial production, we used strain screening and evaluated pH, carbon source concentration, and culture conditions of submerged cultures in normal and baffled shake flasks. The higher productivity culture conditions in shake flasks were selected for evaluation in a pneumatic bioreactor, using modified BAF media with a 10 g/L glucose, pH 5.5, 25 °C, and a volumetric oxygen transfer coefficient (K_La) of 36 h⁻¹. Under those conditions less biomass (12–37 %) was produced in the pneumatic bioreactor compared with the baffled shake flasks. This approach shows that L. trichodermophora can generate a large biomass concentration and constitute the biotechnological foundation of its mycelia mass production.     

Production and Mass Transfer Characteristics of Non-Newtonian Biopolymers for Biomedical Applications

ABSTRACT:?

The market for microbial biopolymers is currently expanding to include several emerging biomedical applications. Specifically, these applications are drug delivery and wound healing. A fundamental understanding of the key fermentation parameters is necessary in order to optimize the production of these biopolymers. Considering that most microbial biopolymer systems exhibit non-Newtonian rheology, oxygen mass transfer can be an important parameter to optimize and control. In this article, we present a critical review of recent advances in rheological and mass transfer characteristics of selected biopolymers of commercial interest in biomedical applications.