Bioreactor Research Articles

Evaluation of gradual adaptation of mixed microalgae consortia cultivation using textile wastewater via fed batch operation

This study investigated mixed microalgae consortia cultivation in a fed batch reactor using textile wastewater. The results showed 95% of total phosphorus (TP) and 70% of total nitrogen (TN) depletion during the operational period. Algal biomass growth, pollutant removal, and biomass constituents were examined for five cycles of the fed batch operation. The length of the cycles decreased from 30 days to 10 days as the cycle repeated, which implied gradual adaptation of microalgae to textile wastewater. Color of textile wastewater was removed in the range of 68–72% in all the cycles experimented. Microbial algal cultivation with textile wastewater would be a feasible approach for remediation and resource recovery purpose.

Ethyl tert-butyl ether (EtBE) degradation by an algal-bacterial culture obtained from contaminated groundwater

EtBE is a fuel oxygenate that is synthesized from (bio)ethanol and fossil-based isobutylene, and replaces the fossil-based MtBE. Biodegradation of EtBE to harmless metabolites or end products can reduce the environmental and human health risks after accidental release. In this study, an algal-bacterial culture enriched from contaminated groundwater was used to (i) assess the potential for EtBE degradation, (ii) resolve the EtBE degradation pathway and (iii) characterize the phylogenetic composition of the bacterial community involved in EtBE degradation in contaminated groundwater. In an unamended microcosm, algal growth was observed after eight weeks when exposed to a day-night light cycle. In the fed-batch reactor, oxygen produced by the algae Scenedesmus and Chlorella was used by bacteria to degrade 50 μM EtBE replenishments with a cumulative total of 1250 μM in a day/night cycle (650 lux), over a period of 913 days. The microbial community in the fed-batch reactor degraded EtBE, using a P450 monooxygenase and 2-hydroxyisobutyryl-CoA mutase, to tert-butyl alcohol (TBA), ethanol and CO2 as determined using 13C nuclear magnetic resonance spectroscopy (NMR) and gas chromatography. Stable isotope probing (SIP) with 13C6 labeled EtBE in a fed-batch vessel showed no significant difference in community profiles of the 13C and 12C enriched DNA fractions, with representatives of the families Halomonadaceae, Shewanellaceae, Rhodocyclaceae, Oxalobacteraceae, Comamonadaceae, Sphingomonadaceae, Hyphomicrobiaceae, Candidatus Moranbacteria, Omnitrophica, Anaerolineaceae, Nocardiaceae, and Blastocatellaceae. This is the first study describing micro-oxic degradation of EtBE by an algal-bacterial culture. This algal-bacterial culture has advantages compared with conventional aerobic treatments: (i) a lower risk of EtBE evaporation and (ii) no need for external oxygen supply in the presence of light. This study provides novel leads towards future possibilities to implement algal-bacterial consortia in field-scale groundwater or wastewater treatment.

High-yield production of spider short-chain insecticidal neurotoxin Tx4(6–1) in Pichia pastoris and bioactivity assays in vivo

Short-chain insecticidal neurotoxin Tx4(6–1) from the spider Phoneutria nigriventer can be prepared by reversed-phase high-performance liquid-chromatography (HPLC) fractionation of PhTx4, but this is difficult and represents an obstacle preventing analyses of its insecticidal activity against agricultural insect pests. Herein, we performed secretory expression of recombinant Tx4(6–1) using Pichia pastoris strain X33 as the host, and screened transformants using enzyme-linked immunosorbent assay (ELISA). In flasks, ∼5 mg/l rTx4(6–1) was expressed as a secreted protein following induction with methanol, and this was increased to 45 mg/l rTx4(6–1) in a fed-batch reactor. Approximately 4 mg of high-purity rTx4(6–1) was purified from a 400 ml fed-batch culture supernatant by Ni+-nitriloacetic acid affinity chromatography, followed by carboxymethyl (CM) sepharose ion-exchange chromatography. Purified rTx4(6–1) was determined by mass spectrometry (MS) analysis, revealing a molecular weight (MW) of 7660.5 Da, larger than the expected size due to O-linked glycosylation. Insect bioactivity tests of rTx4(6–1)-treated fifth-instar silkworm larvae (Bombyx mori Linnaeus) showed neurotoxin symptoms such as contraction paralysis, abdominal contraction, and mouth movement syndrome, with a half lethal dose at 12 h post-injection of ∼4.5–8.5 μg/g body weight. Dietary toxicity was not observed in silkworm larvae.

Toward a Comprehensive and Efficient Robust Optimization Framework for (Bio)chemical Processes

Model-based design principles have received considerable attention in biotechnology and the chemical industry over the last two decades. However, parameter uncertainties of first-principle models are critical in model-based design and have led to the development of robustification concepts. Various strategies have been introduced to solve the robust optimization problem. Most approaches suffer from either unreasonable computational expense or low approximation accuracy. Moreover, they are not rigorous and do not consider robust optimization problems where parameter correlation and equality constraints exist. In this work, we propose a highly efficient framework for solving robust optimization problems with the so-called point estimation method (PEM). The PEM has a fair trade-off between computational expense and approximation accuracy and can be easily extended to problems of parameter correlations. From a statistical point of view, moment-based methods are used to approximate robust inequality and equality constraints for a robust process design. We also apply a global sensitivity analysis to further simplify robust optimization problems with a large number of uncertain parameters. We demonstrate the performance of the proposed framework with two case studies: (1) designing a heating/cooling profile for the essential part of a continuous production process; and (2) optimizing the feeding profile for a fed-batch reactor of the penicillin fermentation process. According to the derived results, the proposed framework of robust process design addresses uncertainties adequately and scales well with the number of uncertain parameters. Thus, the described robustification concept should be an ideal candidate for more complex (bio)chemical problems in model-based design.

Optimization of CH 4 Removal from Diluted Emissions and Continuous PHB Production by Methylocystis hirsuta: Towards GHG Biorefineries

This work aimed at optimizing the removal of diluted CH4 emissions (4 % v/v) by decoupling the actual gas residence time from the overall empty bed residence time (EBRT) in a continuous bubble column bioreactor (BCB) inoculated with Methylocystis hirsuta. Different EBRTs (120, 60, 30 and 15 min) were tested along with six internal gas-recycling ratios (QR/Q = 0, 2, 3, 6, 10 and 15, where QR is the recycling gas flow rate and Q the inlet gas flow rate treated). The results showed a CH4 elimination capacity of 35.2 ± 0.4 g m-3 h-1 (RE=72.9 ± 0.5 %) at an EBRT of 30 min and QR/Q of 15, which was identified as the optimum operating conditions in terms of CH4 abatement. The production of poly-3-hydroxybutyrate (PHB) was initially evaluated batchwise, in experiments limiting micro and macro nutrients such as K, Mn, N, and N with excess of Fe. Nitrogen starvation resulted in the highest PHB accumulation (28.0 ± 1.2 %) batchwise. Based on these results, nine sequential N feast-famine cycles were implemented in the BCB operating at an EBRT of 30 min and a QR/Q of 15. N starvation resulted in a gradual increase in the PHB content from 0.4 ± 0.0 % to 25.7 ± 0.1 % in the first cycle and up to 37.2 ± 2.0 % from the fifth cycle onwards. PHB productivities remained roughly constant during operation under N feast-famine cycles at 1.82-2.23 kg m-3 d-1, which corresponded to specific PHB productivities ranging from 15.9 to 21.6 mg PHB gX-1 h-1.

Comparative Evaluation of a Biotrickling Filter and a Tubular Photobioreactor for the Continuous Abatement of Toluene

Volatile Organic Compounds (VOCs) are among the gas pollutants with a higher detrimental impact on human health and the environment. The increasing awareness of the population on the importance of air quality and the enforcement of stricter environmental regulations regarding the anthropogenic emissions of these hazardous pollutants have triggered the development of cost-efficient, environmentally friendly off-gas treatment technologies. Biological technologies such as biotrickling filters have been consistently proven as well stablished technologies for the treatment of VOC emissions, although the low solubility of oxygen might limit the aerobic degradation when treating high concentrations of VOCs. In this context, the synergic effects between microalgae and bacteria represent a sustainable platform to promote the simultaneous abatement of VOCs and CO2 by increasing the dissolved oxygen concentration as a result of the photosynthetic process. In this study, a conventional biotrickling filter (BTF) and an innovative tubular photobioreactor (TPBR) inoculated with a microalgal-bacterial consortium, and interconnected to an external absorption, column were comparatively evaluated for toluene removal. Operating parameters such as the gas residence time (GRT) and the mineral salt medium renewal rate were optimized in order to boost toluene removal. In this sense, both bioreactors were capable of achieving removals > 85 % at a GRT of 45 s and a medium renewal rate of 800 mL d-1. Maximum toluene elimination capacities of ~21 g m-3 h-1 were recorded in both the BTF and the TPBR. Despite the satisfactory performance of both systems, the photobioreactor presented a competitive advantage due to the capacity of algal biomass to fix the produced CO2, considerably reducing the emissions of this greenhouse gas, while increasing the concentration of dissolved oxygen in the cultivation broth up to 7.7 mg O2 L-1.

Mathematical model based optimization of enzymatic aldol addition of propanal to formaldehyde

A multi-enzyme route for the production of an atorvastatin side-chain was proposed. The approach includes the consecutive double aldol addition of acetaldehyde to the phenylacetamide amino-protected propanal by 2-deoxyribose-5-phosphate aldolase (DERA), the oxidation by ketoreductase, the lipase-catalysed acylation and amino group deprotection by penicillin G-acylase. To explore the feasibility of the process, the DERA reaction was studied into detail. Based on the kinetic and stability studies, a mathematical model was developed and validated in a batch and fed-batch reactor. The highest productivity was obtained in repetitive batch reactor (229.1 g/(L day)). The highest final product concentration of 124 g/L was obtained in fed-batch reactor. The mathematical model-based optimisation provided insight into the possibilities for process metrics improvement. Further, the second step was explored. The results showed that the DERA reaction and the oxidation reaction can be carried out as the multi-enzyme one pot process in the sequential manner.

High cell density fed-batch production of insecticidal recombinant ribotoxin hirsutellin A from Pichia pastoris

BackgroundThe fungal ribotoxin hirsutellin A (HtA) exhibits strong insecticidal activity; however, efficient systems for expressing recombinant HtA (rHtA) are lacking. Here, we established an efficient heterologous expression system to produce large amounts of rHtA.ResultsRecombinant Pichia pastoris transformants with high levels of secretory rHtA were screened, and in a fed-batch reactor, rHtA was secreted at levels up to 80 mg/l following methanol induction, which was more than sixfold higher than that in shake flasks. Approximately 7 mg of highly pure rHtA was obtained from 300 ml of fed-batch culture supernatant by Ni+-nitriloacetic acid affinity chromatography and CM Sepharose ion-exchange chromatography. Mass spectrometry results revealed rHtA as a native N-terminal non-glycosylated monomeric protein with a molecular weight of 15.3 kDa. Purified rHtA exhibited excellent thermal and protease stability and dose-dependent cytotoxicity to Sf9 insect cells and insecticidal activity against Galleria mellonella larvae.ConclusionsThis is the first report of rHtA expression in P. pastoris. The rHtA was expressed at a high level under high-cell-density fed-batch fermentation and was efficiently purified using a two-step purification method. Purified rHtA exhibited thermal and protease stability, as well as appropriate bioactivities. Our results indicate that fed-batch production by P. pastoris is an efficient method to produce functional rHtA.

High-Throughput Micro Bioreactor Development for Biopharmaceuticals

High-Throughput Micro Bioreactor Development for Biopharmaceuticals

A study of model adaptation in iterative real-time optimization of processes with uncertainties

In real-time optimization, plant-model mismatch can be handled by adding bias and gradient correction terms to the model-based optimization problem in order to meet the first-order necessary conditions of optimality. However, since these correction terms do not ensure the satisfaction of the second-order condition of optimality upon convergence, the model that is used in the optimization can be inadequate. In the framework of iterative modifier-adaptation, this paper proposes to only use effective model parameter updates to ensure and to speed up the convergence to the process optimum. Additionally, this paper shows that model adequacy can and should be enforced explicitly in model parameter adaptation. By means of a simulation study of maximizing the product yield in a fed-batch reactor, we demonstrate that the proposed model adaptation procedure computes model parameters which make the iterative real-time optimization with modifier-adaptation converge faster and more reliably to the plant optimum.

Biosurfactant assisted degradation of high ring number PAHs using a two-stage CSTR/fully submerged fixed-film bioreactors in series

Polycyclic aromatic hydrocarbons (PAHs) are released into the environment through natural activities such as forest fires and incomplete combustion of wood and decay of vegetable matter. Anthropogenic activities that release PAHs into the environment include activities such as combustion of coal and petroleum product processing. PAHs are not easily accessible to bacteria for degradation due to their low solubility in water. Among the notorious PAH are the high ring number PAHs (ring-number 4 - 7) that are extremely difficult to degrade due to their hydrophobicity which renders them insoluble in water. One such compound, fluoranthene (Flu), is a four ring PAH classified as a High Molecular Weight (HMW) compound. Due to the difficulty to degrade these compounds, it is necessary to find novel and environmentally compatible methods for treating them. In this study, PAH degrading organisms isolated from engine oil contaminated soil achieved 92 % removal of Flu in a fixed-film bioreactor operated at retention time of 1.19 h at 0.86 L.h-1 under fully submerged conditions. The predominant species of biosurfactant producing bacteria in the reactor were determined to be dominated by Pseudomonas aeruginosa using 16S rRNA genotype fingerprinting analysis.

CFD Simulation of Cross-Flow Filtration

The aim of the present work is to study the transfer processes in a cross-flow filtration cell, in order to determine the conditions for stable and efficient operation of a side-stream filtration module, integrated with a bioreactor. The current interest in membrane integrated bioreactors is connected with the pursuit of energy and cost efficiency in a wide area of industrial applications, including wastewater treatment, food industry, pharmaceutical industry, and fuel production. A numerical CFD model is employed, based on previous experience with experimental concentration of antioxidants, such as polyphenols and flavonoids from extracts of natural products by nanofiltration. The geometry under investigation is a 3D model of the experimental flat-sheet cell with tangential orientation of the feed inlet. The swirling turbulent flow in the feed channel is favourable for reducing the concentration polarization layer on the membrane surface and preventing fouling. The main factors, affecting the filtration process, are the shear stress distribution and the concentration profiles in the vicinity of the membrane surface. The CFD models of mass transfer in cross-flow nanofiltration are scarce and there are none for the reference experimental filtration cell. The present CFD simulation reveals the concentration distribution in the feed channel. It complements previous data for the flow pattern with new knowledge on the mass transfer there, directed to understanding and control of the concentration polarization phenomenon. The numerical study uses the tools of ANSYS Fluent R13, based on the finite volume method for solving the Reynolds-Averaged Navier-Stokes (RANS) equations. The obtained results are analysed in rapport with available experimental data.

Cr(VI) remediation with inorganic as(III) as an electron sink immobilized in a ceramic bead packed bed biofilm reactor

Ceramic bead media Biofilm Reactor Performance was evaluated for Cr(VI) reduction utilizing As(III) as electron source with mixed culture of chemoautotrophic bacteria. The inoculant used was isolated from cow dip in Tzaneen (Limpopo Province) and WWTW Brits Northwest South Africa. Preliminary studies show that these bacteria can remove Cr(VI) up to 99 %, and oxidizing As(III) up to 90 %. A bench-scale, ceramic bead media biofilm reactor was operated until steady-state conditions was approached under a range of influent Cr(VI) concentrations ranging from (30-200) mg/L and As(III) concentration ranging from (50-340) mg/L in the ratio of 1:1.7. Hydraulic detention time of 5 hour and 17 hours was observed for optimum Cr(VI) reduction and As(III) oxidation. Result suggests that As(III) was indeed utilized as an electron donor for complete Cr(VI) removal. Parameters such as pH, ORP, temperature and dissolved oxygen concentration was seen optimal at average of 7.1, -156 mV, 31 oC and 0.92 mg/O2L. The steady-state Cr(VI) reduction efficiency was affected by the influent Cr(VI) and As(III) concentration and hydraulic detention time. Higher Cr(VI) concentration was seen in the effluent when the influent concentration was increase above 100 mg/Cr(VI)L and 170 mg/As(III)L. Similarly, at lower hydraulic detention time, Cr(VI) reduction As(III)oxidation were affected. The bioreactor showed strong resilience by recovering from Cr(VI) and As(III) overloading through reduction in influent Cr(VI) and As(III) concentrations.

Biological Degradation of Pyrene and Fluoranthene in Porous Media and Attached Growth Systems by Biosurfactant Producing Bacteria

The dissolution and degradation of higher ring-number polycyclic aromatic compounds were tested in batch and attached growth medium using soil biopile and fixed-film bioreactor systems. Bioavailability of polycyclic aromatic hydrocarbons (PAHs) decreases with increasing ring-number. This means high ring-number compounds will survive longer in the environment and will therefore have a longer-term impact. In the current study, pyrene and fluoranthene were degraded using a biosurfactant producing culture of Pseudomonas aeruginosa grown in fixed-bed and soil biopile experiments. High ring-number PAHs prove to be recalcitrant in nature due to their hydrophobicity and poor partitioning to soil media fixed biomass in biofilm reactors. The use of biosurfactant could facilitate faster dissolution of the PAH and thus would increase bioavailability of the PAH compounds to PAH degrading bacteria. The culture used in this study was obtained from engine oil contaminated soil, that grow on HMW PAHs and are capable of degrading PAHs were enriched, isolated and characterize. The isolated microbial cultures were further characterised 16S rDNA gene sequencing and genotype fingerprinting. During baseline study, over 90 % fluoranthene was degraded as a model compound in biofilm reactor system. Over 88 % pyrene was degraded in soil biopiles. In both cases, increased biosurfactant dose improved degradation rate up to the point where the PAH concentration became inhibitory to biomass growth and PAH degradation.

Bioreactors Pull Out Some of the Stops

Bioreactors Pull Out Some of the Stops

Improving the Energy Efficiency of the ATAD System through Redesign using Integration Superstructure

The aim of this study is application of an approach for solution of two-stage stochastic optimization problem for design of heat-integrated Autothermal Thermophilic Aerobic Digestion (ATAD) bioreactors system for municipal wastewater treatment operating under uncertainties. It incorporates in a common superstructure of two models of heat integration with one heat storage tank and two heat storage tanks for storing the heat used for preheating the incoming into the ATAD system cold raw sludge. On the first stage of the approach heat exchanger networks areas and the volumes of the heat storage tanks are determined. On the second stage - the flows rates represented by respective heating and cooling times are determined. As an optimization criterion - the annual capital costs for needed for the purpose of redesign of the ATAD system equipment as heat exchangers, heat storage tanks and pumps is determined as well as operating costs related with energy consumption of used pumps. The optimization problem is solved using genetic algorithm. Implementation of the proposed approach results in reduction of the depth of the thermal shock and achievement of sustainability improvement of the ATAD system.

High-level expression and purification of active scorpion long-chain neurotoxin BjαIT from Pichia pastoris

As an insect-selective neurotoxin, scorpion long-chain BjαIT is a promising prospect for insecticidal application; however, the difficulty of obtaining natural BjαIT represents the major obstacle preventing analysis of its insecticidal activity against agricultural insect pests. Here, we screened recombinant Pichia pastoris transformants showing high levels of secretory recombinant (r)BjαIT. Secreted rBjαIT was expressed at levels as high as 340 mg/L following methanol induction in a fed-batch reactor, with ∼21 mg of pure rBjαIT obtained from 200-mL fed-batch culture supernatant by Ni2+-nitriloacetic acid affinity chromatography and CM Sepharose ion-exchange chromatography. Injection of purified rBjαIT induced neurotoxicity symptoms in locust (Locusta migratoria) larvae, and the half-lethal dose of rBjαIT for locusts at 24-h post-injection ranged from 11 to 14 μg/g body weight. These results demonstrated that large amounts of active rBjαIT were efficiently prepared from P. pastoris, suggesting this system as efficacious for determining rBjαIT insecticidal activity against other agricultural insect pests.

Dynamic self-optimizing control for unconstrained batch processes

In this paper, we consider near-optimal operation for a class of unconstrained batch processes using the self-optimizing control (SOC) methodology. The existing static SOC approach is extended to the dynamic case by means of a static reformulation of the dynamic optimization problem. However, the dynamic SOC problem is posed as a structure-constrained controlled variable (CV) selection problem, which is different from the static cases. A lower-block triangular structure is specified for the combination matrix, H, to allow for optimal operation whilst respecting causality. A new result is that the structure-constrained SOC problem still results in a convex formulation, which has an analytic solution where the optimal CVs associated with discrete time instants are solved separately. In addition, the inputs are directly determined based on current CV functions for on-line utilization. A fed-batch reactor and a batch distillation column are used to demonstrate the usefulness of the proposed approach.

One-Pot Multienzyme Cofactors Recycling (OPME-CR) System for Lactose and Non-natural Saccharide Conjugated Polyphenol Production.

A one-pot multienzyme cofactors recycling (OPME-CR) system was designed for the synthesis of UDP-α-d-galactose, which was combined with LgtB, a β-(1,4) galactosyltransferase from Neisseria meningitidis, to modify various polyphenol glycosides. This system recycles one mole of ADP and one mole of UDP to regenerate one mole of UDP-α-d-galactose by consuming two moles of acetylphosphate and one mole of d-galactose in each cycle. The ATP additionally used to generate UDP from UMP was also recycled at the beginning of the reaction. The engineered cofactors recycling system with LgtB efficiently added a d-galactose unit to a variety of sugar units such as d-glucose, rutinose, and 2-deoxy-d-glucose. The temperature, pH, incubation time, and divalent metal ions for the OPME-CR system were optimized. The maximum number of UDP-α-d-galactose regeneration cycles (RCmax) was 18.24 by fed batch reaction. The engineered system generated natural and non-natural polyphenol saccharides efficiently and cost-effectively.

High solid fed-batch butanol fermentation with simultaneous product recovery: Part II-process integration.

In these studies, liquid hot water (LHW) pretreated and enzymatically hydrolyzed Sweet Sorghum Bagasse (SSB) hydrolyzates were fermented in a fed-batch reactor. As reported in the preceding paper, the culture was not able to ferment the hydrolyzate I in a batch process due to presence of high level of toxic chemicals, in particular acetic acid released from SSB during the hydrolytic process. To be able to ferment the hydrolyzate I obtained from 250 g L-1 SSB hydrolysis, a fed-batch reactor with in situ butanol recovery was devised. The process was started with the hydrolyzate II and when good cell growth and vigorous fermentation were observed, the hydrolyzate I was slowly fed to the reactor. In this manner the culture was able to ferment all the sugars present in both the hydrolyzates to acetone butanol ethanol (ABE). In a control batch reactor in which ABE was produced from glucose, ABE productivity and yield of 0.42 g L-1 h-1 and 0.36 were obtained, respectively. In the fed-batch reactor fed with SSB hydrolyzates, these productivity and yield values were 0.44 g L-1 h-1 and 0.45, respectively. ABE yield in the integrated system was high due to utilization of acetic acid to convert to ABE. In summary we were able to utilize both the hydrolyzates obtained from LHW pretreated and enzymatically hydrolyzed SSB (250 g L-1 ) and convert them to ABE. Complete fermentation was possible due to simultaneous recovery of ABE by vacuum. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:967-972, 2018.