Outdoor production of microalgae
Vree, Jeroen H. de - \ 2016
Wageningen University. Promotor(en): Rene Wijffels, co-promotor(en): Rouke Bosma; Maria Barbosa. - Wageningen : Wageningen University - ISBN 9789462578784 - 179
algae - algae culture - design - bioreactors - photobioreactors - modeling - algen - algenteelt - ontwerp - bioreactoren - fotobioreactoren - modelleren
This thesis describes the production of microalgae under outdoor conditions, for this research was done at pilot scale. Microalgae are an interesting alternative to currently used sources for bulk commodities as food, feed and chemicals. Research activities within the field are shattered; different reactor systems are investigated at different locations while the systems are operated with different species. The shattered activities prevent a consensus to be reached within the scientific community on the reactor system that has the best performance. Selecting the best performing reactor system will bring the algae industry to the next level. In this PhD thesis different reactor designs were compared on a single location while using the same species in all systems. For this purpose the microalgal pilot facility AlgaePARC (Production And Research Centre) was designed and is described within this thesis. Followed by a comparison of the different reactor designs while using for each comparison a different operational strategy. Operational strategies investigated were chemostat operation and turbidostat operation. During chemostat operation a fixed daily dilution rate is applied to the reactor system, biomass concentrations vary as a result of the applied dilution rate and light conditions. During turbidostat operation the biomass concentration within a system is fixed and as a result of the set biomass concentration and light conditions the daily dilution rate varies. Findings from laboratory scale to pilot scale experiments are extrapolated to indicate the potential of microalgae production at a commercial scale. For these extrapolations mathematical models should be used, which require microalgae species specific input parameters. In this thesis input parameters for two industrially relevant microalgae species were obtained and reported. Finally a techno-economic evaluation was developed to indicate the potential of microalgae as a bulk commodity and to pinpoint focal points for future research.
Microalgae production in a biofilm photobioreactor
Blanken, Ward - \ 2016
Wageningen University. Promotor(en): Rene Wijffels, co-promotor(en): Marcel Janssen. - Wageningen : Wageningen University - ISBN 9789462578425 - 234
algae - algae culture - biofilms - bioreactors - growth - production costs - biomass - artificial lighting - photosynthesis - carbon dioxide - algen - algenteelt - biofilms - bioreactoren - groei - productiekosten - biomassa - kunstmatige verlichting - fotosynthese - kooldioxide
Microalgae can be used to produce high-value compounds, such as pigments or high value fatty acids, or as a feedstock for lower value products such as food and feed compounds, biochemicals, and biofuels. In order to produce these bulk products competitively, it is required to lower microalgae production cost. Production costs could be reduced by employing microalgae biofilms as a production platform. The main advantages of microalgae biofilms are a direct harvest of concentrated microalgae paste, and the uncoupling of the hydraulic retention time from the microalgal retention time. The latter allows to decrease the liquid volume or to employ dilute waste streams. To successfully employ biofilms, however, it is required that microalgal biofilms can be cultivated at high productivity and high photosynthetic efficiency. The aim of this thesis was to optimize the productivity of microalgal biofilms.
Light energy drives microalgal growth. Sunlight is free and abundant, but sunlight intensity varies over the day and the seasons. This makes it impossible to maintain optimal production conditions throughout the day. These fluctuations in irradiance can be prevented by applying artificial lighting. Although, artificial lighting will supply a constant light intensity and thus increase productivity and simplify process control, it will also increase microalgae production cost. A quantitative evaluation of lighting costs and energy requirement was still missing and this was the topic of Chapter 2. The costs related to artificial lighting were identified as 25.3 $ per kilogram of dry-weight biomass, with only 4% to 6% of the electrical energy required to power the lamps eventually stored as chemical energy in microalgal biomass. Energy loss and increased production cost may be acceptable for the production of high value products, but in general they should be avoided.
In Chapter 3, a photobioreactor design based on a rotating biological contactor (RBC) was introduced and used as a production platform for microalgal biomass cultivated in a biofilm. In the photobioreactor, referred to as the Algadisk, microalgae grow in biofilm on vertical rotating disks partially submerged in water with dissolved nutrients. The objective was to evaluate the potential of the Algadisk photobioreactor, and identify the window of operation of the process with respect to the effects of disk roughness, disk rotation speed and CO2 concentration. These parameters were evaluated in relation to biomass productivity, photosynthetic efficiency, and the long-term cultivation stability of the production process.
The mesophilic green microalga Chlorella sorokiniana was used as a model organism. In the lab-scale Algadisk reactor, a productivity of 20.1 ±0.7 gram per m2 disk surface per day and a biomass yield on light of 0.9 ±0.04 gram dry weight biomass per mol photons were obtained. This productivity could be retained over 21 weeks without re-inoculation. To obtain maximal and stable productivity it was important that the disk surface provides a structure that allows biomass retention on the disk after harvest. The retained biomass acts as inoculum for the new biofilm and is therefore essential for quick biofilm regrowth. Most important process parameters were CO2 supply, temperature, and pH. Although deviations of these parameters from the optimal conditions resulted in productivity loss, the system quickly recovered when optimal conditions were restored. These results exhibit an apparent opportunity to employ the Algadisk photobioreactor and biofilm systems in general at large scale for microalgae biomass production provided CO2 supply is adequate.
In order to better understand the process conditions inside the biofilm a model was developed in the further chapters. These mathematical models were calibrated and validated with dedicated experiments. In Chapter 4 first a general applicable kinetic model was developed able to predict light limited microalgal growth. This model combines a mathematical description for photoautotrophic sugar production with a description for aerobic chemoheterotrophic biomass growth. The model is based on five measurable biological parameters which were obtained from literature for the purpose of this study. The model was validated on experiments described in literature for both Chlorella sorokiniana and Chlamydomonas reinhardtii. The specific growth rate was initially predicted with a low accuracy, which was most likely caused by simplifications in the light model and inaccurate parameter estimations. When optimizing the light model and input parameters the model accuracy was improved and validated. With this model a reliable engineering tool became available to predict microalgal growth in photobioreactors. This microalgal growth model was included in the biofilm growth models introduced in Chapters 5 and 6.
In Chapter 5 microalgal biofilms of Chlorella sorokiniana were grown under simulated day-night cycles at high productivity and high photosynthetic efficiency. The experimental data under day/night cycles were used to validate a microalgal biofilm growth model. For this purpose the light limited microalgal growth model from Chapter 4 was extended to include diurnal carbon-partitioning and maintenance under prolonged dark conditions. This new biofilm growth model was then calibrated and validated experimentally. Based on these experiments and model simulations no differences in the light utilization efficiency between diurnal and continuous light conditions were identified. Indirectly this shows that biomass lost overnight represents sugar consumption for synthesis of new functional biomass and maintenance related respiration. This is advantageous, as this result shows that it is possible to cultivate microalgae at high photosynthetic efficiencies on sunlight and that the night does not negatively impact overall daily productivity. Long periods of darkness resulted in reduced maintenance related respiration.
Based on simulations with the validated biofilm growth model it could be determined that the photosynthetic efficiency of biofilm growth is higher than that of suspension growth. This is related to the fact that the maintenance rate in the dark zones of the biofilm is lower compared to that in the dark zones of suspension cultures, which are continuously mixed with the photic zone.
In Chapter 3 it was identified that concentrated CO2 streams are required to obtain high productivities. However, over-supplying CO2 results into loss of CO2 to the environment and is undesirable for both environmental and economic reasons. In Chapter 6 the phototrophic biofilm growth model from Chapter 5 was extended to include CO2 and O2 consumption, production, and diffusion. The extended model was validated in growth experiments with CO2 as limiting substrate. Based on the validated model the CO2 utilization and productivity in biofilm photobioreactors were optimized by changing the gas flow rate, the number of biofilm reactors in series, and the gas composition. This resulted in a maximum CO2 utilization efficiency of 96% by employing flue gas, while the productivity only dropped 2% compared to non-CO2 limited growth. In order to achieve this 25 biofilm reactors units, or more, must be operated in series. Based on these results we conclude that concentrated CO2 streams and plug flow behaviour of the gaseous phase over the biofilm surface are essential for high CO2 utilization efficiencies and high biofilm productivity.
In Chapter 7 the implications of these studies for the further development of biofilm photobioreactors was discussed in the light of current biofilm photobioreactor designs. Design elements of state of the art biofilm photobioreactors, were combined into a new conceptual biofilm photobioreactor design. This new design combines all advantages of phototrophic biofilms minimizing the amount of material required. Further improvements by means of process control strategies were suggested that aim for maximal productivity and maximal nutrient utilization efficiency. These strategies include: control of the biofilm thickness, control of the temperature, and optimized nutrient supply strategies.
Biotechnological removal of H2S and thiols from sour gas streams under haloalkaline conditions
Roman, P. - \ 2016
Wageningen University. Promotor(en): Albert Janssen, co-promotor(en): Martijn Bijmans. - Wageningen : Wageningen University - ISBN 9789462577336 - 193
desulfurization - biogas - bioreactors - methane - sulfides - oxidation - ontzwaveling - biogas - bioreactoren - methaan - sulfiden - oxidatie
Biological removal of H2S from sour gas streams became popular in recent years because of high process efficiency and low operational costs. To expand the scope of these processes to gas streams containing volatile organosulfur compounds, like thiols, it is necessary to provide new insights into their impact on overall biodesulfurization process. In the current thesis multidisciplinary investigations are performed, such as investigations of inhibitory effects of organic sulfur compounds on sulfide-oxidizing biomass by biochemical and enzyme studies; modelling of the process performance and biological pathways; preparation of analytical tools for measuring unstable sulfur compounds under the process conditions; following the microbial dynamics. The research described in this thesis increases the understanding of the underlying processes occurring in biological gas desulfurization systems when organosulfur compounds are present in the feed gas and provides solutions how to cope with these compounds in full-scale gas biodesulfurization installations.
Bio-induced solid selenium for recovery from water
Hageman, S.P.W. - \ 2015
Wageningen University. Promotor(en): Cees Buisman, co-promotor(en): Renata van der Weijden; Fons Stams. - Wageningen : Wageningen University - ISBN 9789462575103 - 156
selenium - waste water - waste water treatment - recovery - toxicity - bioreactors - selenium - afvalwater - afvalwaterbehandeling - terugwinning - toxiciteit - bioreactoren
Selenium in the form of selenate or selenite in wastewater needs to be removed due to its potential toxicity in the environment. Also, selenium is a valuable element that is used in several industries and current selenium resources are likely to be exhausted in less than 50 years. Waste streams containing selenium can therefore be used as a source of selenium. This requires conversion of the selenium in wastewater into a form that can be recovered. Biologically induced selenate reduction to recoverable selenium has the advantage that it uses the selective reduction capacities of biomass and a renewable electron donor.
To improve the recoverability of selenium the conversion of selenate to selenite was seen as an interesting opportunity. Selenite is more reactive than selenate and can be removed in a second step. As described in Chapter 2, it proved possible to convert selenate to mainly selenite at a low electron donor concentration.
Another method which is reviewed in this thesis is direct biological reduction of selenate to elemental selenium. After reduction the solids can be removed by a liquid solid separation process. Previously amorphous selenium particles were produced, which hampered recovery. In this research it is demonstrated that at a higher temperature, around 40 - 50°C, and at a higher pH, around pH 8 - 9, a more hexagonal selenium structure can be produced (Chapter 3). Crystalline acicular selenium particles of different sizes were thus obtained. This implies that selenium particles formation can be controlled and that selenium particles can grow. Large selenium particles make the separation process economic.
To grow larger selenium particles, a long-term experiment was performed at 50°C (Chapter 4). The reduction rate was poor, but selenium acicular particles were produced. These particles were also detected as clusters. These clusters open up new recovery opportunities. With Eerbeek sludge the optimal conditions for selenate conversion are around pH=7 and 30°C. To enlarge the selenium particles it is strongly recommended to use a different sludge since the optimal conditions with Eerbeek sludge do not match the conditions needed for acicular particle formation.
When selenate is converted to selenite, the selenite can be precipitated by sulphide to form selenium sulphide. Emmtec sludge was used to reduce the sulphur compounds to sulphide, leaving selenium as the sole remaining element. This process was performed at T=30°C and a pH between 6 and 7. The selenium thus recovered had a crystalline hexagonal structure (revealed by x-ray diffraction) and the particles were as large as 125µm3.
Future research on the two routes that are explored in this thesis can give insights into selenium reduction mechanisms and the formation of large selenium particles. The recoverability of biological selenium particles has also been improved (as discussed in this thesis). In conclusion, this thesis has resulted in a new, bio-selective, renewable selenium recovery method via selenium sulphide.
Anaerobic oxidation of methane : evaluation of alternative conditions
Suarez Zuluaga, D.A. - \ 2015
Wageningen University. Promotor(en): Cees Buisman, co-promotor(en): Jan Weijma. - Wageningen : Wageningen University - ISBN 9789462574823 - 131
microorganisms - methane - oxidation - sulfates - reduction - bioreactors - micro-organismen - methaan - oxidatie - sulfaten - reductie - bioreactoren
Microorganisms capable of performing anaerobic oxidation of methane (AOM) coupled to sulphate reduction have high doubling times which make their enrichment difficult. However, due to higher energy gain, they might be rapidly enriched using alternative electron acceptors. In chapter 2, it was shown that up to 50 times higher conversion rates were obtained with thiosulphate when compared to sulphate. However, it was also presented that thiosulphate was not be exclusively used by microorganisms that reduce it, but that it was also disproportionated into sulphate and sulphide (Chapter 2).
In Chapter 3, a 5 litre membrane bioreactor was fed not only with methane and sulphate but also with acetate and thiosulphate. As previous experiments using these additional substrates had allowed to obtain either faster conversion rates or enrichment of methanotrophic microorganisms; it was expected that AOM rates in the reactor would increase relatively fast. However, the microorganisms that were enriched were not AOM related. They microbial community that showed the highest activity rates in the reactor was comprised by thiosulphate disproportionated bacteria and green sulphur bacteria. The former disproportionated thiosulphate into sulphate and sulphide while the latter converted the sulphide into elemental sulphur.
Chapter 4, unlike the previous chapters, focused on studying the occurrence of AOM in a fresh water ecosystem. Such system was located next to a natural gas source which captured methane for domestic purposes. It was presented how, with the different electron acceptors added, AOM and trace methane oxidation occurred. However, net AOM was only measured in the presence of sulphate as electron acceptor. Furthermore, the microorganisms that were enriched in the presence of methane and sulphate were also detected.
There are several hypotheses which attempted to explain the AOM coupled to sulphate reduction. One of them indicates that it is a process that involves two microorganisms working in a syntrophic relationship. The first microorganism would convert the methane into carbon dioxide and pass the electrons to the second one. Consequently, the second microorganism would convert the sulphate into hydrogen sulphide. In such a structure, the way that electrons are released by the conversion performed by the first microorganism is unknown. It is possible, that electrons are not transfer via electron shuttles or chemical compounds, but that they are transferred directly from one microorganism to the other. A methodology which could be used to determine if the AOM consortia uses direct electron transfer mechanisms was evaluated in Chapter 5. Different kinds of granular biomass were used for this evaluation and, the granule types that would potentially be capable of using direct electron transfer mechanisms were detected.
Strain improvement of oleaginous microalgae
Jaeger, L. de - \ 2015
Wageningen University. Promotor(en): Gerrit Eggink; Rene Wijffels, co-promotor(en): Dirk Martens. - Wageningen : Wageningen University - ISBN 9789462574847 - 200
algen - biomassa - oliën - productiviteit - opbrengsten - transcriptomica - triacylglycerol lipase - bioreactoren - transformatie - mutanten - algenteelt - biomassa productie - algae - biomass - oils - productivity - yields - transcriptomics - triacylglycerol lipase - bioreactors - transformation - mutants - algae culture - biomass production
The increasing world population and living standards have enlarged the demand for food, feed, and for chemicals. Traditional fossil fuel based commodities need to be replaced, not only because these resources are finite, but also to relieve the impact of carbon emission and pollution, resulting from fossil fuel derived processes. Much attention is on using plants to produce sustainable, renewable alternatives to petrochemical based processes. Palm oil is the crop with the highest lipid yield known today, but the production of palm oil causes deforestation on a large scale. Microalgae are a promising platform for the production of sustainable commodity products. A commodity product that can be produced in microalgae is triacylglycerol (TAG). The TAG molecules that are accumulated in microalgae are comparable to the TAG profiles of commonly used vegetable oils, and can directly be applied for edible oil as well as for biodiesel production. Currently, microalgae derived products have proven to be functional and a potential replacement for conventional crops. However, microalgae derived products, especially TAGs, are not economically feasible yet. In order to make microalgal derived products a reality we need to decrease the production costs by smart technological solutions, biological understanding and metabolic engineering.
To get more insight in the lipid accumulation mechanism of microalgae, and to define targets for future strain improvement strategies, transcriptome sequencing of the oleaginous microalgae Neochloris oleoabundans was done. This oleaginous microalga can be cultivated in fresh water as well as salt water. The possibility to use salt water gives opportunities for reducing production costs and fresh water footprint for large scale cultivation.
In chapter 2 the lipid accumulation pathway was studied to gain insight in the gene regulation 24 hours after nitrogen was depleted. Oil accumulation is increased under nitrogen depleted conditions in a comparable way in both fresh and salt water. The transcriptome sequencing revealed a number of genes, such as glycerol-3-phosphate acyltransferase and via glycerol-3-phosphate dehydrogenase, that are of special interest and can be targeted to increase TAG accumulation in microalgae. NMR spectroscopy revealed an increase in proline content in saline adapted cells, which was supported by up regulation of the genes involved in proline biosynthesis. In addition to proline, the ascorbate-glutathione cycle seems to be of importance for successful osmoregulation by removal of reactive oxygen species in N. oleoabundans, because multiple genes in this pathway were upregulated under salt conditions. The mechanism behind the biosynthesis of compatible osmolytes in N. oleoabundans can be used to improve salt resistance in other industrially relevant microalgal strains.
Another very promising candidate for TAG production is the oleaginous green microalga Scenedesmus obliquus.
In chapter 3, UV mutagenesis was used to create starchless mutants, since no transformation approach was available for this species, due to its rigid and robust cell wall. All five starchless mutants that were isolated from over 3500 screened mutants, showed an increased triacylglycerol productivity. All five starchless mutants showed a decreased or completely absent starch content. In parallel, an increased TAG accumulation rate was observed for the starchless mutants and no substantial decrease in biomass productivity was perceived. The most promising mutant (Slm1) showed an increase in TFA productivity of 41% at 4 days after nitrogen depletion and reached a TAG content of 49.4% (%CDW).
In chapter 4 the Slm1 strain was compared to the wild type strain using photobioreactors. In the wild type, TAG and starch accumulated simultaneously during initial nitrogen starvation, and starch was subsequently degraded and likely converted into TAG. The Slm1 did not produce starch and the carbon and energy acquired from photosynthesis was partitioned towards TAG synthesis. This resulted in an increase of the maximum TAG content in Slm1 to 57% (%CDW) compared to 45% (%CDW) in the wild type. Furthermore, it increased the maximum yield of TAG on light by 51%, from 0.144 in the wild type to 0.217 g TAG mol-1 photon-1 in the Slm1 mutant. No differences in photosynthetic efficiency between the Slm1 mutant and the wild type were observed, indicating that the mutation specifically improved carbon partitioning towards TAG and the photosynthetic capacity was not affected.
To identify the mutation that caused the starchless phenotype of Slm1 the transcriptome of both the wild type and the Slm1 mutant was sequenced as described in chapter 5. A single nucleotide polymorphism (SNP) was discovered in the small subunit of the starch biosynthesis rate-controlling enzyme ADP-glucose pyrophosphorylase, which resulted in the introduction of a STOP codon in the messenger RNA of the enzyme. The characterization of the mutation increases the understanding of carbon partitioning in oleaginous microalgae, leading to a promising target for future genetic engineering approaches to increase TAG accumulation in microalgae.
To use the insight that is gained in chapters 2-5 for metabolic engineering of TAG accumulation and carbon partitioning, a metabolic engineering toolbox is required. However, the development of transformation protocols for new and less well studied industrially relevant microalgae is challenging. In chapter 6, a simple and effective tool for the optimization of transformation protocols is proposed. Optimal voltage settings were determined for five microalgae: C. reinhardtii, Chlorella vulgaris, N. oleoabundans, S. obliquus, and Nannochloropsis sp. This method can be used to speed up the screening process for species that are susceptible for transformation and to successfully develop transformation strategies for industrially relevant microalgae, which lack an efficient transformation protocol.
In addition to the increase in productivity, improving the quality in terms of fatty acid composition of TAG molecules would be desired as well. For example, the accumulation of stearic acid rich TAG molecules is of special interest, because of the improved structural properties. The lipid accumulating starchless mutant of the model species C. reinhardtii BAFJ5 was used as model species in chapter 7, since genetic toolbox is well established for this species. In this chapter, stearoyl-ACP desaturase (SAD), is silenced by artificial microRNA. The mRNA levels for SAD were reduced after the silencing construct was induced. In one of the strains, the reduction in SAD mRNA resulted in a doubling of the stearic acid content in triacylglycerol molecules, which shows that increasing the fraction of stearic acid in TAG is possible. Furthermore, we hypothesize that in addition to direct conversion in the chloroplast, C. reinhardtii is able to redirect stearic acid from the chloroplast to the cytosol and convert it to oleic acid in the endoplasmic reticulum by stearoyl-CoA desaturase.
In chapter 8, an outlook is given on microalgal strain improvement strategies for the future, reflecting on the results obtained in this thesis. Also a roadmap is suggested to get genetically modified microalgal derived products on the market. The results presented in this thesis, provide a significant improvement in the understanding of TAG accumulation and carbon partitioning in oleaginous microalgae. Furthermore, improved microalgal strains with increased TAG accumulation or improved TAG fatty acid composition under nitrogen depleted conditions were generated. In addition, an outlook is presented in which the major bottlenecks are presented in future industrial applications of microalgae.
Effect of Methanethiol Concentration on Sulfur Production in Biological Desulfurization Systems under Haloalkaline Conditions
Roman, P. ; Veltman, R. ; Bijmans, M.F.M. ; Keesman, K.J. ; Janssen, A.J.H. - \ 2015
Environmental Science and Technology 49 (2015)15. - ISSN 0013-936X - p. 9212 - 9221.
natron-alkaline conditions - sulfide oxidation - oxidizing bacteria - aqueous-solutions - soda lakes - lab-scale - bioreactors - solubility - kinetics - polysulfides
Bioremoval of H2S from gas streams became popular in recent years because of high process efficiency and low operational costs. To expand the scope of these processes to gas streams containing volatile organic sulfur compounds, like thiols, it is necessary to provide new insights into their impact on overall biodesulfurization process. Published data on the effect of thiols on biodesulfurization processes are scarce. In this study, we investigated the effect of methanethiol on the selectivity for sulfur production in a bioreactor integrated with a gas absorber. This is the first time that the inhibition of biological sulfur formation by methanethiol is investigated. In our reactor system, inhibition of sulfur production started to occur at a methanethiol loading rate of 0.3 mmol L–1 d–1. The experimental results were also described by a mathematical model that includes recent findings on the mode of biomass inhibition by methanethiol. We also found that the negative effect of methanethiol can be mitigated by lowering the salinity of the bioreactor medium. Furthermore, we developed a novel approach to measure the biological activity by sulfide measurements using UV-spectrophotometry. On the basis of this measurement method, it is possible to accurately estimate the unknown kinetic parameters in the mathematical model.
Modeling studies of biological gas desulfurization under haloalkaline conditions
Klok, J.B.M. - \ 2015
Wageningen University. Promotor(en): Albert Janssen, co-promotor(en): Karel Keesman. - Wageningen : Wageningen University - ISBN 9789462572980 - 158
biogas - aardgas - ontzwaveling - sulfiden - oxidatie - bioreactoren - wiskundige modellen - simulatiemodellen - biogas - natural gas - desulfurization - sulfides - oxidation - bioreactors - mathematical models - simulation models
Biogas, synthesis and natural gas streams often require treatment because of the presence of gaseous hydrogen sulphide (H2S). About 25 years ago, a biotechnological gas treatment process was developed as an alternative to the conventionally applied technologies. This process is known as the Thiopaq process and offers a number of advantages compared to the existing physical-chemical processes. Depending on the process conditions, H2S is oxidized to elemental bio-sulfur (90-94 mol%) and sulphate (6-10 mol%). In order to enable cost effective large scale applications, the selectivity for sulfur production should be increased to more than 97 mol%. Hence, a better understanding of the combined effect of abiotic and biological reaction kinetics and the relation to hydrodynamic characteristics is required.
The first part of this PhD study focuses on biological reaction kinetics and biological pathways for sulphide oxidation that occur in the process at haloalkaline conditions. It was found that two different sulfide oxidizing enzyme systems are present in haloalkaline sulfide oxidizing bacteria. It has been hypothesized that the different enzymatic routes are determined by the process conditions. Both enzyme systems were taken into account to propose and validate a new physiological mathematical model that can handle multi-substrates and multi-products.
In the second part of the thesis, this model was evaluated via a normalized sensitivity method and it was demonstrated that certain key parameters affect the activity of the biomass at different substrate levels. Furthermore, from CSTR simulations it has been demonstrated that non-linear effects are of importance when scaling up from lab-scale to full-scale industrial units.
Finally, the developed kinetic models have been incorporated in a full-scale biodesulfurization model that includes the effects of turbulent flow regimes and mass transfer of oxygen. This enables us to better understand the overall process. Moreover, the model can also be used as a tool to design model-based control strategies which will lead to better overall process performance, i.e. maximize sulfur production and minimize chemical consumption rates.
Bioflocculation of wastewater organic matter at short retention times
Faust, L. - \ 2014
Wageningen University. Promotor(en): Huub Rijnaarts, co-promotor(en): Hardy Temmink. - Wageningen : Wageningen University - ISBN 9789462571716 - 153
afvalwaterbehandeling - uitvlokking - bioreactoren - geactiveerd slib - organische verbindingen - biopolymeren - energieterugwinning - waste water treatment - flocculation - bioreactors - activated sludge - organic compounds - biopolymers - energy recovery
PAT for PER.C6® perfusion cultivation
Mercier, S.M. - \ 2014
Wageningen University. Promotor(en): Rene Wijffels, co-promotor(en): Mathieu Streefland. - Wageningen : Wageningen University - ISBN 9789462571396 - 155
celcultuur vaccins - vaccinontwikkeling - procesontwerp - procesbewaking - procesoptimalisatie - bioreactoren - celkweek - cell culture vaccines - vaccine development - process design - process control - process optimization - bioreactors - cell culture
BioHydrogen : hydrogen from light and organic waste
Hoekema, S. - \ 2014
Wageningen University. Promotor(en): Rene Wijffels, co-promotor(en): Marcel Janssen; Hans Tramper. - Wageningen : Wageningen University - ISBN 9789462571051 - 228
organisch afval - waterstof - licht - bioproceskunde - bioreactoren - energie - organic wastes - hydrogen - light - bioprocess engineering - bioreactors - energy
Effect of dissolved oxygen concentration on the bioflocculation process in high loaded MBRs
Faust, L. ; Temmink, B.G. ; Zwijnenburg, A. ; Kemperman, A.J.B. ; Rijnaarts, H. - \ 2014
Water Research 66 (2014). - ISSN 0043-1354 - p. 199 - 207.
afvalwaterbehandeling - uitvlokking - bioreactoren - waste water treatment - flocculation - bioreactors - waste-water treatment - extracellular polymeric substances - activated-sludge flocs - improved energy recovery - membrane bioreactor - size distribution - biofilm structure - stability - sewage - performance
High-loaded membrane bioreactors (HL-MBRs), i.e. MBRs which are operated at extremely short sludge and hydraulic retention times, can be applied to flocculate and concentrate sewage organic matter. The concentrated organics can be used for energy recovery, or for the production of more valuable organic chemicals. Little is known about the effect of the dissolved oxygen concentration (DO) on this bioflocculation process. To examine this effect, two HL-MBRs were operated, respectively at a low (1 mg L-1) and a higher (4 mg L-1) DO. The higher DO resulted in a better flocculation efficiency, i.e. 92% of the colloidal COD in the sewage flocculated compared to 69% at the lower DO. The difference was attributed to a higher microbial production of extracellular polymeric substances at a DO of 4 mg L-1 and to more multivalent cations (calcium, iron and aluminium) being distributed to the floc matrix. In addition, the HL-MBR that was operated at a DO of 4 mg L-1 gave a bigger mean floc size, a lower supernatant turbidity, better settleability and better membrane filterability than the HL-MBR that was operated at a DO of 1 mg L-1.
Sulfide response analysis for sulfide control using a pS electrode in sulfate reducing bioreactors
Villa Gomez, D.K. ; Cassidy, J. ; Keesman, K.J. ; Sampaio, R.M. ; Lens, P.N.L. - \ 2014
Water Research 50 (2014). - ISSN 0043-1354 - p. 48 - 58.
afvalwaterbehandeling - bioreactoren - sulfiden - waste water treatment - bioreactors - sulfides - anaerobic-digestion process - acid-mine drainage - waste-water treatment - stirred-tank reactor - zns precipitation - hydrogen-sulfide - control design - reduction - bacteria - culture
Step changes in the organic loading rate (OLR) through variations in the influent chemical oxygen demand (CODin) concentration or in the hydraulic retention time (HRT) at constant COD/SO4 2- ratio (0.67) were applied to create sulfide responses for the design of a sulfide control in sulfate reducing bioreactors. The sulfide was measured using a sulfide ion selective electrode (pS) and the values obtained were used to calculate proportional-integral-derivative (PID) controller parameters.
Step changes in the organic loading rate (OLR) through variations in the influent chemical oxygen demand (CODin) concentration or in the hydraulic retention time (HRT) at constant COD/SO42- ratio (0.67) were applied to create sulfide responses for the design of a sulfide control in sulfate reducing bioreactors. The sulfide was measured using a sulfide ion selective electrode (pS) and the values obtained were used to calculate proportional-integral -derivative (PID) controller parameters. The experiments were performed in an inverse fluidized bed bioreactor with automated operation using the LabVIEW software version 2009 (R). A rapid response and high sulfide increment was obtained through a stepwise increase in the CODin, concentration, while a stepwise decrease to the HRT exhibited a slower response with smaller sulfide increment. Irrespective of the way the OLR was decreased, the PS response showed a time-varying behavior due to sulfide accumulation (HRT change) or utilization of substrate sources that were not accounted for (CODin change). The pS electrode response, however, showed to be informative for applications in sulfate reducing bioreactors. Nevertheless, the recorded pS values need to be corrected for pH variations and high sulfide concentrations (>200 mg/L). (C) 2013 Elsevier Ltd. All rights reserved.
Evaluation of tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots for the production of geraniol, the first committed step in terpenoid indole alkaloid pathway
Ritala, A. ; Dong, L. ; Imseng, N. ; Seppanen-Laakso, T. ; Vasilev, N. ; Krol, A.R. van der; Rischer, H. ; Maaheimo, H. ; Virkki, A. ; Brandli, J. ; Schillberg, S. ; Eibl, R. ; Bouwmeester, H.J. ; Oksman-Caldentey, K.M. - \ 2014
Journal of Biotechnology 176 (2014). - ISSN 0168-1656 - p. 20 - 28.
catharanthus-roseus - isoprenoid biosynthesis - plastidial pathways - essential oils - key enzyme - monoterpene - cultures - synthase - cells - bioreactors
The terpenoid indole alkaloids are one of the major classes of plant-derived natural products and are well known for their many applications in the pharmaceutical, fragrance and cosmetics industries. Hairy root cultures are useful for the production of plant secondary metabolites because of their genetic and biochemical stability and their rapid growth in hormone-free media. Tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) hairy roots, which do not produce geraniol naturally, were engineered to express a plastidtargeted geraniol synthase gene originally isolated from Valeriana officinalis L. (VoGES). A SPME-GC–MS screening tool was developed for the rapid evaluation of production clones. The GC–MS analysis revealed that the free geraniol content in 20 hairy root clones expressing VoGES was an average of 13.7 g/g dry weight (DW) and a maximum of 31.3 g/g DW. More detailed metabolic analysis revealed that geraniol derivatives were present in six major glycoside forms, namely the hexose and/or pentose conjugates of geraniol and hydroxygeraniol, resulting in total geraniol levels of up to 204.3 g/g DW following deglycosylation. A benchtop-scale process was developed in a 20-L wave-mixed bioreactor eventually yielding hundreds of grams of biomass and milligram quantities of geraniol per cultivation bag.
IPV v2.0 : upgrading the established inactivated polio vaccine production process
Thomassen, Y.E. - \ 2014
Wageningen University. Promotor(en): Rene Wijffels, co-promotor(en): W.A.M. Bakker; L.A. van der Pol. - Wageningen : Wageningen University - ISBN 9789461738561 - 231
poliomyelitis - geïnactiveerde vaccins - vaccinontwikkeling - bioreactoren - poliomyelitis - inactivated vaccines - vaccine development - bioreactors
The first vaccine against poliovirus (PV), the causative agent of poliomyelitis, was developed in the 1950s by Jonas Salk. The vaccine (IPV) consists of an injected dose of purified and inactivated wild-type PVs (all three serotypes). Soon after this discovery, at the Rijks Instituut voor de Volksgezondheid (RIV) in Bilthoven, an industrial-scale production process for IPV was developed based on micro-carrier technology and primary monkey kidney cells. In the 1970s, the manufacturing of IPV was scaled up to 300-L by the development of well-controlled bioreactors for cell culture, the so-called “Bilthoven Units” (originally used for bacterial fermentations). In 2004, the Vero cell line was introduced to replace the then used tertiary monkey kidney cells followed by a scale-up from 700 to 1,500-L (from two 350-L to two 750-L bioreactors). IPV manufacturing has been part of the regular vaccine manufacturing activities in Bilthoven ever since the establishment of the IPV production process.
With polio eradication on our doorstep, the World Health Organization (WHO) is pursuing a new IPV based on non-wild-type strains to increase the biosafety of vaccine manufacturing. In addition, and due to the pending cessation of oral polio vaccines (OPV), the global demand for affordable IPV is increasing. To accommodate these questions two research programs were started at the Netherlands Vaccine Institute (now Institute for Translational Vaccinology). One concerned optimization of the current conventional IPV production process, the other the manufacturing of an affordable sIPV, an IPV based on the attenuated Sabin PV strains normally used for OPV production. The technology of the sIPV production process, developed in the latter project, is also aimed to be transferred to developing countries manufacturers (Chapter 2).
From the substantial history in polio vaccine production in Bilthoven, a valuable dataset has been generated. Data from over 50 batches at two different production scales (700-L and 1,500-L) were analyzed using multivariate data analysis (MVDA). This statistical method is stimulated by the ICH (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use) to improve scientific understanding of production processes for troubleshooting and improved process control. The initial explorative analysis, performed on single unit operations, indicated consistent manufacturing. Known outliers (e.g., rejected batches) were identified using principal component analysis (PCA). The source of operational variation was pinpointed to variation of input such as cell- and virus culture media. Other relevant process parameters were in control and, using this manufacturing data, could not be correlated to product quality attributes. The gained knowledge of the IPV production process, not only from the MVDA, but also from digitalizing the available historical data, has proven to be useful for troubleshooting, understanding limitations of available data and seeing the opportunity for improvements (Chapter 3).
One of the gaps in the data was located in the product quantification during processing. The available assay used for determining the D-antigen concentration in in-process samples had high variability. A so-called fast ELISA was developed and qualified for analysis of polio D-antigen. The original 20h-protocol was optimized by minimizing the total incubation time to 1h, and by replacing the signal reagent 3,3',5,5'-tetramethylbenzidine by a chemiluminogenic signal reagent with a theoretical low intrinsic background and high dynamic range. This fast D-antigen ELISA was suitable for measurement of D-antigen concentrations in the different matrixes present during the different unit-operations in the production process (Chapter 4).
To accommodate research to improve and optimize the current cIPV production process an up-to-date lab-scale version encompassing the legacy inactivated polio vaccine production process was set-up based on the knowledge obtained during the MVDA of historical data. This lab-scale version was designed to be representative of the large scale, meaning a scale-down model, to allow experiments for process optimization that can be readily applied to manufacturing scale. Initially the separate unit operations were scaled-down at setpoint. Subsequently, the unit operations were applied successively in a comparative manner to large-scale manufacturing. This allows the assessment of the effects of changes in one unit operation to the consecutive units at small-scale. The developed scale-down model for cell and virus culture (2.3-L) presents a feasible model with its production scale counterpart (750-L) when operated at setpoint. Also, the scale-down models for the DSP unit operations clarification, concentration, size exclusion chromatography, ion exchange chromatography, and inactivation are in agreement with the manufacturing scale. The small-scale units can be used separately, as well as sequentially, to study variations and critical product quality attributes in the production process. Finally, it has been shown that the scale-down unit operations can be used consecutively to prepare trivalent vaccine at lab-scale with comparable characteristics to the product produced at manufacturing scale (Chapter 5).
The upcoming of disposables in GMP manufacturing triggered the study on alternatives for the clarification unit (Chapter 5) and alternative Wave-type, bioreactors (Chapter 6). This type of bioreactors makes use of sterilized disposable bags, which could be beneficial in a GMP environment, for example to reduce the cleaning validation burden, or to facilitate change-over to another product using the same equipment. Wave-type bioreactors make use of vertical (standard rocking motion-type) or both vertical and horizontal displacement (CELL-tainer®(CELLution Biotech BV)) for mixing instead of an impeller, which is used in stirred tank reactors. Using the design of experiments (DoE) approach, models for the mixing times in both the CELL-tainer®and the BIOSTAT®CultiBag RM (Sartorius Stedim Biotech) bioreactor (standard rocking motion-type) were developed. The conditions for cultivation of Vero cells in the CELL-tainer®bioreactor were chosen based on comparable mixing times. Vero cells growing adherent to Cytodex 1 microcarriers were cultivated in the CELL-tainer®and in the BIOSTAT®CultiBag RM. Vero cell growth in both bioreactors was comparable with respect to the growth characteristics and main metabolite production and consumption rates. Additionally, poliovirus production in both bioreactors was shown to be similar.
In view of WHOs pursuit towards an IPV manufacturing process with increased biosafety, the development of sIPV was taken up. Prior to large scale production of clinical lots, an initial proof of principle study was done (Chapter 2). Starting from the conventional IPV (cIPV) production process, minimal adaptations, such as lower virus cultivation temperature, were implemented. Also, the selected disposable filter unit (Chapter 5) was implemented. To quickly prepare sIPV clinical lots and show proof-of-principle of sIPV in human, no further process optimization and/or modernization was done. sIPV was produced at industrial scale followed by formulation of both plain and aluminium adjuvanted sIPV (Chapter 7). The final products met the quality criteria, were immunogenic in rats, showed no toxicity in rabbits and could be released for testing in the clinic. While an immunogenic product, both in animals as in humans was prepared, the product yield was extremely low and further process development will be needed to obtain an affordable sIPV.
Especially the yield of Sabin PV type 2 after ion exchange chromatography was low. To determine if this effect could be due to a difference in the isoelectric point (pI) of the poliovirus a method for pImeasurement of live virus was developed (Chapter 8). A method for analyzing biological hazardous components (biological safety level 2) was set up for the capillary isoelectric focusing-whole column imaging detection (CIEF-WCID) analyzer. This method is based on closed circuits. Subsequently, the pI’s of complete intact polioviruses were determined. The polioviruses that were analyzed are the commonly used viruses for the production of IPV - Mahoney (type 1), MEF-1 (type 2), and Saukett (type 3) - as well as for OPV - Sabin types 1, 2, and 3. The determined pI's were 6.2 for Mahoney, 6.7 for MEF-1, and 5.8 for Saukett. The pI's of Sabin types 1, 2, and 3 viruses were 7.4, 7.2, and 6.3, respectively. With a pIof 7.2, Sabin PV type 2 is prone to self-aggregation at the pH used during chromatography (pH 7.0). Self-aggregation was thus suggested to be the main cause of low product yield and prevention of this self-aggregation was suggested to be the main focus point for process optimization.
Besides optimization of the downstream processing, optimization of the upstream processing, i.e. increased virus yields after cell and virus culture was studied (Chapter 9). Vero cells were grown adherent to microcarriers (Cytodex 1; 3 g L-1) using animal component free media in stirred-tank type bioreactors. Different strategies for media refreshment, daily media replacement (semi-batch), continuous media replacement (perfusion) and recirculation of media, were compared with batch cultivation. Cell densities increased using a feed strategy from 1 × 106cells mL-1during batch cultivation to 1.8, 2.7 and 5.0 × 106cells mL-1during semi-batch, perfusion and recirculation, respectively. The effects of these different cell culture strategies on subsequent poliovirus production were investigated. Increased cell densities allowed up to 3 times higher D-antigen levels when compared with that obtained from batch-wise Vero cell culture. However, the cell specific D-antigen production was lower when cells were infected at higher cell densities. This cell density effect is in good agreement with observations for different cell lines and virus types. From the evaluated alternative culture methods, application of a semi-batch mode of operations allowed the highest cell specific D-antigen production. The increased product yields that can easily be reached using these higher cell density cultivation methods, showed the possibility for better use of bioreactor capacity for the manufacturing of polio vaccines to ultimately reduce vaccine cost per dose. Further, the use of animal-component-free cell- and virus culture media shows opportunities for modernization of human viral vaccine manufacturing.
To assess the affordability of sIPV the manufacturing costs were determined (Chapter 10). The sIPV manufacturing costs, when produced as described in Chapter 7, indicate the requirement for process optimizations. However, the manufacturing costs can be reduced at least a factor 2 when implementing the upstream processing optimization, ACF media and a semi-batch process, as described in Chapter 9. Assuming improvements in downstream processing will result in a process with yields comparable to the cIPV process costs can be further lowered. In addition, a scale-up from two 350-L bioreactors to two 1,000-L bioreactors would nearly halve the manufacturing costs resulting in a more than cost competitive sIPV. These costs analysis showed that an affordable sIPV is feasible.
Nutrient removal and microalgal biomass production on urine in a short light-path photobioreactor
Tuantet, K. ; Temmink, B.G. ; Zeeman, G. ; Janssen, M.G.J. ; Wijffels, R.H. ; Buisman, C.J.N. - \ 2014
Water Research 55 (2014). - ISSN 0043-1354 - p. 162 - 174.
waterzuivering - bioreactoren - fosfaten - stikstof - terugwinning - nieuwe sanitatie - biomassa productie - urine - algenteelt - biobased economy - water treatment - bioreactors - phosphates - nitrogen - recovery - new sanitation - biomass production - urine - algae culture - biobased economy - chlorella-sorokiniana - spirulina-platensis - anaerobic treatment - black water - phosphorus - management - efficiency - vulgaris - biofuels
Due to the high nitrogen and phosphorus content, source-separated urine can serve as a major nutrient source for microalgae production. The aim of this study was to evaluate the nutrient removal rate and the biomass production rate of Chlorella sorokiniana being grown continuously in urine employing a short light-path photobioreactor. The results demonstrated, for the first time, the possibility of continuous microalgae cultivation in human urine. The lowest dilution factor successfully employed was a factor of 2 (50% v/v urine). Microalgae dominated a smaller bacterial population and were responsible for more than 90% of total nitrogen and phosphorus removal.
Activity and viability of methanogens in anaerobic digestion of unsaturated and saturated long-chain fatty acids
Sousa, D.Z. ; Salvador, A.F. ; Ramos, J. ; Guedes, A.P. ; Barbosa, S. ; Stams, A.J.M. ; Alves, M.M. ; Pereira, M.A. - \ 2013
Applied and Environmental Microbiology 79 (2013)14. - ISSN 0099-2240 - p. 4239 - 4245.
16s ribosomal-rna - microbial communities - calcium addition - waste-water - oleic-acid - bacteria - inhibition - sludge - bioreactors - diversity
Lipids can be anaerobically digested to methane, but methanogens are often considered to be highly sensitive to the long-chain fatty acids (LCFA) deriving from lipids hydrolysis. In this study, the effect of unsaturated (oleate [C18:1]) and saturated (stearate [C18:0] and palmitate [C16:0]) LCFA toward methanogenic archaea was studied in batch enrichments and in pure cultures. Overall, oleate had a more stringent effect on methanogens than saturated LCFA, and the degree of tolerance to LCFA was different among distinct species of methanogens. Methanobacterium formicicum was able to grow in both oleate- and palmitate-degrading enrichments (OM and PM cultures, respectively), whereas Methanospirillum hungatei only survived in a PM culture. The two acetoclastic methanogens tested, Methanosarcina mazei and Methanosaeta concilii, could be detected in both enrichment cultures, with better survival in PM cultures than in OM cultures. Viability tests using live/dead staining further confirmed that exponential growth-phase cultures of M. hungatei are more sensitive to oleate than are M. formicicum cultures; exposure to 0.5 mM oleate damaged 99% ± 1% of the cell membranes of M. hungatei and 53% ± 10% of the cell membranes of M. formicicum. In terms of methanogenic activity, M. hungatei was inhibited for 50% by 0.3, 0.4, and 1 mM oleate, stearate, and palmitate, respectively. M. formicicum was more resilient, since 1 mM oleate and >4 mM stearate or palmitate was needed to cause 50% inhibition on methanogenic activity
Algenteeltsystemen in praktijkkassen in Nederland
Hemming, S. ; Sapounas, A. ; Voogt, W. - \ 2013
teeltsystemen - algenteelt - glastuinbouw - bioreactoren - biobased economy - cropping systems - algae culture - greenhouse horticulture - bioreactors - biobased economy
In de tuinbouwpraktijk experimenteren een aantal telers met de combinatie van algen en traditionele gewassen. Deze poster biedt een kort overzicht van de gebruikte systemen.
Innovative landfill bioreactor systems for municipal solid waste treatment in East Africa aimed at optimal energy recovery and minimal greenhouse gas emissions
Salukele, F.M. - \ 2013
Wageningen University. Promotor(en): Wim Rulkens, co-promotor(en): Joost van Buuren. - S.l. : s.n. - ISBN 9789461736338 - 190
afvalverwerking - bioreactoren - stortterreinen - milieutechnologie - milieufactoren - oost-afrika - waste treatment - bioreactors - landfills - environmental technology - environmental factors - east africa
Landfilling is currently the dominant disposal method for municipal solid waste (MSW) in developing countries. Approximately 50% of the MSW generated in East Africa is disposed in landfills. Low costs and availability of land have made landfilling the most common waste management option in East Africa. Two main aspects associated with landfills are the landfill gas potential (LFG) and the greenhouse gas emission. A desk study into the development and application of landfill systems for treating MSW have indicated that the operation of landfills as bioreactors is an interesting and viable option for MSW management. The main objective of the thesis was to develop and describe landfill bioreactor (LFB) basedtreatment systems suitable for MSW in East African cities. MSW collected in these cities is characterized dominantly by a high content of organic material and a high moisture content. It is expected that a more sophisticated and modern form of landfill such as a LFB will become important as a treatment system for MSW in East Africa on the short and middle term. For this purpose, four innovative landfill bioreactor system options which are technically feasible and resource-recovery oriented that match the conditions of East African cities have been developed. These innovative system options of landfills operated as bioreactors were identified, elaborated and evaluated based on literature information regarding the construction and performance of landfill bioreactors in highly industrialized western countries and characteristics of MSW in East Africa, experimental research on pilot plant scale and desk studies regarding biological conversion of the waste, and modeling of the biodegradation rates and biogas production of MSW. The four system options were also evaluated by means of a semi-mathematical calculation model regarding their investment and operation costs, land space requirement, leachate treatment costs and savings, LFG generation and LFG collection and utilization costs and benefits, airspace recovery, greenhouse gas accounting and global warming avoidance.The results with respect to the evaluation were compared with a controlled dumpsite for MSW as currently applied in East Africa. All four modifications of the LFB show great advantages with respect to landfill size, amount of biogas collected and reduction of the emission of greenhouse gases.The innovative system options proposed in this thesis are useful and helpful for decision makers in making the choice of MSW disposal suitable for the East-African cities
Synthesis of galacto-oligosaccharides with ß-galactosidases
Warmerdam, A. - \ 2013
Wageningen University. Promotor(en): Remko Boom, co-promotor(en): Anja Janssen. - S.l. : s.n. - ISBN 9789461735621 - 171
oligosacchariden - bèta-galactosidase - productie - synthese - immobilisatie - bioreactoren - lactose - oligosaccharides - beta-galactosidase - production - synthesis - immobilization - bioreactors - lactose
Galacto-oligosaccharides (GOS) are generally enzymatically synthesized with β-galactosidases. GOS are of interest because of their prebiotic effects on human health. They are mainly applied in infant nutrition, because of their resemblance to human milk oligosaccharides, but they are also applied in e.g. dairy products and beverages.
β-Galactosidases synthesize GOS from lactose through transgalactosylation: instead of only using water as acceptor (as in hydrolysis), they can use carbohydrates as acceptor. In this way, GOS with a degree of polymerization up to ten can be formed. The ratio of hydrolysis over transgalactosylation depends on the substrate concentration, temperature, and the source of the enzyme.
A β-galactosidase preparation from Bacillus circulans, called Biolacta N5, is known to produce high GOS yields compared to enzymes from other sources. The aim of this thesis was to obtain more insight on the mechanism of GOS production with Biolacta N5 and to investigate how the GOS production process can be optimized.
Biolacta N5 consists of four β-galactosidase isoforms, β-gal-A, β-gal-B, β-gal-C, and β-gal-D, which were purified and characterized in chapter 2. At low substrate concentrations, these isoforms differ in hydrolysis and transgalactosylation activity. β-Gal-D seems the best isoform for GOS production, followed by β-gal-C and β-gal-B, and β-gal-A showed the least GOS formation. By studying the thermodynamics of lactose conversion with isothermal titration calorimetry (ITC), the differences in behavior were confirmed, although the interpretation of the results of ITC was quite difficult dealing with a complex mixture of reactions. In contrast to the selectivity at low substrate concentrations, the selectivity of the isoforms hardly differed at high lactose concentrations. These conditions are usually used for industrial GOS production. Only β-gal-A produced slightly more galactose. The initial GOS formation rates indicated that β-gal-A and β-gal-B are the best isoforms for GOS production.
In chapter 3, the effect of high concentrations was further studied on the behavior of the complete Biolacta N5 preparation. High concentrations of reacting and non-reacting carbohydrates were added to the oNPG activity assay with Biolacta N5. Small carbohydrates were found to act as acceptor in the reaction, which resulted in an increased reaction rate. The rate of the limiting step of the reaction, i.e. the binding of the galactose residue with the acceptor, is increased, and therewith the release of the product is faster. At the same time, the additives cause molecular crowding, which results in a higher affinity between the enzyme and the substrate.
In chapter 4, a kinetic model was developed to quantify the effects of lactose, glucose, galactose, and oligosaccharides on the oNPG converting activity of the β-galactosidases from B.circulans, Aspergillus oryzae and Kluyveromyces lactis. Using multiple substrates simultaneously yields more information than using only lactose or oNPG, because of the competition between the substrates. Three main differences were found that explain why Biolacta N5 produces higher GOS yields than other β-galactosidases: (i) it had a higher reaction rate constant of using lactose or oligosaccharides as substrate relative to water as acceptor (so it had a very low relative hydrolysis rate); and (ii) it also had a high reaction rate with galactose as acceptor, whereas (iii) the other two enzymes are strongly inhibited by galactose. The reaction rate constants indicate that β-gal-A is the most active isoforms in GOS production; however, also its hydrolysis rate is highest. Many of the rate constants increase with increasing molecular weight of the isoforms.
Chapter 5 reports on the stability of Biolacta N5 at various temperatures in buffer, and in systems with initially 5.0 and 30% (w/w) lactose. Samples were taken in time and analyzed for oNPG converting activity. The oNPG converting activity was corrected for the presence of lactose, glucose, galactose, and oligosaccharides with the mechanistic model from chapter 4. The stability, expressed with the half-life time, of the enzyme was found to strongly increase with initial lactose concentrations. At high substrate concentration, higher temperatures can be used for GOS production than was presumed feasible based on stability measurements in diluted solutions.
Biolacta N5 is still active after one batch run of GOS production, but in a batch process the enzyme is wasted after the reaction. For this reason, the use of immobilized enzyme in a continuous packed bed reactor (PBR) was investigated in terms of productivity in chapter 6. The carbohydrate composition of the product in both systems was comparable. The half-life time of the immobilized enzyme at a lactose concentration of 33% (w/w) and 50ºC was approximately 90 days. The enzymatic productivity using immobilized enzyme in a PBR may be six times higher than that using free enzyme in a batch reactor. When striving for an equal volumetric productivity of both systems, the volume of a PBR can be much smaller than that of a batch reactor, depending on the enzyme dosage and running time of the one batch.
Chapter 7 discusses various alternatives for process optimization. One option for a higher GOS productivity is to use an enzyme preparation that contains only β-gal-A and β-gal-B. A somewhat higher oligosaccharide yield can be obtained when initially using a mixture of lactose with a better acceptor molecule. This results in a changed oligosaccharide composition and less lactose in the final product. The sustainability of GOS production in a PBR with immobilized enzyme and 33% (w/w) lactose seems to be similar in terms of exergy to that in a batch reactor with free enzyme and 60% (w/w) lactose.