Effects of operating conditions on biogas production in an anaerobic digestion system of the food and beverage industry.

BACKGROUND: Food residuals (FR) were anaerobically biotransformed to produce biogases (e.g. methane and hydrogen), and different pre-treatment conditions, including particle size, oil content, pH and salt content, were controlled in this study. The bio-solids of a municipal solid waste (MSW) from a wastewater treatment plant were added to assess its effect on anaerobic transformation efficiency and gas yields. RESULTS: The breaking of FR and the application of MSW were effective in enhancing the transformation efficiency and yield of biogases. The energy transfer efficiency value of the combined FRs used in this study was probably 23%. However, it can be very cost effective to apply arbitrary proportions to treat two types of FR in the anaerobic digestion tank of a wastewater treatment plant. It was also found that the alkalinity and pH value were two major parameters that controlled the success of the transformation. About 0.16-0.17 kg of alkalinity was needed during the anaerobic digestion of 1 kg dry FR, but this requirement was decreased by the treatment applying MSW. Olive oil had higher reducing rates when used as a substitute for heat-oxidized oil to study the effect of oil content on methylation. CONCLUSION: The conditions for anaerobic digestion established in this study were practical for the digestion of FR in wastewater treatment plants in Taiwan. However, we nonetheless found that it was cost effective to use arbitrary proportions for both types of FR and integrate the anaerobic digestion process used in wastewater treatment plants. © 2020 Society of Chemical Industry.

Limited life cycle and cost assessment for the bioconversion of lignin-derived aromatics into adipic acid.

Lignin is an abundant and heterogeneous waste byproduct of the cellulosic industry, which has the potential of being transformed into valuable biochemicals via microbial fermentation. In this study, we applied a fast-pyrolysis process using softwood lignin resulting in a two-phase bio-oil containing monomeric and oligomeric aromatics without syringol. We demonstrated that an additional hydrodeoxygenation step within the process leads to an enhanced thermochemical conversion of guaiacol into catechol and phenol. After steam bath distillation, Pseudomonas putida KT2440-BN6 achieved a percent yield of cis, cis-muconic acid of up to 95 mol% from catechol derived from the aqueous phase. We next established a downstream process for purifying cis, cis-muconic acid (39.9 g/L) produced in a 42.5 L fermenter using glucose and benzoate as carbon substrates. On the basis of the obtained values for each unit operation of the empirical processes, we next performed a limited life cycle and cost analysis of an integrated biotechnological and chemical process for producing adipic acid and then compared it with the conventional petrochemical route. The simulated scenarios estimate that by attaining a mixture of catechol, phenol, cresol, and guaiacol (1:0.34:0.18:0, mol ratio), a titer of 62.5 (g/L) cis, cis-muconic acid in the bioreactor, and a controlled cooling of pyrolysis gases to concentrate monomeric aromatics in the aqueous phase, the bio-based route results in a reduction of CO2 -eq emission by 58% and energy demand by 23% with a contribution margin for the aqueous phase of up to 88.05 euro/ton. We conclude that the bio-based production of adipic acid from softwood lignins brings environmental benefits over the petrochemical procedure and is cost-effective at an industrial scale. Further research is essential to achieve the proposed cis, cis-muconic acid yield from true lignin-derived aromatics using whole-cell biocatalysts.

Technoeconomic analysis of semicontinuous bioreactor production of biopharmaceuticals in transgenic rice cell suspension cultures.

Biopharmaceutical protein production using transgenic plant cell bioreactor processes offers advantages over microbial and mammalian cell culture platforms in its ability to produce complex biologics with simple chemically defined media and reduced biosafety concerns. A disadvantage of plant cells from a traditional batch bioprocessing perspective is their slow growth rate which has motivated us to develop semicontinuous and/or perfusion processes. Although the economic benefits of plant cell culture bioprocesses are often mentioned in the literature, to our knowledge no rigorous technoeconomic models or analyses have been published. Here we present technoeconomic models in SuperPro Designer® for the large-scale production of recombinant butyrylcholinesterase (BChE), a prophylactic/therapeutic bioscavenger against organophosphate nerve agent poisoning, in inducible transgenic rice cell suspension cultures. The base facility designed to produce 25 kg BChE per year utilizing two-stage semicontinuous bioreactor operation manufactures a single 400 mg dose of BChE for $263. Semicontinuous operation scenarios result in 4-11% reduction over traditional two-stage batch operation scenarios. In addition to providing a simulation tool that will be useful to the plant-made pharmaceutical community, the model also provides a computational framework that can be used for other semicontinuous or batch bioreactor-based processes.

Mesenchymal stromal cell therapy: progress in manufacturing and assessments of potency.

Mesenchymal stromal cell (MSC) therapies have been pursued for a broad spectrum of indications but mixed reports on clinical efficacy have given rise to some degree of skepticism regarding the effectiveness of this approach. However, recent reports of successful clinical outcomes and regulatory approvals for graft-versus-host disease, Crohn's disease and critical limb ischemia have prompted a shift in this perspective. With hundreds of clinical trials involving MSCs currently underway and an increasing demand for large-scale manufacturing protocols, there is a critical need to develop standards that can be applied to processing methods and to establish consensus assays for both MSC processing control and MSC product release. Reference materials and validated, uniformly applied tests for quality control of MSC products are needed. Here, we review recent developments in MSC manufacturing technologies, release testing and potency assays. We conclude that, although MSCs hold considerable promise clinically, economies of scale have yet to be achieved although numerous bioreactor technologies for scalable production of MSCs exist. Additionally, rigorous disease-specific product testing and comprehensive understanding of mechanisms of action, which are linked to relevant process and product release potency assays, will be required to ensure that these therapies continue to be successful.

Accurate and cost-effective prediction of HBsAg titer in industrial scale fermentation process of recombinant Pichia pastoris by using neural network based soft sensor.

In the current work, the attempt was made to apply best-fitted artificial neural network (ANN) architecture and the respective training process for predicting final titer of hepatitis B surface antigen (HBsAg), produced intracellularly by recombinant Pichia pastoris Mut+ in the commercial scale. For this purpose, in large-scale fed-batch fermentation, using methanol for HBsAg induction and cell growth, three parameters of average specific growth rate, biomass yield, and dry biomass concentration-in the definite integral form with respect to fermentation time-were selected as input vectors; the final concentration of HBsAg was selected for the ANN output. Used dataset consists of 38 runs from previous batches; feed-forward ANN 3:5:1 with training algorithm of backpropagation based on a Bayesian regularization was trained and tested with a high degree of accuracy. Implementing the verified ANN for predicting the HBsAg titer of the five new fermentation runs, excluded from the dataset, in the full-scale production, the coefficient of regression and root-mean-square error were found to be 0.969299 and 2.716774, respectively. These results suggest that this verified soft sensor could be an excellent alternative for the current relatively expensive and time-intensive analytical techniques such as enzyme-linked immunosorbent assay in the biopharmaceutical industry.

A chicken bioreactor for efficient production of functional cytokines.

BACKGROUND: The global market for protein drugs has the highest compound annual growth rate of any pharmaceutical class but their availability, especially outside of the US market, is compromised by the high cost of manufacture and validation compared to traditional chemical drugs. Improvements in transgenic technologies allow valuable proteins to be produced by genetically-modified animals; several therapeutic proteins from such animal bioreactors are already on the market after successful clinical trials and regulatory approval. Chickens have lagged behind mammals in bioreactor development, despite a number of potential advantages, due to the historic difficulty in producing transgenic birds, but the production of therapeutic proteins in egg white of transgenic chickens would substantially lower costs across the entire production cycle compared to traditional cell culture-based production systems. This could lead to more affordable treatments and wider markets, including in developing countries and for animal health applications. RESULTS: Here we report the efficient generation of new transgenic chicken lines to optimize protein production in eggs. As proof-of-concept, we describe the expression, purification and functional characterization of three pharmaceutical proteins, the human cytokine interferon α2a and two species-specific Fc fusions of the cytokine CSF1. CONCLUSION: Our work optimizes and validates a transgenic chicken system for the cost-effective production of pure, high quality, biologically active protein for therapeutics and other applications.

Characterization and cost-benefit analysis of automated bioreactor-expanded mesenchymal stem cells for clinical applications.

BACKGROUND: Expanding quantities of mesenchymal stem cells (MSCs) sufficient to treat large numbers of patients in cellular therapy and regenerative medicine clinical trials is an ongoing challenge for cell manufacturing facilities. STUDY DESIGN AND METHODS: We evaluated options for scaling up large quantities of bone marrow-derived MSCs (BM-MSCs) using methods that can be performed in compliance with Good Manufacturing Practices (GMP). We expanded BM-MSCs from fresh marrow aspirate in αMEM supplemented with 5% human platelet lysate using both an automated cell expansion system (Quantum, Terumo BCT) and a manual flask-based method using multilayer flasks. We compared MSCs expanded using both methods and assessed their differentiation to adipogenic and osteogenic tissue, capacity to suppress T-cell proliferation, cytokines, and growth factor secretion profile and cost-effectiveness of manufacturing enough BM-MSCs to administer a single dose of 100 × 106 cells per subject in a clinical trial of 100 subjects. RESULTS: We have established that large quantities of clinical-grade BM-MSCs manufactured with an automated hollow-fiber bioreactor were phenotypically (CD73, CD90, CD105) and functionally (adipogenic and osteogenic differentiation and cytokine and growth factor secretion) similar to manually expanded BM-MSCs. In addition, MSC manufacturing costs significantly less and required less time and effort when using the Quantum automated cell expansion system over the manual multilayer flasks method. CONCLUSION: MSCs manufactured by an automated bioreactor are physically and functionally equivalent to the MSCs manufactured by the manual flask method and have met the standards required for clinical application.

Resolving mismatches in the flexible production of ethanol and butanol from eucalyptus wood with vacuum fermentation.

In flexible ethanol-butanol plants, low tolerance to butanol by solventogenic clostridia (and resulting dilute fermentation) results in considerable number of empty fermentors whenever production focuses on ethanol. This research identified scenarios in which vacuum fermentation (in-situ vacuum recovery) may be applied to solve this problem. We conducted ethanol (Saccharomyces cerevisiae) and ABE (Clostridium beijerinckii NCIMB 8052) batch vacuum fermentations of eucalyptus hydrolysates according to the distribution of sugars in a flexible plant. Based on the experiments and performance targets set for the ABE fermentation, we simulated a flexible plant that processes 1000 dry t eucalyptus/day using pretreatment and enzymatic hydrolysis steps with moderate solids loading (15% w/w). The simulation showed that the number of fermentation tanks can decrease by 62% (eliminating 10 idle tanks, 3748 m3 each) by applying vacuum recovery only to the fermentation of mixed (cellulose + hemicellulose) hydrolysates to ABE. We concluded that this configuration can result in savings of up to 2 MMUS$/year in comparison with flexible plants having only conventional batch fermentors, and additional cost savings are expected from reduced wastewater footprint.

Biosynthesis of butyric acid by Clostridium tyrobutyricum.

Butyric acid (C3H7COOH) is an important chemical that is widely used in foodstuffs along with in the chemical and pharmaceutical industries. The bioproduction of butyric acid through large-scale fermentation has the potential to be more economical and efficient than petrochemical synthesis. In this paper, the metabolic pathways involved in the production of butyric acid from Clostridium tyrobutyricum using hexose and pentose as substrates are investigated, and approaches to enhance butyric acid production through genetic modification are discussed. Finally, bioreactor modifications (including fibrous bed bioreactor, inner disk-shaped matrix bioreactor, fibrous matrix packed in porous levitated sphere carriers), low-cost feedstocks, and special treatments (including continuous fermentation with cell recycling, extractive fermentation with solvent, using different artificial electron carriers) intended to improve the feasibility of commercial butyric acid bioproduction are summarized.

Integrated continuous biomanufacturing platform with ATF perfusion and one column chromatography operation for optimum resin utilization and productivity.

A new integrated continuous biomanufacturing platform for continuous production of antibodies at fixed cell volumes and cell concentrations for extended periods with immediate capture is presented. Upstream antibody production has reached technological maturity, however, the bottleneck for continuous biomanufacturing remains the efficient and cost-effective capture of therapeutic antibodies in an initial chromatography step. In this study, the first successful attempt at using one-column continuous chromatography (OCC) for the continuous capture of therapeutic antibodies produced through alternating tangential flow perfusion is presented. By performing upstream media optimizations, the upstream perfusion rate was reduced to one vessel volume per day (vv/d), increasing antibody titer and reducing the volume of perfusate. In addition, process improvements were performed to increase productivity by 80% over previously reported values. In addition, a real-time method for evaluating column performance to make column switching decisions was developed. This improved productivity coupled with the use of a single-column improved process monitoring and control in OCC compared to multi-column systems. This approach is the first report on using a single column for the implementation of an integrated continuous biomanufacturing platform and offers a cost-effective and flexible platform process for the manufacture of therapeutic proteins.

Optimization of a biotechnological multiproduct batch plant design for the manufacture of four different products: A real case scenario.

In this work a biotechnological multiproduct batch plant that manufactures four different recombinant proteins for human application is described in some detail. This batch plant design is then optimized with regards to the size of equipment using a mixed-integer linear programming (MILP) formulation recently developed by us in order to find a hypothetical new biotechnological batch plant based on the information of real known processes for the production of the four recombinant protein products. The real plant was divided for practical purposes into two sub-processes or facilities: a fermentation facility and a purification facility. Knowing the specific steps conforming the downstream processing of each product, size, and time factors were computed and used as parameters to solve the aforementioned MILP reformulation. New constraints were included to permit the selection of some equipment-such as centrifuges and membrane filters-in a discrete set of sizes. For equipment that can be built according to customer needs-such as reactors-the original formulation was retained. Computational results show the ability of this optimization methodology to deal with real data giving reliable solutions for a multi-product batch plant composed of 44 unit operations in a relatively small amount of time showing that in the case studied it is possible to save up to a 66% of the capital investment in equipment given the cost data used. Biotechnol. Bioeng. 2017;114: 1252-1263. © 2017 Wiley Periodicals, Inc.

Toward high solids loading process for lignocellulosic biofuel production at a low cost.

High solids loadings (>18 wt%) in enzymatic hydrolysis and fermentation are desired for lignocellulosic biofuel production at a high titer and low cost. However, sugar conversion and ethanol yield decrease with increasing solids loading. The factor(s) limiting sugar conversion at high solids loading is not clearly understood. In the present study, we investigated the effect of solids loading on simultaneous saccharification and co-fermentation (SSCF) of AFEX™ (ammonia fiber expansion) pretreated corn stover for ethanol production using a xylose fermenting strain Saccharomyces cerevisiae 424A(LNH-ST). Decreased sugar conversion and ethanol yield with increasing solids loading were also observed. End-product (ethanol) was proven to be the major cause of this issue and increased degradation products with increasing solids loading was also a cause. For the first time, we show that with in situ removal of end-product by performing SSCF aerobically, sugar conversion stopped decreasing with increasing solids loading and monomeric sugar conversion reached as high as 93% at a high solids loading of 24.9 wt%. Techno-economic analysis was employed to explore the economic possibilities of cellulosic ethanol production at high solids loadings. The results suggest that low-cost in situ removal of ethanol during SSCF would significantly improve the economics of high solids loading processes. Biotechnol. Bioeng. 2017;114: 980-989. © 2016 Wiley Periodicals, Inc.

Cost comparison of full-scale water reclamation technologies with an emphasis on membrane bioreactors.

The paper assesses the costs of full-scale membrane bioreactors (MBRs). Capital expenditures (CAPEX) and operating expenses (OPEX) of Spanish MBR facilities have been verified and compared to activated sludge plants (CAS) using water reclamation treatment (both conventional and advanced). Spanish MBR facilities require a production of 0.6 to 1.2 kWh per m3, while extended aeration (EA) and advanced reclamation treatment require 1.2 kWh per m3. The energy represents around 40% of the OPEX in MBRs. In terms of CAPEX, the implementation costs of a CAS facility followed by conventional water reclamation treatment (physical-chemical + sand filtration + disinfection) ranged from 730 to 850 €.m-3d, and from 1,050 to 1,250 €.m-3d in the case of advanced reclamation treatment facilities (membrane filtration) with a capacity of 8,000 to 15,000 m3d-1. The MBR cost for similar capacities ranges between 700 and 960 €.m-3d. This study shows that MBRs that have been recently installed represent a cost competitive option for water reuse applications for medium and large capacities (over 10,000 m3d-1), with similar OPEX to EA and conventional water reclamation treatment. In terms of CAPEX, MBRs are cheaper than EA, followed by advanced water reclamation treatment.

Energy, cost and design aspects of coarse- and fine-bubble aeration systems in the MBBR IFAS process.

With the MBBR IFAS (moving bed biofilm reactor integrated fixed-film activated sludge) process, the biomass required for biological wastewater treatment is either suspended or fixed on free-moving plastic carriers in the reactor. Coarse- or fine-bubble aeration systems are used in the MBBR IFAS process. In this study, the oxygen transfer efficiency (OTE) of a coarse-bubble aeration system was improved significantly by the addition of the investigated carriers, even in-process (∼1% per vol-% of added carrier material). In a fine-bubble aeration system, the carriers had little or no effect on OTE. The effect of carriers on OTE strongly depends on the properties of the aeration system, the volumetric filling rate of the carriers, the properties of the carrier media, and the reactor geometry. This study shows that the effect of carriers on OTE is less pronounced in-process compared to clean water conditions. When designing new carriers in order to improve their effect on OTE further, suppliers should take this into account. Although the energy efficiency and cost effectiveness of coarse-bubble aeration systems can be improved significantly by the addition of carriers, fine-bubble aeration systems remain the more efficient and cost-effective alternative for aeration when applying the investigated MBBR IFAS process.

Cost minimization in a full-scale conventional wastewater treatment plant: associated costs of biological energy consumption versus sludge production.

Energy consumption and sludge production minimization represent rising challenges for wastewater treatment plants (WWTPs). The goal of this study is to investigate how energy is consumed throughout the whole plant and how operating conditions affect this energy demand. A WWTP based on the activated sludge process was selected as a case study. Simulations were performed using a pre-compiled model implemented in GPS-X simulation software. Model validation was carried out by comparing experimental and modeling data of the dynamic behavior of the mixed liquor suspended solids (MLSS) concentration and nitrogen compounds concentration, energy consumption for aeration, mixing and sludge treatment and annual sludge production over a three year exercise. In this plant, the energy required for bioreactor aeration was calculated at approximately 44% of the total energy demand. A cost optimization strategy was applied by varying the MLSS concentrations (from 1 to 8 gTSS/L) while recording energy consumption, sludge production and effluent quality. An increase of MLSS led to an increase of the oxygen requirement for biomass aeration, but it also reduced total sludge production. Results permit identification of a key MLSS concentration allowing identification of the best compromise between levels of treatment required, biological energy demand and sludge production while minimizing the overall costs.

A scalable low-cost cGMP process for clinical grade production of the HIV inhibitor 5P12-RANTES in Pichia pastoris.

In the continued absence of an effective anti-HIV vaccine, approximately 2 million new HIV infections occur every year, with over 95% of these in developing countries. Calls have been made for the development of anti-HIV drugs that can be formulated for topical use to prevent HIV transmission during sexual intercourse. Because these drugs are principally destined for use in low-resource regions, achieving production costs that are as low as possible is an absolute requirement. 5P12-RANTES, an analog of the human chemokine protein RANTES/CCL5, is a highly potent HIV entry inhibitor which acts by achieving potent blockade of the principal HIV coreceptor, CCR5. Here we describe the development and optimization of a scalable low-cost production process for 5P12-RANTES based on expression in Pichia pastoris. At pilot (150 L) scale, this cGMP compliant process yielded 30 g of clinical grade 5P12-RANTES. As well as providing sufficient material for the first stage of clinical development, this process represents an important step towards achieving production of 5P12-RANTES at a cost and scale appropriate to meet needs for topical HIV prevention worldwide.

High-EPA Biomass from Nannochloropsis salina Cultivated in a Flat-Panel Photo-Bioreactor on a Process Water-Enriched Growth Medium.

Nannochloropsis salina was grown on a mixture of standard growth media and pre-gasified industrial process water representing effluent from a local biogas plant. The study aimed to investigate the effects of enriched growth media and cultivation time on nutritional composition of Nannochloropsis salina biomass, with a focus on eicosapentaenoic acid (EPA). Variations in fatty acid composition, lipids, protein, amino acids, tocopherols and pigments were studied and results compared to algae cultivated on F/2 media as reference. Mixed growth media and process water enhanced the nutritional quality of Nannochloropsis salina in laboratory scale when compared to algae cultivated in standard F/2 medium. Data from laboratory scale translated to the large scale using a 4000 L flat panel photo-bioreactor system. The algae growth rate in winter conditions in Denmark was slow, but results revealed that large-scale cultivation of Nannochloropsis salina at these conditions could improve the nutritional properties such as EPA, tocopherol, protein and carotenoids compared to laboratory-scale cultivated microalgae. EPA reached 44.2% ± 2.30% of total fatty acids, and α-tocopherol reached 431 ± 28 µg/g of biomass dry weight after 21 days of cultivation. Variations in chemical compositions of Nannochloropsis salina were studied during the course of cultivation. Nannochloropsis salina can be presented as a good candidate for winter time cultivation in Denmark. The resulting biomass is a rich source of EPA and also a good source of protein (amino acids), tocopherols and carotenoids for potential use in aquaculture feed industry.

Dynamic control of nutrient-removal from industrial wastewater in a sequencing batch reactor, using common and low-cost online sensors.

On-line control of the biological treatment process is an innovative tool to cope with variable concentrations of chemical oxygen demand and nutrients in industrial wastewater. In the present study we implemented a simple dynamic control strategy for nutrient-removal in a sequencing batch reactor (SBR) treating variable tank truck cleaning wastewater. The control system was based on derived signals from two low-cost and robust sensors that are very common in activated sludge plants, i.e. oxidation reduction potential (ORP) and dissolved oxygen. The amount of wastewater fed during anoxic filling phases, and the number of filling phases in the SBR cycle, were determined by the appearance of the 'nitrate knee' in the profile of the ORP. The phase length of the subsequent aerobic phases was controlled by the oxygen uptake rate measured online in the reactor. As a result, the sludge loading rate (F/M ratio), the volume exchange rate and the SBR cycle length adapted dynamically to the activity of the activated sludge and the actual characteristics of the wastewater, without affecting the final effluent quality.

Optimisation of a simple method to transiently transfect a CHO cell line in high-throughput and at large scale.

Transient expression of heterologous proteins in mammalian systems is a powerful way to generate protein reagents quickly. However, it has historically suffered from poor yields in comparison to methods where the recombinant gene is stably integrated into the genome and high expressing clones isolated. Transient methods have been well described for HEK-based systems. In this paper we show the use of a design of experiments (DoE) approach to quickly analyse the effect of a range of different parameters on protein expression from a CHO-based transient system. We show that this system is amenable to a very simple transfection procedure by independent direct addition of DNA and transfection reagent to the culture vessel. In addition we show that expression can be improved by reducing the temperature of the culture conditions post-transfection. The process is demonstrated to be transferrable from 3 ml cultures in deep 24-well plates through cultures in CultiFlask Bioreactors, shake flasks and up to 25 L culture in Wave Bioreactors. Data are shown to illustrate the utility of the system with a number of different classes of protein.

Towards the commercialization of Botryococcus braunii for triterpenoid production.

Botryococcus braunii can accumulate unusually high levels of triterpenoid hydrocarbons making it a potential source of high value chemicals. However, its commercial application is hampered by its slow growth and lack of large-scale studies of triterpenoid hydrocarbon production. This study investigated hydrocarbon production in two race B of B. braunii strains, Overjuyo-3 and Kossou-4, at 25 °C in 500 L open tanks under artificial lighting in modified BG11 medium over 60 days. Maximum growth was reached by 40 days with Overjuyo-3 producing more biomass (3.05 g L(-1)) than Kossou-4 (2.55 g L(-1)). However, Kossou-4 produced more oil (0.75 g L(-1)) and triterpenoid hydrocarbons (C30-C34; 50 % of oil weight) compared to 0.63 g L(-1) of oil in Overjuyo-3 with triterpenoid hydrocarbons making up 29 % of oil weight. This research demonstrates for the first time that large-scale production of high value triterpenoid hydrocarbon for commercial application is feasible with Kossou-4 strain.