Solid-state fermentation: a continuous process for fungal tannase production

Truly continuous solid-state fermentations with operating times of 2-3 weeks were conducted in a prototype bioreactor for the production of fungal (Penicillium glabrum) tannase from a tannin-containing model substrate. Substantial quantities of the enzyme were synthesized throughout the operating periods and (imperfect) steady-state conditions seemed to be achieved soon after start-up of the fermentations. This demonstrated for the first time the possibility of conducting solid-state fermentations in the continuous mode and with a constant noninoculated feed. The operating variables and fermentation conditions in the bioreactor were sufficiently well predicted for the basic reinoculation concept to succeed. However, an incomplete understanding of the microbial mechanisms, the experimental system, and their interaction indicated the need for more research in this novel area of solid-state fermentation.     

Characterisation of optimum cultural environmental conditions for the production of high numbers of Metarhizium anisopliae blastospores with enhanced ecological fitness

The aim of this study was the production of high numbers of M. anisopliae blastospores with enhanced germination efficiency under conditions of water-stress (ecological fitness). Different nitrogen sources and concentrations were screened for their ability to induce blastospore production while keeping pH and water activity (a_w) at fixed levels (6.8, and 0.98 a_w, respectively). After optimum nitrogen status was determined (cornsteep solid (CS) + yeast extract (YE); or cottonseed flour (CF) + YE), the effect of interaction of nitrogen source, pH (3.5, 5, 6.8, 8, 9 and 10) and solutes for a_w adjustment (KCl, NaCl, PEG 200) on blastospore production, endogenous polyol content (glycerol, erythritol, arabitol and mannitol) and total protein, were determined. For both ionic (NaCl, KCl) and non-ionic solutes (PEG 200), optimum blastospore production (between 4×10⁷ and 2×10⁸ blastospores ml^-1) and growth occurred in the pH range 6.8-8, with the CF + YE nitrogen profile giving higher yields than CS + YE. Optimum conditions for high erythritol and total protein endogenous concentrations (40.37-73.44 and 14.33-18.90 mg g^-1 fresh weight, respectively) occurred between pH 6.8 and 8 by ionic a_w modification (KCl, NaCl) and with the CF + YE nitrogen profile. Germination of blastospores produced under these cultural conditions was between 62 and 89% under conditions of water-stress (0.96 a_w). On the other hand, blastospores with lower amounts of erythritol and total protein content had decreased germination (8-67%). These results could have significant implications for developing a liquid fermentation medium for the production of high numbers of fungal propagules with enhanced efficacy under non-optimum environmental conditions.     

Integration of mixing, heat transfer, and biochemical reaction kinetics in anaerobic methane fermentation

An extensive investigation of anaerobic methane fermentation requires identifying the relationship between the physical environment and biological process. In this study, a computational fluid dynamics (CFD) technique was used to characterize bacterial fermentation mechanisms intertwined with mixing and heat transfer in anaerobic digesters. The results demonstrate that the methane yield remains almost unchanged while the energy efficiency decreases with increasing mixing power in a complete‐mix digester, and that the energy output increases nonlinearly with the increase in heating energy in a plug‐flow digester. The CFD method can be applied to other bioreactors to gain valuable insights into their behavior as well. Integrating flow and temperature with kinetic behavior for anaerobic digestion not only solves the controversy about how mixing influences the digestive process, but also assists in optimizing the digester design and increasing the efficiency of energy conversion, and additionally, provides a reference for improving the mixing guidelines recommended by the U.S. Environmental Protection Agency. Biotechnol. Bioeng. 2012; 109: 2864–2874. © 2012 Wiley Periodicals, Inc.     

Statistical optimization of process variables for the large-scale production of Metarhizium anisopliae conidiospores in solid-state fermentation

Optimization of conidial production was achieved by response surface methodology (RSM), a powerful mathematical approach widely applied in the optimization of fermentation process, using the three substrates; rice, barley and sorghum at variable pH, moisture content and yeast extract concentrations. These three factors were found to be important, affecting Metarhizium anisopliae spore production. A 2(3) full factorial central composite design and RSM were applied to determine the optimal concentration of each variable. A second-order polynomial was determined by the multiple regression analysis of the experimental data. Moisture content of 75.68% for sorghum, 73.21% for barley and 22.34% for rice produced optimal results. Maximal conidial yield was recorded for rice at a pH of 7.01; at 7.06 for sorghum and at 6.76 for barley.     

A dual-substrate steady-state model for biological hydrogen production in an anaerobic hydrogen fermentation process

Biological hydrogen production from anaerobic waste fermentation possesses potential benefits in simultaneously reducing organic wastes and generating sustainable energy sources. Three kinetic-based steady-state models for anaerobic fermentation of multiple substrates, including glucose and peptone, were evaluated. Experimental results obtained from a continuous stirred tank reactor (CSTR) were primarily used for model evaluation. The dual-substrate steady-state model developed and the associated kinetic parameters estimated in this study successfully described the anaerobic growth of hydrogen-producing bacteria. The model was able to capture the general trends of consumption of substrates and accumulation of products, including formate, acetate, butyrate, and hydrogen, at dilution rates (D) between 0.06 and 0.69/h. According to the model, the adverse effects of endogeneous and peptone metabolism on net hydrogen production can be minimized by increasing D. For the operational conditions of D > 0.69/h, however, substantial washout of hydrogen-producing bacteria from the CSTR was observed, and it resulted in a rapid drop in hydrogen production rate as well.     

Evaluating the importance of residual effects from previous years' treatment on the efficiency of different strategies for control of Sahelian grasshoppers with Metarhizium anisopliae var. acridum

A model for the residual effect of previous years' treatment with the mycoinsecticide Metarhizium anisopliae var. acridum has been constructed based on field data from treatment of two different grasshopper species in east Niger. This model was incorporated in an earlier developed simulation model to evaluate control strategies for the grasshopper Oedaleus senegalensis in West Africa and the model system was used to assess the potential importance of second year's residual effects for the efficiency of different treatment strategies. It has earlier been hypothesized that the persistence of M. anisopliae and the ability of this agent to impose long-term control on grasshopper populations through secondary cycling in some cases might render the use of M. anisopliae more efficient than the use of chemical insecticides like fenitrothion. Results show that this effect is possible if M. anisopliae is used in a repetitive treatment strategy where control operations are concentrated in the main millet production areas.     

An efficient and scalable process for helper-dependent adenoviral vector production using polyethylenimine-adenofection

Safety requirements for adenoviral gene therapy protocols have led to the development of the third generation of vectors commonly called helper-dependent adenoviral vectors (HDVs). HDVs have demonstrated a high therapeutic potential; however, the poor efficiency and reliability of the actual production process hampers further large-scale clinical evaluation of this new vector. The current HDV production methods involve a preliminary rescue step through transfection of adherent cell cultures by an HDV plasmid followed by a helper adenovirus (HV) infection. Amplification by serial co-infection of complementary cells allows an increase in the HDV titer. Using a HEK293 FLP/frt cell system in suspension culture, an alternative protocol to the current transfection/infection procedure was evaluated. In this work, the adenofection uses the HDV plasmid linked to the HV with the help of polyethylenimine (PEI) and has shown to outperform standard protocols by producing higher HDV yield. The influence of complex composition on the HDV production was examined by a statistical design. The optimized adenofection and amplification conditions were successively performed to generate HDV at the 3 L bioreactor scale. Following only two serial co-infection passages, up to 1.44 x 10(8) HDV infectious units/mL of culture were generated, which corresponded to 26% of the total particles produced. This production strategy, realized in cell suspension culture, reduced process duration and therefore the probability of vector recombination by introducing a cost-effective transfection protocol, ensuring production of high-quality vector stock.     

A two-step fermentation process for efficient production of penta-N-acetyl-chitopentaose in recombinant Escherichia coli

The nodC gene from Mesorhizobium loti was cloned into E. coli, leading to production of chitin oligosaccharides (COs)—mainly penta-N-acetyl-chitopentaose. A two-step fermentation procedure was then developed which gave 930 mg CO/L with a productivity of 37 mg/l·h.     

Laboratory and field investigation of a mixture of Metarhizium acridum and Neem seed oil against the Tree Locust Anacridium melanorhodon melanorhodon (Orthoptera: Acrididae)

The in vitro compatibility of Metarhizium acridum strain IMI 330189 with different concentrations of Neem seed oil Azadirachta indica A. Juss. was investigated under laboratory conditions. Water, diesel and ground nut oil dilutions of M. acridum were inoculated into semi-synthetic culture medium with Neem oil and incubated for 10 days at 20, 28, and 34°C. Fungus vegetative growth and conidia production were estimated and compatibility calculated according to the in vitro classification model [T]. Field tests were also conducted during two successive years, where fourth instar nymphs of the Tree Locust, Anacridium melanorhodon melanorhodon, were sprayed with Metarhizium, Metarhizium/Neem mixture, Neem, or Malathion, on a plantation of Acacia senegal. Samples of the treated nymphs were taken 24 h after application, placed in cages and their mortality observed daily for 3 weeks. The compatibility test revealed that at 28°C, all Neem concentrations below 2% were compatible with M. acridum and concentrations of 2 and 2.5% were moderately toxic, while at 20°C, 1.0% Neem was not compatible with the fungus. In the field, Metarhizium+Neem resulted in 74 and 92% mortality during two successive years compared to 64 and 83% for Metarhizium alone. These findings clearly reveal that Neem concentration on the mixture could be increased to just under 1.0% Neem without negative impact on the fungus and that mixing Metarhizium with small quantities of Neem oil accelerates locust mortality and increases efficacy.     

The microbiological basis of process control in methanogenic fermentation of soluble wastes

The essential requirement for anaerobic digestion of industrial wastes is that the process should operate reliably at high performance. In the digestion of dilute, soluble wastes it is necessary to retain the active biomass within the digester at short liquid retention times for the process to be economically feasible and this is reflected in digester design. Performance of digesters can only be assessed by interpretation of measurable parameters such as pH₂, E_h, pH, volatile fatty acid concentrations, temperature, gas production, biomass content and feed rate and composition. The effects of changes in these parameters on the microbiology of methanogenic digestion and the application of this knowledge in control of the process is discussed.     

Development of an industrializable fermentation process for propionic acid production

Propionic acid (PA) is a short-chain fatty acid with wide industrial application including uses in pharmaceuticals, herbicides, cosmetics, and food preservatives. As a three-carbon building block, PA also has potential as a precursor for high-volume commodity chemicals such as propylene. Currently, most PA is manufactured through petrochemical routes, which can be tied to increasing prices and volatility due to difficulty in demand forecasting and feedstock availability. Herein described are research advancements to develop an industrially feasible, renewable route to PA. Seventeen Propionibacterium strains were screened using glucose and sucrose as the carbon source to identify the best platform strain. Propionibacterium acidipropionici ATCC 4875 was selected as the platform strain and subsequent fermentation optimization studies were performed to maximize productivity and yield. Fermentation productivity was improved three-fold to exceed 2 g/l/h by densifying the inoculum source. Byproduct levels, particularly lactic and succinic acid, were reduced by optimizing fermentor headspace pressure and shear. Following achievement of commercially viable productivities, the lab-grade medium components were replaced with industrial counterparts to further reduce fermentation costs. A pure enzymatically treated corn mash (ECM) medium improved the apparent PA yield to 0.6 g/g (PA produced/glucose consumed), but it came at the cost of reduced productivity. Supplementation of ECM with cyanocobalamin restored productivity to near lab-grade media levels. The optimized ECM recipe achieved a productivity of 0.5 g/l/h with an apparent PA yield of 0.60 g/g corresponding to a media cost <1 USD/kg of PA. These improvements significantly narrow the gap between the fermentation and incumbent petrochemical processes, which is estimated to have a manufacturing cost of 0.82 USD/kg in 2017.     

Anaerobic hydrogen production with an efficient carrier-induced granular sludge bed bioreactor

A novel bioreactor containing self-flocculated anaerobic granular sludge was developed for high-performance hydrogen production from sucrose-based synthetic wastewater. The reactor achieved an optimal volumetric hydrogen production rate of approximately 7.3 L/h/L (7,150 mmol/d/L) and a maximal hydrogen yield of 3.03 mol H2/mol sucrose when it was operated at a hydraulic retention time (HRT) of 0.5 h with an influent sucrose concentration of 20 g COD/L. The gas-phase hydrogen content and substrate conversion also exceeded 40 and 90%, respectively, under optimal conditions. Packing of a small quantity of carrier matrices on the bottom of the upflow reactor significantly stimulated sludge granulation that can be accomplished within 100 h. Among the four carriers examined, spherical activated carbon was the most effective inducer for granular sludge formation. The carrier-induced granular sludge bed (CIGSB) bioreactor was started up with a low HRT of 4-8 h (corresponding to an organic loading rate of 2.5-5 g COD/h/L) and enabled stable operations at an extremely low HRT (up to 0.5 h) without washout of biomass. The granular sludge was rapidly formed in CIGSB supported with activated carbon and reached a maximal concentration of 26 g/L at HRT = 0.5 h. The ability to maintain high biomass concentration at low HRT (i.e., high organic loading rate) highlights the key factor for the remarkable hydrogen production efficiency of the CIGSB processes.     

Advances in bioreactor control for production of biotherapeutic products

Advanced control strategies are well established in chemical, pharmaceutical, and food processing industries. Over the past decade, the application of these strategies is being explored for control of bioreactors for manufacturing of biotherapeutics. Most of the industrial bioreactor control strategies apply classical control techniques, with the control system designed for the facility at hand. However, with the recent progress in sensors, machinery, and industrial internet of things, and advancements in deeper understanding of the biological processes, coupled with the requirement of flexible production, the need to develop a robust and advanced process control system that can ease process intensification has emerged. This has further fuelled the development of advanced monitoring approaches, modeling techniques, process analytical technologies, and soft sensors. It is seen that proper application of these concepts can significantly improve bioreactor process performance, productivity, and reproducibility. This review is on the recent advancements in bioreactor control and its related aspects along with the associated challenges. This study also offers an insight into the future prospects for development of control strategies that can be designed for industrial-scale production of biotherapeutic products.     

林可霉素生产中三级种子罐的发酵工艺优化

【背景】林可霉素是一种在临床应用上占有重要地位的林可酰胺类抗生素,关于调控发酵生产中三级种子罐相关参数优化林可霉素发酵工艺的研究较少。【目的】优化林可霉素发酵工艺,提高林可霉素发酵效价及市场竞争力。【方法】对林可霉素生产中三级种子罐的培养基和接种量及三级种子移种菌龄进行优化。【结果】在三级种子罐培养基中葡萄糖、淀粉、玉米浆、黄豆饼粉和硫酸铵浓度分别为64.0、5.0、15.0、14.5和3.5g/L,三级种子罐接种量为25%及三级种子移种菌龄为60h的优化条件下,林可霉素四级发酵效价高达7883U/mL,比优化前效价提高了10%。【结论】对林可霉素生产中三级种子罐相关参数进行调控,初步优化了林可霉素发酵工艺,提高了发酵效价,为优化林可霉素发酵工艺提供了新思路。     

发酵生物制氢研究进展

综述了近年来发酵生物制氢领域的研究进展?在菌种方面,除了对现有产氢菌种的深入研究外,还采用生物学,分子生物学及生物信息学手段建立产氢菌种库;在氢酶的研究方面,已逐步从基因确定、功能研究拓展到基因工程构建高效产氢菌研究：而在与废弃生物质处理相结合的反应过程方面,研究主要集中在利用不同种类的废弃物的产氢和高效产氢反应器上。此外,还初步总结了目前对发酵制氢可行性和经济性的评价,并对其发展方向提出了新的看法。     

Fed-batch fermentation for production of Kluyveromyces marxianusFII 510700 cultivated on a lactose-based medium

A strain of Kluyveromyces marxianus was grown in batch culture in lactose-based media at varying initial lactose concentrations (10–60 g L^–1) at 30°C, pH 5.0, dissolved oxygen concentrations greater than 20%. Increasing the concentration of mineral salts three-fold at 40 g L^–1 and 60 g L^–1 initial lactose concentration showed only a small increase in the yield of biomass, from 0.38 g g^–1 to 0.41 g g^–1, indicating that the initial batch cultures were not significantly nutrient- (mineral salts)-limited. A relatively high biomass concentration (105 g L^–1) was obtained in fed-batch culture following extended lactose feeding. An average specific growth rate (0.27 h^–1), biomass yield (0.38 g g^–1) and overall productivity (2.9 g L^–1 h^–1) were obtained for these fed-batch conditions. This fed-batch protocol provides a strategy for achieving relatively high concentrations and productivities of K. marxianus on other lactose-based substrate streams (e.g., whey) from the dairy industry.     

Fed-batch bioreactor process scale-up from 3-L to 2,500-L scale for monoclonal antibody production from cell culture

This case study focuses on the scale-up of a Sp2/0 mouse myeloma cell line based fed-batch bioreactor process, from the initial 3-L bench scale to the 2,500-L scale. A stepwise scale-up strategy that involved several intermediate steps in increasing the bioreactor volume was adopted to minimize the risks associated with scale-up processes. Careful selection of several available mixing models from literature, and appropriately applying the calculated results to our settings, resulted in successful scale-up of agitation speed for the large bioreactors. Consideration was also given to scale-up of the nutrient feeding, inoculation, and the set-points of operational parameters such as temperature, pH, dissolved oxygen, dissolved carbon dioxide, and aeration in an integrated manner. It has been demonstrated through the qualitative and the quantitative side-by-side comparison of bioreactor performance as well as through a panel of biochemical characterization tests that the comparability of the process and the product was well controlled and maintained during the process scale-up. The 2,500-L process is currently in use for the routine clinical production of Epratuzumab in support of two global Phase III clinical trials in patients with lupus. Today, the 2,500 L, fed-batch production process for Epratuzumab has met all scheduled batch releases, and the quality of the antibody is consistent and reproducible, meeting all specifications, thus confirming the robustness of the process.     

Industrial media and fermentation processes for improved growth and protease production by Tetrahymena thermophila BIII

Tetrahymena thermophila was cultivated on industrial by-product media. The composition of the best medium (with milk proteins) was optimised by a central composite design for growth and protease secretion. The optimal combination [1.07% (w/v) of yeast extract and 0.99% (w/v) of skimmed milk] improved biomass production by 46%. In a fermentation strategy, the pH must be regulated to produce no cell damage, lengthening the stationary phase and resulting in a more abundant protease production. To increase cell concentration and protease secretion, a continuous culture with cell recycling by microfiltration was successfully tested on ciliated protozoa. Journal of Industrial Microbiology & Biotechnology (2000) 24, 285–290. Received 28 July 1999/ Accepted in revised form 20 January 2000     

发酵过程多尺度解析与调控的研究进展

工业发酵科学致力于实现高产量、高转化率和高生产强度的相对统一。通过从分子、细胞和反应器进行发酵过程多尺度解析与调控,实施全局与动态的优化与控制能够确保发酵过程高效、转化定向、过程稳定和系统有序。本文从发酵微生物代谢途径动力学模型、细胞代谢特性、发酵提取相耦合与反应器设计四个方面,总结和讨论发酵过程多尺度解析与调控的研究进展。整合分析发酵过程不同尺度特征并且针对性地开展多尺度整合调控是实现高效工业微生物发酵的重要策略。