Non-growth associated production of enzymes in solid state fermentation system: Its mathematical description for two enzymes produced by Bacillus licheniformis M27

Logistic model, based on more general and realistic assumptions, has been derived to express the production of two non-growth associated enzymes by Bacillus licheniformis M27 in a solid state fermentation system. The model explained the production of alpha-amylase and neutral protease with correlation coefficients ranging between 0.974 and 0.985 in basal and standardized wheat bran media. It is apparent from the values of parameters in the model that the rate constant in standardized medium was lower (0.15 l/h^?1) than in the basal medium (0.32 l/h^?1), though higher maximum enzyme titres (1.7 times) were observed in the former medium. The data thus indicate dependence of enzyme titres on the maximum biomass formed. The model represents a significant advance in model formulation as it recognizes and takes care of all other products (enzymes etc) formed during fermentation. The model may prove useful in optimizing product synthesis, design of bioreactor and determination of harvest time, especially due to its adequacy and efficiency. Models for predicting product formation in solid state fermentation system are scarce and confined to fungal fermentations. No such model for bacterial solid state fermentation system was available earlier.     

Solid-state (solid-substrate) food/beverage fermentations involving fungi

“Solid-substrate” fermentation developed in the Orient is a very useful fermentation method. It is presently used to produce a variety of foods, beverages and related products. Solid-substrate fermentation products utilizing fungi including soy sauce, miso and tempe, ontjom, sake, and bread have been produced for centuries at the home and village level. They are examples of economical methods of preserving and improving the flavor, texture and nutritive values of cereal/legume substrates. “Solid-substrate” fermentation is also applied to animal products such as milk to produce Roquefort and Camembert cheeses which diversify the food flavors available to man “Solid-substrate”fermentation has certain advantages. The substrate is concentrated; the product can be extracted with relatively small quantities of solvent; the product can be easily dehydrated; moisture level can be controlled favoring the desired organisms; enzyme concentration is generally higher than is submerged fermentation; product concentration is generally higher than in submerged cultures; it is the only technique that yields true mushroom fruiting bodies and it can be used not only for production of crude enzyme concentrates (koji) but also for raising the protein content of high starch substrates. It also can be used to increase the content of vitamins at low cost. Disadvantages of “solid-substrates”from the modern industrial processing view point are the greater difficulty of handling solid substrate and the greater difficulty of controlling the fermentation parameters, temperature, pH and oxygen, and rate of microbial growth compared with liquid submerged fermentations.     

Production of pectinesterase and polygalacturonase by Aspergillus niger in submerged and solid state systems

Production of pectinesterase and polygalacturonase by Aspergillus niger was studied in submerged and solid-state fermentation systems. With pectin as a sole carbon source, pectinesterase and polygalacturonase production were four and six times higher respectively in a solid state system than in a submerged fermentation system and required a shorter time for enzyme production. The addition of glucose increased pectinesterase and polygalacturonase production in the solid state system but in submerged fermentation the production was markedly inhibited. A comparison of enzyme productivities showed that those determined for pectinesterase and polygalacturonase with pectin as a carbon source were three and five times higher by using the solid state rather than the submerged fermentation system. The productivities of the two enzymes were affected by glucose in both fermentation systems. The membranes of cells from the solid state fermentation showed increased levels of C18:1, C16:0 and C18:0 fatty acids. Differences in the regulation of enzyme synthesis by Aspergillus niger depended on the fermentation system, favoring the solid state over the submerged fermentation for pectinase production. Received 12 May 1997/ Accepted in revised form 19 September 1997     

Comparative study of protease production in solid substrate fermentation versus submerged fermentation

Attempts have been made to compare solid substrate fermentation (SSF) with submerged fermentation for the production of proteases by Bacillus amyloliquefaciens. In submerged fermentation it produced 800,000 units of enzyme under optimal conditions in a 20-litre fermentor whereas in solid substrate, it produced 250,000 units/g. Owing to the simplicity and easiness of operation of SSF and for applications like unhairing, biodetergents and bating the former would be advantageous for the production of proteases.     

Challenges for mass production of nematodes in submerged culture

Nematodes of Steinernema and Heterorhabditis genera are used as agents in insect biocontrol programs. They are associated with specific bacteria which are also involved in the mechanism of pathogenicity and which are consumed by nematodes as living food. S. feltiae has various developmental stages in its life cycle, including four juvenile stages, adults and the free living form. During mating, males coil themselves around the female, which is around 1 cm long. Successful commercialization of nematode-bacteria biocontrol products depends on the ability to produce sufficient quantities of these products at competitive prices for a full pest control program. This could be feasible if high cell density submerged cultures are designed and implemented; however, major problems related to nematodes mass production in a bioreactor remain unsolved due to the lack of knowledge about the physiological aspects of the nematode, bacteria and nematode-bacteria association, interaction between the three phases present in the bioreactor (liquid, gas, nematodes-bacteria), possibility of mating under hydrodynamic stress conditions, etc. We have found that the two most important engineering aspects to take into account the mass propagation of nematodes are oxygen transfer rate and hydrodynamics to allow mating and to avoid mechanical damage of juveniles in stage 2.This article focuses on several aspects related to the fermentation system such as kinetics of growth, shear stress, hydrodynamics fields in the bioreactor and oxygen demand. Also, results published by other groups, together with those of our own, will be discussed in relation to the main challenges found during the fermentation process.     

Production of Hydrophobins from fungi

Hydrophobins (HPs) are industrially important surface active, amphipathic proteins produced by fungi. There are many applications reported for HPs in the literature notably as, agents for enhancing bioavailability of water insoluble drugs, food stabilizers, antifouling agents for biomedical devices like catheters, fusion partner for recombinant proteins for purification, low friction coatings on biomaterials, immobilizing enzymes in biosensors, etc. However, there are limitations for industrial scale production of HPs. Various methods have been reported for their production e.g. use of wild fungi from natural hydrophobic environments, use of modified bioreactors for submerged and solid state fermentation and recombinant homologous as well as heterologous microbes. Knowing the industrial importance of HPs many reviews have been published focusing on technical and medical applications of these proteins; however there is no comprehensive overview of HP production in the literature. This review summarizes the efforts made to improve yields of HPs by various bioprocesses and also highlights the strategies designed to overcome problems of low yield of HPs.     

Screening of tannin acyl hydrolase (E.C.3.1.1.20) producing tannery effluent fungal isolates using simple agar plate and SmF process

Industrially important tannase producing fungi were isolated from tannery effluent using simple agar plate method. The isolates were screened by submerged fermentation using auto-controlled bioreactor. The colony diameter on the solid surface media shows high correlation with quantitative production of tannase. The isolate Aspergillus niger shows maximum production of both extracellular and intracellular enzyme.     

Solid-state fermentation--are there any biotechnological advantages?

Solid-state fermentation (SSF) has developed in eastern countries over many centuries, and has enjoyed broad application in these regions to date. By contrast, in western countries the technique had to compete with classical submerged fermentation and, because of the increasing pressure of rationalisation and standardisation, it has been widely superseded by classical submerged fermentation since the 1940s. This is mainly because of problems in engineering that appear when scaling up this technique. However, there are several advantages of SSF, for example high productivities, extended stability of products and low production costs, which say much about such an intensive biotechnological application. With increasing progress and application of rational methods in engineering, SSF will achieve higher levels in standardisation and reproducibility in the future. This can make SSF the preferred technique for special fields of application such as the production of enzymes and food.     

Combined submerged and solid substrate fermentation for the bioconversion of lignocellulose

A novel two-stage bioreactor has been designed for a combined submerged (SF) and solid substrate fermentation (SSF) of wheat straw. The straw was pretreated with steam, and cellulases from the culture fluid of Trichoderma reesei were adsorbed on it for increased bioconvertibility. SSF was conducted in the top part of the bioreactor by inoculating the straw with a 36-h mycelial culture of T. reesei, or Coriolus versicolor. In the bottom part of the fermenter, Endomycopsis fibuliger was grown in SF. The SF liquor was recirculated through the SSF stage at 24 h intervals to remove glucose and other metabolites that may inhibit growth, and to maintain optimum moisture level and temperature. The removed glucose and other metabolites provided nutrients for the yeast in the SF stage. The combined fermentation resulted in overall higher biomass yield, increased bioconversion, increased cellulase production, and increased digestibility compared with single SSF or SF.     

中国食（药）用真菌发酵工程研究进展

通过回顾食(药)用真菌生产工艺的研究历史,阐述了现代食(药)用真菌发酵工程包括液体发酵和固体发酵的研究状况,着重指出新型与多样性固体发酵工程的创建和发展可为人类提供取之不尽、用之不竭的传统生产工艺难以获得的珍贵目的产物。     

传统与未来的碰撞：食品发酵工程技术与应用进展

随着生物技术的飞速发展,作为食品生物工程的主要组成部分,食品发酵工程技术不断升级,在传统发酵食品的菌种、发酵过程、产品品质得到改善的同时,生物制造的功能食品组分、未来食品等新型产品也应运而生。首先概述了由生物技术和信息技术的进步带来的食品发酵研究手段与生产方式的多层面变革,并重点阐释了利用食品合成生物学设计构建细胞工厂的思路和方法,以及食品生物工程在微生物分析、过程工程和分离工程方面的智能化进程。其次,介绍了现代食品生物工程技术在改善传统发酵食品品质及安全性、生产功能食品组分、添加剂和酶制剂、创制未来食品和开发新型益生食品方面的应用进展。最后,对全球和我国食品发酵产业面临的挑战和未来发展趋势进行了总结和展望,以期为食品发酵的技术革新和工业化应用提供参考。     

Application of an airlift bioreactor to the nystatin biosynthesis

Pilot plant studies were performed using a concentric-tube airlift bioreactor of 2.5 m³ fermentation volume. The results have proven the relative merits of such a system in the biosynthesis of nystatin, produced by Streptomyces noursei, in submerged aerobic cultivation and batch operation mode. The results were compared to those obtained in a pilot-scale stirred tank bioreactor of 3.5 m³ fermentation volume. The fermentation processes in the two fermentation devices were similar with respect to substrate utilization, biomass production and nystatin biosynthesis. In the riser section, the dissolved oxygen concentration was higher than that in the downcomer. The volumetric oxygen mass transfer coefficient was dependent on the rheological behaviour of the biosynthesis liquids, which was not constant during the fermentation process. The total energy consumption for nystatin production in the airlift bioreactor was 56% of that in the stirred tank, while the operating costs represented 78% of those in the stirred tank bioreactor.     

Rheological properties of chromosomal and plasmid DNA during alkaline lysis reaction

The experimental apparatus for the simultaneous L-lactic acid fermentation by Rhizopus oryzae immobilized in calcium alginate beads and product separation process was set up in which a three-phase fluidized-bed bioreactor was used as a fermentor and an external electrodialyzer as a separator, and a pump was applied to recycle the fermentation broth between the bioreactor and the separator. The L-lactic acid produced in the fermentor was separated in the separator, product inhibition was alleviated without any addition of alkali or alkali salts and the product purification process could be simplified. The specific productivity and the yield in electrodialysis fermentation (ED-F) process operated in continuous feeding mode were almost the same as that in CaCO₃-buffered fermentation process. A mathematical model of L-lactic acid production in ED-F process was also suggested, in which the model equations for the bioreactor and the electrodialyzer were combined to describe the simultaneous fermentation and product separation. The model predictions were in good agreement with the experimental data.     

Production of polymalic acid and malic acid by Aureobasidium pullulans fermentation and acid hydrolysis

Malic acid is a dicarboxylic acid widely used in the food industry and also a potential C4 platform chemical that can be produced from biomass. However, microbial fermentation for direct malic acid production is limited by low product yield, titer, and productivity due to end‐product inhibition. In this work, a novel process for malic acid production from polymalic acid (PMA) fermentation followed by acid hydrolysis was developed. First, a PMA‐producing Aureobasidium pullulans strain ZX‐10 was screened and isolated. This microbe produced PMA as the major fermentation product at a high‐titer equivalent to 87.6 g/L of malic acid and high‐productivity of 0.61 g/L h in free‐cell fermentation in a stirred‐tank bioreactor. Fed‐batch fermentations with cells immobilized in a fibrous‐bed bioreactor (FBB) achieved the highest product titer of 144.2 g/L and productivity of 0.74 g/L h. The fermentation produced PMA was purified by adsorption with IRA‐900 anion‐exchange resins, achieving a ～100% purity and a high recovery rate of 84%. Pure malic acid was then produced from PMA by hydrolysis with 2 M sulfuric acid at 85°C, which followed the first‐order reaction kinetics. This process provides an efficient and economical way for PMA and malic acid production, and is promising for industrial application. Biotechnol. Bioeng. 2013; 110: 2105–2113. © 2013 Wiley Periodicals, Inc.     

Controlled gas environments in industrial fermentations

The gas environment is of major importance in controlling aerobic fermentation processes for the manufacture of microbial products. Oxygen and carbon dioxide levels in gas-liquid equilibria affect productivity and energy consumption in such processes and appear to be implicated in the regulation of microbial metabolism. Gas-liquid transfer has been intensively studied by many investigators for Newtonian and non-Newtonian fluids, primarily in terms of oxygen-limitation in biomass and product formation. More recentreports show that microbial growth and product formation are affected by levels of oxygen and carbon dioxide in the gas environment, suggesting that microbial metabolism may be directed towards specific products by the control of such environments. High product concentrations may also be obtained by solid substrate fermentations with mycelial organisms cultured on semi-solid agricultural products at low moisture contents. Such methods are commonly used in the Orient for the manufacture of enzymes and traditional fermented foods and could probably be extended to other microbial products. This review covers fundamental aspects of engineering research in microbial processes that suggest applications for controlled gas environments in submerged culture and solid substrate fermentations of potential industrial interest.     

Enhancement glucose oxidase production by solid-state fermentation of Aspergillus niger on polyurethane foams using mussel processing wastewaters

In the present work, studies of glucose oxidase (GOD) production in solid-state fermentation on polyurethane foams, using mussel processing wastewaters (MPW) as culture media, were carried out. Initially, the results generated in this experimental modality were compared with those that were obtained in conventional submerged fermentation. Later on, cultures were tested in solid state with fed-batch performance, defining suitable conditions of operation. These conditions were finally corroborated in experiments with a bioreactor of own design, in which GOD production was improved 13 times regarding to the initial cultures.     

Continuous production of citric acid from sugarcane molasses using a combination of submerged immobilized and surface stabilized cultures of Aspergillus niger KCU 520

Summary A high performance fermentation process for the continuous production of citric acid from sugarcane molasses by using the combination of submerged calcium alginate-immobilized and surface-stabilized cultures of Aspergillus niger KCU 520 in a continuous flow horizontal bioreactor is described. The citric acid productivity was dependent on the dilution rate with an optimum value of 0.015/h. Presaturation of fermentation medium with sterile air, in addition to surface aeration, before feeding into the bioreactor enhanced the citric acid productivity. The highest productivity, citric acid product concentration and yield obtained were 1.7 kg M^–3h^–1, 110kg M^–3 and 91% respectively. The cultures were continuously used for 30 days without any apparent loss in citric acid productivity.     

影响灵芝三萜类物质液态发酵合成的关键因素研究进展

灵芝是我国名贵的食药两用型菌类,具有广泛的药用价值,其三萜类物质为灵芝中最主要的药理活性物质之一。灵芝液态发酵因具有生长周期短、环境条件可控、目标产物质量稳定及易实现规模化制备等特点而成为获得灵芝三萜类物质最有前景的方式。灵芝三萜代谢途径、发酵工艺及参数、溶解氧控制等是影响灵芝三萜类物质液态发酵合成的关键因素。本文总结了灵芝三萜生物合成的代谢途径和相关的酶(基因)、液态发酵方式和发酵参数调节的溶解氧控制这3个层面对灵芝三萜类物质生物合成的影响,并对今后的研究方向进行了展望,为液态培养灵芝三萜类物质调控及高产提供参考,也为下一步研究提供借鉴。     

Bioprocessing of differentiated plant in vitro systems

Plant cells contain a wide range of interesting secondary metabolites, which are used as natural pigments and flavoring agents in foods and cosmetics as well as phyto‐pharmaceutical products. However, conventional industrial extraction from whole plants or parts of them is limited due to environmental and geographical issues. The production of secondary metabolites from in vitro cultures can be considered as alternative to classical technologies and allows a year‐round cultivation in the bioreactor under optimal conditions with constant high‐level quality and quantity. Compared to plant cell suspensions, differentiated plant in vitro systems offer the advantage that they are genetically stable. Moreover, the separation of the biomass from culture medium after fermentation is much easier. Nevertheless, several investigations in the literature described that differentiated plant in vitro systems are instable concerning the yield of the target metabolites, especially in submerged cultivations. Other major problems are associated with the challenges of cultivation conditions and bioreactor design as well as upscaling of the process. This article reviews bioreactor designs for cultivation of differentiated plant in vitro systems, secondary metabolite production in different bioreactor systems as well as aspects of process control, management, and modeling and gives perspectives for future cultivation methods.     

Investigation of the bacitracin biosynthesis in an airlift bioreactor

Results of pilot plant studies using an external-loop airlift bioreactor (170 l fermentation volume, liquid height-to-riser diameter: 27, loop-to-tower cross-section-area: 0.1225) have proven the relative merits of such a system in the bacitracin biosynthesis produced by the Bacillus licheniformis submerged aerobic cultivation. The results were compared to those obtained in a pilot-scale stirred-tank bioreactor with the same values of k_La. Excepting the aeration rate of 0.2 vvm, the fermentation process performed at 0.5 vvm and 1/0 vvm, respectively, unfolded similarly in the two fermentation devices with respect to the cell mass production, substrate utilization and bacitracin production during the fermentation process. In the riser section of the airlift bioreactor, the dissolved oxygen levels were higher, while in the downcomer section they were lower than those realized in the stirred tank bioreactor. Power requirements of the airlift fermenter were by 17–64% lower than those for a mechanically agitated system, depending on the aeration rates, which led to an important energy saving. Moreover, the lack of mechanical devices in the airlift system provides safety and a more gentle environment for the cultivation of microorganisms.