Succinate synthesis and excretion by Penicillium simplicissimum under aerobic and anaerobic conditions

Succinate is an interesting chemical for industries producing food and pharmaceutical products, surfactants, detergents and biodegradable plastics. Succinate is produced mainly by a mixed-acid fermentation process using anaerobically growing bacteria. However, succinate excretion is also widespread among fungi. In this article we report results on the intracellular concentration and the excretion of succinate by Penicillium simplicissimum under aerobic and anaerobic conditions. The intracellular concentration of succinate increased slightly with the specific growth rate and strongly if the respiratory chain was inhibited by sodium azide or anaerobic conditions (N(2)). A strong increase of succinate excretion was observed if the respiratory chain was inhibited. It is suggested that succinate synthesis under functional (sodium azide) or environmental (N(2)) anaerobic conditions occurs via the reductive part of the tricarboxylic acid cycle. Succinate is then excreted because the oxidative part of the tricarboxylic acid cycle is inactive. A possible role of succinate synthesis in the regeneration of NAD ('fumarate respiration') is discussed.     

Fermentation for future food systems: Precision fermentation can complement the scope and applications of traditional fermentation

Modern biotechnology holds great potential for expanding the scope of fermentation to create novel foods and improve the sustainability of food production.

The growing human population and global warming pose an impending threat for global food security (Linder, 2019). This has prompted a critical re‐examination of the food supply chain from producers to consumers in order to increase the overall efficiency of food production, storage and transport. Much research in plant science consequently aims to increase production with new, high‐yield crop, fruit and vegetable varieties better adapted to changing climatic conditions. Yet, there is also much room for improving food safety by minimising food losses and recycling waste, valorising by‐products, improving nutritional value and increasing storage time. This is where fermentation comes in as a cost‐efficient, versatile and proven technology that extends the shelf life of food products and enhances their nutritional content. Moreover, there is enormous potential in fermentation to further increase efficiency and product range and even create new food products from non‐food biomass.

… there is enormous potential in fermentation to further increase efficiency and product range and even create new food products from non‐food biomass.

In a broader sense, fermentation can be defined as the cultivation of microorganisms such as bacteria, yeasts and fungi to break down complex molecules into simpler ones, notably organic acids, alcohols or esters. In a practical sense, it is one of the oldest food processing technologies to increase storage life along with cooking, smoking or air‐drying: fermentation was already fully industrialised for producing beer and bread millennia ago in ancient Mesopotamia and Egypt. It is also an elegant and simple technology as these microorganisms do most of the work without much human involvement.Louis Pasteur’s discovery that microorganisms cause fermentation laid the basis for further improvement of the technology from traditional spontaneous fermentation to the use of defined starter cultures. Fermentation is now widely used to produce alcoholic beverages, bread and pastry, dairy products, pickled vegetables, soy sauce and so on. More recent advances based on genomics and synthetic biology include precision and biomass fermentation to produce specific compounds for the food and chemical industry or medicinal use. This is not the limit though: when combined with genomics, fermentation has even greater potential for creating novel foods and other products.     

Formation and Fate of Fermentation Products in Hot Spring Cyanobacterial Mats

The fate of representative fermentation products (acetate, propionate, butyrate, lactate, and ethanol) in hot spring cyanobacterial mats was investigated. The major fate during incubations in the light was photoassimilation by filamentous bacteria resembling Chloroflexus aurantiacus. Some metabolism of all compounds occurred under dark aerobic conditions. Under dark anaerobic conditions, only lactate was oxidized extensively to carbon dioxide. Extended preincubation under dark anaerobic conditions did not enhance anaerobic catabolism of acetate, propionate, or ethanol. Acetogenesis of butyrate was suggested by the hydrogen sensitivity of butyrate conversion to acetate and by the enrichment of butyrate-degrading acetogenic bacteria. Accumulation of fermentation products which were not catabolized under dark anaerobic conditions revealed their importance. Acetate and propionate were the major fermentation products which accumulated in samples collected at temperatures ranging from 50 to 70°C. Other organic acids and alcohols accumulated to a much lesser extent. Fermentation occurred mainly in the top 4 mm of the mat. Exposure to light decreased the accumulation of acetate and presumably of other fermentation products. The importance of interspecies hydrogen transfer was investigated by comparing fermentation product accumulation at a 65°C site, with naturally high hydrogen levels, and a 55°C site, where active methanogenesis prevented significant hydrogen accumulation. There was a greater relative accumulation of reduced products, notably ethanol, in the 65°C mat.     

维生素B12的生物合成、发酵生产与应用

维生素B12(VB12)是一种重要的动物和人类营养因子, 广泛应用于饲料、食品和医药卫生领域。中国已成为全球VB12的主要产地, 2007年产量为27 t, 占全球总产量的77%。VB12是目前已发现的最大、最复杂的维生素分子, 化学合成极其困难, 所有VB12产品均采用生物发酵制备其主体结构。VB12主要由古生菌和一些真细菌通过有氧或厌氧两种途径合成, 工业上主要采用费式丙酸菌(Propionibacterium freudenrechii）和脱氮假单胞菌(Pseudomonas denitrificans)进行发酵生产。综述了VB12的基本性质, 生物合成途径, 以及发酵生产工艺, 并对VB12的应用与市场前景作了分析。     

维生素B12的生物合成、发酵生产与应用

维生素B12(VB12)是一种重要的动物和人类营养因子, 广泛应用于饲料、食品和医药卫生领域。中国已成为全球VB12的主要产地, 2007年产量为27 t, 占全球总产量的77%。VB12是目前已发现的最大、最复杂的维生素分子, 化学合成极其困难, 所有VB12产品均采用生物发酵制备其主体结构。VB12主要由古生菌和一些真细菌通过有氧或厌氧两种途径合成, 工业上主要采用费式丙酸菌(Propionibacterium freudenrechii）和脱氮假单胞菌(Pseudomonas denitrificans)进行发酵生产。综述了VB12的基本性质, 生物合成途径, 以及发酵生产工艺, 并对VB12的应用与市场前景作了分析。     

Redox potential control and applications in microaerobic and anaerobic fermentations

Many fermentation products are produced under microaerobic or anaerobic conditions, in which oxygen is undetectable by dissolved oxygen probe, presenting a challenge for process monitoring and control. Extracellular redox potentials that can be detected conveniently affect intracellular redox homeostasis and metabolism, and consequently control profiles of fermentation products, which provide an alternative for monitoring and control of these fermentation processes. This article reviews updated progress in the impact of redox potentials on gene expression, protein biosynthesis and metabolism as well as redox potential control strategies for more efficient production of fermentation products, taking ethanol fermentation by the yeast Saccharomyces under microaerobic conditions and butanol production by the bacterium Clostridium under anaerobic conditions as examples.     

On-line measurement of gas production rates

Gas evolution rates represent an important variable to track in biological and certain electrochemical processes. Accurate gas flow rate sensors exist for gas streams possessing a pressure head, such as when pressurized air or oxygen is delivered to a fermentation process. However, these devices impose pressure heads that can inhibit gas production and, therefore, yield false measurements. Examples of effected processes would include electrochemical production of a gas at the electrode (e.g., electrolysis) or anaerobic fermentation (e.g., anaerobic production of methane). In this work, we present an on-line gas measurement technique that measures on-line gas production from an anaerobic microbial process that is continuously fed simulated food waste over a 6-month period. Commentary is given on the sensor's accuracy and ease of use within the context of long-term operation, ability to measure both low and high gas production rates, as well as its potential for process control and system-health monitoring.     

酱香大曲中地衣芽孢杆菌及其特征风味代谢产物的分析研究

从酱香型酒生产用的高温大曲中筛选到三株产酱香的革兰氏阳性杆菌,有芽孢,兼性厌氧。经形态学、生理生化特性和16S rDNA序列分析,鉴定其为地衣芽孢杆菌(Bacillus licheniformis)。初步探索这三株细菌液态培养的最适条件,以麸皮浸出汁为培养基,150 r/min 55℃发酵6d,产生明显的酱香香气。从发酵液中检测到的乙偶姻、四甲基吡嗪和呋喃扭尔,是产酱香细菌发酵产生的特征性产物。     

Inorganic types of fermentation and anaerobic respirations in the evolution of energy-yielding metabolism

We proposed long ago the following sequence as one of the main pathways in the evolution of energy-yielding metabolism: fermentation→nitrate fermentation→nitrate respiration→oxygen respiration. In the present report our concept is presented in a more general form: (1) fermentation→ →(2) fermentation with H₂ release→(3) inorganic types of fermentation→(4) anaerobic respirations →(5) oxygen respiration, based upon recent biological and physical information. The energy-yielding efficiency increased gradually together with the evolution. (2) is characterized by the participation of ferredoxin, (3) by the establishment of electron transfer chain, and (4) by the participation of cytochrome and oxidative phosphorylation. The close relationship between the primary structure of ferredoxins of anaerobic bacteria and that of a cytochrome (cytochromec ₃) was demonstrated. It reveals that the transition from inorganic types of fermentation to anaerobic respirations was direct and accompanied by the transition from ferredoxins to cytochromes, and it further supports our concept that the cytochrome system, and consequently the oxidative phosphorylation, were induced at this evolutionary step. Our concept based upon biological observations is consistent with a physical theory recently proposed by M. Shimizu.     

Traditional healthful fermented products of Japan

A variety of fermentation products, such as foods containing probiotic bacteria, black rice vinegar (kurosu), soy sauce (shoyu), soybean-barley paste (miso), natto and tempeh, are sold in food stores in Japan. These fermented food products are produced by traditional methods that exploit mixed cultures of various non-toxic microorganisms. These microorganisms include lactic acid bacteria, acetic acid bacteria, sake yeast, koji molds and natto bacteria. Many traditional fermented foods have been studied and their effects on metabolism and/or immune system have been demonstrated in animal and/or human cells. This review summarizes the scientific basis for the effects of these traditional food products, which are currently produced commercially in Japan.     

Changes in lactic acid bacteria and components of Awa-bancha by anaerobic fermentation

ABSTRACT

Awa-bancha is a post-fermented tea produced in Naka and Kamikatsu, Tokushima, Japan. We investigated the lactic acid bacteria in each stage of production of Awa-bancha and evaluated the relationships with the components. Lactic acid bacteria were isolated from tea leaves cultured with de Man, Rogosa, and Sharpe (MRS) agar plates, and the species were identified by homology of the 16 S rRNA gene and multiplex polymerase chain reaction (PCR) of the recA gene to distinguish the Lactobacillus plantarum group. As a result, a variety of species were isolated from the raw tea leaves, and Lactobacillus pentosus was isolated most frequently after anaerobic fermentation. Regarding the tea leaf components, organic acids, such as lactic acid, increased, free amino acids decreased, and catechins changed owing to anaerobic fermentation. Our results suggest that the microbial flora mainly composed of L. pentosus is important in the anaerobic fermentation process for flavor formation of Awa-bancha.     

The role of transport processes in survival of lactic acid bacteria, Energy transduction and multidrug resistance

Lactic acid bacteria play an essential role in many food fermentation processes. They are anaerobic organisms which obtain their metabolic energy by substrate phosphorylation. In addition three secondary energy transducing processes can contribute to the generation of a proton motive force: proton/substrate symport as in lactic acid excretion, electrogenic precursor/product exchange as in malolactic and citrolactic fermentation and histidine/histamine exchange, and electrogenic uniport as in malate and citrate uptake in Leuconostoc oenos. In several of these processes additional H+ consumption occurs during metabolism leading to the generation of a pH gradient, internally alkaline. Lactic acid bacteria have also developed multidrug resistance systems. In Lactococcus lactis three toxin excretion systems have been characterized: cationic toxins can be excreted by a toxin/proton antiport system and by an ABC-transporter. This cationic ABC-transporter has surprisingly high structural an d functional analogy with the human MDR1-(P-glycoprotein). For anions an ATP-driven ABC-like excretion systems exist.     

固体发酵法固定工业废水处理优势菌的研究

为实现优势菌种的工程应用,由焦化废水处理站污泥筛选优势好氧菌A和兼性厌氧菌F,使用谷壳g、豆皮d为载体,采用固体发酵法进行固定化产品制备。初步研究表明固体发酵法固定菌种具有可行性,主要调控因素有温度、初始含水率、工业废水比例、pH、通风量和发酵时间。以pH8．0、温度好氧菌30℃（兼性厌氧菌35℃）、接种量1mL茴液儋田定化载体,初始含水量80％,固体发酵时间5d,低于60℃鼓风干燥,制备固定化产品。保存三个月,谷壳固定化产品好氧菌八的硝化能力和兼性厌氧菌R的反硝化能力不随保存时间的延长而改变;豆皮固定化产品氏、Fd降解能力随保存时问延长下降12％左右。以实验室岔／O工艺系统验证固定化产品处理焦化废水效果,缺氧池中无NO3-N积累,反硝化作用明显。氐和气对氰化物的去除率均达99％以上;对苯酚的去除效果气（98．84％）优于氏（79．6％）;但对NH4＋-N的去除率氏（75．46％）优于Ag（62．55％）。     

Lessons from the cow: What the ruminant animal can teach us about consolidated bioprocessing of cellulosic biomass

Consolidated bioprocessing (CBP) of cellulosic biomass is a promising source of ethanol. This process uses anaerobic bacteria, their own cellulolytic enzymes and fermentation pathways that convert the products of cellulose hydrolysis to ethanol in a single reactor. However, the engineering and economics of the process remain questionable. The ruminal fermentation is a very highly developed natural cellulose-degrading system. We propose that breakthroughs developed by cattle and other ruminant animals in cellulosic biomass conversion can guide future improvements in engineered CBP systems. These breakthroughs include, among others, an elegant and effective physical pretreatment; operation at high solids loading under non-aseptic conditions; minimal nutrient requirements beyond the plant biomass itself; efficient fermentation of nearly all plant components; efficient recovery of primary fermentation end-products; and production of useful co-products. Ruminal fermentation does not produce significant amounts of ethanol, but it produces volatile fatty acids and methane at a rapid rate. Because these alternative products have a high energy content, efforts should be made to recover these products and convert them to other organic compounds, particularly transportation fuels.     

Development of a Micromethod for Identification of Anaerobic Bacteria

A microprocedure was described for determining the carbohydrate fermentation patterns of 48 anaerobic bacteria at one time in microtiter plates. The cultures were transferred into agar-filled wells of microtiter plates with a replicator inside an anaerobic glove box. Fermentation was measured both with a colorimetric indicator and with a small pH electrode. The method was approximately 97% accurate. It would be most useful for laboratories that need to identify large numbers of anaerobes at one time.     

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.     

Determining the impacts of fermentative bacteria on wollastonite dissolution kinetics

Silicate minerals can be a source of calcium and alkalinity, enabling CO₂ sequestration in the form of carbonates. For this to occur, the mineral needs to be first dissolved in an acidifying process such as the biological process of anaerobic fermentation. In the present study, the main factors which govern the dissolution process of an alkaline silicate mineral (wollastonite, CaSiO₃) in an anaerobic fermentation process were determined. Wollastonite dissolution kinetics was measured in a series of chemical batch experiments in order to be able to estimate the required amount of alkaline silicate that can neutralize the acidifying fermentation process. An anaerobic fermentation of glucose with wollastonite as the neutralizing agent was consequently performed in a fed-batch reactor. Results of this experiment were compared with an abiotic (control) fed-batch reactor in which the fermentation products (i.e. organic acids and alcohols) were externally supplied to the system at comparable rates and proportions, in order to provide chemical conditions similar to those during the biotic (fermentation) experiment. This procedure enabled us to determine whether dissolution of wollastonite was solely enhanced by production of organic acids or whether there were other impacts that fermentative bacteria could have on the mineral dissolution rate. The established pH profiles, which were the direct indicator of the dissolution rate, were comparable in both experiments suggesting that the mineral dissolution rate was mostly influenced by the quantity of the organic acids produced.     

Activity and identity of fermenting microorganisms in full-scale biological nutrient removing wastewater treatment plants

Hydrolysis and fermentation are of key importance in biological nutrient removal (BNR) wastewater treatment plants as they provide polyphosphate-accumulating organisms and denitrifying bacteria with carbon and energy sources (e.g. short chain fatty acids). Little information, however, exists about the microbiology of the microorganisms involved in hydrolysis and fermentation. In this study, fermentation of monosaccharides was found to be a universal process taking place in all full-scale BNR plants investigated, where glucose and other monosaccharides were consumed and fermented during anaerobic conditions. The removal rates of glucose were in the range of 0.05–0.32 mmol gVSS⁻¹ h⁻¹ and only slightly lower than glucose removal under aerobic conditions. The main fermentation products detected were (in descending order) propionic acid, lactic acid, acetic acid and formic acid. The fermentation was diverse, consisting of at least three fermentation metabolisms, including lactic acid (homolactic), mixed acid and propionic acid fermentations. Possible existence of alcohol and/or butyric acid fermentations could not be excluded. Fermentation organisms in Aalborg East treatment plant were identified by using microautoradiography combined with fluorescence in situ hybridization. All microorganisms involved in monosaccharide fermentation belonged to either Gram-positive Firmicutes or Actinobacteria . Most of them were related either to Streptococcus , hybridizing to the oligonucleotide probe Str, or to uncultured Actinobacteria with a phenotype of polyphosphate-accumulating organisms. The fermenting bacteria were widespread in the nine full-scale BNR plants investigated and constituted 3–21% of the total bacterial biovolume.     

Fermentation of plant-based dairy alternatives by lactic acid bacteria

Ethical, environmental and health concerns around dairy products are driving a fast-growing industry for plant-based dairy alternatives, but undesirable flavours and textures in available products are limiting their uptake into the mainstream. The molecular processes initiated during fermentation by lactic acid bacteria in dairy products is well understood, such as proteolysis of caseins into peptides and amino acids, and the utilisation of carbohydrates to form lactic acid and exopolysaccharides. These processes are fundamental to developing the flavour and texture of fermented dairy products like cheese and yoghurt, yet how these processes work in plant-based alternatives is poorly understood. With this knowledge, bespoke fermentative processes could be engineered for specific food qualities in plant-based foods. This review will provide an overview of recent research that reveals how fermentation occurs in plant-based milk, with a focus on how differences in plant proteins and carbohydrate structure affect how they undergo the fermentation process. The practical aspects of how this knowledge has been used to develop plant-based cheeses and yoghurts is also discussed.     

餐厨垃圾厌氧消化处理主要过程的微生物群落结构分析

【背景】厌氧消化是我国餐厨垃圾处理的主要方法,微生物在其处理过程中起到关键作用,但是目前对其不同工艺单元微生物群落结构的研究较少。【目的】通过分析各工艺单元的微生物多样性与群落结构,为改进餐厨垃圾资源化处理技术、提高资源利用效率提供科学依据。【方法】采集某餐厨垃圾处理厂油水分离、厌氧发酵、沼渣脱水等3个工艺单元产生的废液样品,采用16S rRNA基因高通量测序技术,研究其菌群组成、丰度、优势菌群及其与环境因子的相关性。【结果】初始油水分离样品中的微生物群落种类相对较少,而经厌氧发酵和沼渣脱水处理后样品中的微生物群落种类较丰富。在门水平上,厚壁菌门(Firmicutes)在各单元样品中所占平均比例最高,为81.1%,其次为拟杆菌门(Bacteroidetes)和绿弯菌门(Chloroflexi),分别占15.81%和4.59%;在属水平上,相对丰度较高的菌属为乳酸菌属(Lactobacillus)、互营单胞菌属(Syntrophomonas)等。餐厨垃圾处理过程中的部分菌属可能具有资源-环境双重属性,例如在沼渣脱水单元相对丰度高达32.67%的假单胞菌属(Pseudomonas),该菌属中既存在少部分致病菌或条件致病菌,也具有生产聚羟基脂肪酸酯的功能菌。影响各组样品微生物群落组成结构最显著的因子是p H值,其次是总糖的含量。【结论】研究明确了典型餐厨垃圾厌氧消化处理工艺单元的微生物群落结构和多样性,并提出了优化处理工艺、强化资源利用效率的建议。