Microbial production of organic acids: expanding the markets

Microbial production of organic acids is a promising approach for obtaining building-block chemicals from renewable carbon sources. Although some acids have been produced for some time and in-depth knowledge of these microbial production processes has been gained, further microbial production processes seem to be feasible, but large-scale production has not yet been possible. Citric, lactic and succinic acid production exemplify three processes in different stages of industrial development. Although the questions being addressed by current research on these processes are diverging, a comparison is helpful for understanding microbial organic acid production in general. In this article, through analysis of the current advances in production of these acids, we present guidelines for future developments in this fast-moving field.     

Batch cultures of supplemented whey permeate using Lactobacillus helveticus: unstructured model for biomass formation, substrate consumption and lactic acid production

The Luedeking and Piret expression can not account for the cessation of production observed at the end of batch; so an empiric term has been previously added to this equation which accounted in a global way for possible substrate limitations. In the model developed in this work, a carbon substrate limitation appeared explicitly in the production expression. Assuming a sigmoidal variation with time of specific growth rate previously validated, the new production model matched well the entire experimental production kinetics. It has been successfully tested for a wide range of nitrogen supplementations, i.e. from an almost total coupling between growth and production for largely supplemented media, to a high decoupling in case of few available nitrogen. Since all the parameters of this model have an obvious biologic meaning, it may be an unvaluable tool for the comprehension of the phenomenon. The model accounted also well for the variation of the specific production rate versus specific growth rate, avoiding the noise due to the direct differentiation of experimental data.     

生物制氢及其与以海水为介质的高盐有机废水处理的耦合研究

当前,全球能源系统的主体是＂碳基能源＂——石油和煤等。这些不可再生的资源已日渐枯竭,而且大量使用会破坏地球生态系统。因此,用＂氢基能源＂逐步取代＂碳基能源＂已成为发达国家能源战略的首选目标,有的国家甚至将这一目标定在本世纪中叶。对于中国等发展中国家,大力开发生物质能等新的可再生＂碳基能源＂,同时加速发展＂氢基能源＂,争取提前进入氢能时代,才能实现可持续发展,甚至跨越式发展。制氢技术包括非生物制氢和生物制氢。非生物制氢目前已小量生产和应用,生物制氢的研究也有相当长的时间,其中影响生物制氢进入实用的主要因素是能耗和生产成本过高。因此,如果作为一个孤立的技术系统,生物制氢只能作为战略性项目。首先介绍了生物制氢的主要原理、目前限制生物制氢产业化的关键限制因子;提出了从系统论的原理出发,通过技术集成,突破生物制氢成本的＂瓶颈＂,达到环保和资源利用的双重目的,使其提前实用化;最后,重点阐述了以海水为介质的高盐有机废水的生物制氢技术的研究进展,尤其介绍我国在相关方面的研究进展。     

Ultra-sonication application in biodiesel production from heterotrophic oleaginous microorganisms

Utilization of microbial oil for biodiesel production has gained growing interest due to the increase in prices and the shortage of the oils and fats traditionally used in biodiesel production. However, it is still in the laboratory study stage due to the high cost of production. Employing organic wastes as raw materials to grow heterotrophic oleaginous microorganisms for further lipid production to produce biodiesel has been predicted to be a promising method for reducing costs. However, there are many obstacles including the low biodegradability of organic wastes, low lipid accumulation capacity of heterotrophic oleaginous microorganisms while using organic wastes, a great dependence on a high-energy consumption approach for biomass harvesting, utilization of toxic organic solvents for lipid extraction, and large amount of methanol required in trans-esterification and in-situ trans-esterifications. Ultra-sonication as a green technology has been extensively utilized to enhance bio-product production from organic wastes. In this article, ultra-sonication applications in biodiesel production steps with heterotrophic oleaginous microorganisms have been reviewed, and its impact, potential, and limitations on the process have been discussed.     

Studies on cellulosic ethanol production for sustainable supply of liquid fuel in China

As the biggest developing country, China faces a serious challenge in satisfying its need for huge amounts of energy resources, especially for liquid fuel. The Chinese government has recently started a bioethanol project, and has produced about 1 million tons of ethanol fuel from corn and wheat in 2005. As it has the largest population in the world and limited lands for food production, cellulosic ethanol would be a more suitable choice for China. Many research projects in China on biodegradation and biotransformation of lignocellulosics have been carried out. Furthermore, understanding the biodegradation mechanism of lignocellulosics and developing practical processes for ethanol production have been ongoing. After more than 30 years of research, several pilot scale facilities have been set up, and lots of experience has been acquired. However, the calculated production cost of cellulosic ethanol is still higher than that of corn ethanol. To overcome this problem, the biorefinery conception has been introduced into research on lignocellulosics transformation. A corncob biorefinery process has been developed in Shandong University. By combining the cellulase and ethanol production with a xylose-related products production, the total production cost can be reduced. A scale of 50,000-ton/year cellulosic ethanol biorefinery is being planned to be built at Yucheng.     

Enatiomerically pure hydroxycarboxylic acids: current approaches and future perspectives

The growing awareness of the importance of chirality in conjunction with biological activity has led to an increasing demand for efficient methods for the industrial synthesis of enantiomerically pure compounds. Polyhydroxyalkanotes (PHAs) are a family of polyesters consisting of over 140 chiral R-hydroxycarboxylic acids (R-HAs), representing a promising source for obtaining chiral chemicals from renewable carbon sources. Although some R-HAs have been produced for some time and certain knowledge of the production processes has been gained, large-scale production has not yet been possible. In this article, through analysis of the current advances in production of these acids, we present guidelines for future developments in biotechnological processes for R-HA production.     

The role of Chironomidae in energy flow of a lotic ecosystem

Chironomid secondary production was estimated on a species-specific basis for 14 dominant taxa in a third-order woodland stream. Results from this study were used to provide an expalanation for the common observation that benthos secondary production in streams is insufficient to account for levels of fish production,i.e., the ALLEN paradox. Annual chironomid secondary production was 29.7 g dry mass m^–2 and accounted for 80% of the total aquatic insect secondary production. A contribution by chironomids this high has not been reported previously from similar streams and indicates that chironomids are an energetically important group available for fish consumption. Most studies examining chironomid secondary production group all taxa at the family level and calculate secondary production for the entire family using the size-frequency method. This approach violates assumptions of the size-frequency method and will result in inaccurate and unpredictable estimates of chironomid secondary production. The species-specific approach to estimate chironomid production used in this study, combined with non-chironomid production, yielded a benthos annual production rate that exceeded consumption necessary to support fish production.     

Metabolomics-driven approach to solving a CoA imbalance for improved 1-butanol production in Escherichia coli

High titer 1-butanol production in Escherichia coli has previously been achieved by overexpression of a modified clostridial 1-butanol production pathway and subsequent deletion of native fermentation pathways. This strategy couples growth with production as 1-butanol pathway offers the only available terminal electron acceptors required for growth in anaerobic conditions. With further inclusion of other well-established metabolic engineering principles, a titer of 15 g/L has been obtained. In achieving this titer, many currently existing strategies have been exhausted, and 1-butanol toxicity level has been surpassed. Therefore, continued engineering of the host strain for increased production requires implementation of alternative strategies that seek to identify non-obvious targets for improvement. In this study, a metabolomics-driven approach was used to reveal a CoA imbalance resulting from a pta deletion that caused undesirable accumulation of pyruvate, butanoate, and other CoA-derived compounds. Using metabolomics, the reduction of butanoyl-CoA to butanal catalyzed by alcohol dehydrogenase AdhE2 was determined as a rate-limiting step. Fine-tuning of this activity and subsequent release of free CoA restored the CoA balance that resulted in a titer of 18.3 g/L upon improvement of total free CoA levels using cysteine supplementation. By enhancing AdhE2 activity, carbon flux was directed towards 1-butanol production and undesirable accumulation of pyruvate and butanoate was diminished. This study represents the initial report describing the improvement of 1-butanol production in E. coli by resolving CoA imbalance, which was based on metabolome analysis and rational metabolic engineering strategies.     

Succinate production in Escherichia coli

Succinate has been recognized as an important platform chemical that can be produced from biomass. While a number of organisms are capable of succinate production naturally, this review focuses on the engineering of Escherichia coli for the production of four-carbon dicarboxylic acid. Important features of a succinate production system are to achieve an optimal balance of reducing equivalents generated by consumption of the feedstock, while maximizing the amount of carbon channeled into the product. Aerobic and anaerobic production strains have been developed and applied to production from glucose and other abundant carbon sources. Metabolic engineering methods and strain evolution have been used and supplemented by the recent application of systems biology and in silico modeling tools to construct optimal production strains. The metabolic capacity of the production strain, the requirement for efficient recovery of succinate, and the reliability of the performance under scaleup are important in the overall process. The costs of the overall biorefinery-compatible process will determine the economic commercialization of succinate and its impact in larger chemical markets.     

Gellan gum

For decades microbial exopolysaccharides have been invaluable ingredients in the food industry, as well as having many attractive pharmaceutical and chemical applications. Gellan gum is a comparatively new gum elaborated by the Gram-negative bacterium Sphingomonas paucimobilis. Although its physico-chemical properties have been well characterized, the ecology and physiology of Sphingomonas, and the factors influencing the fermentation process for production of this gum have received much less attention. This review focuses on the metabolism and the enzymic activity of this bacterium, as well as the factors that influence gellan production, including process temperature, pH, stirring rate, oxygen transfer, and composition of the production medium. Potential strategies for improving the production process are discussed in the context of processes for the production of other microbial biopolymers, particularly exopolysaccharides. In addition, the importance and potential utility of gellan lyases in modification of gellan and in other applications is critically evaluated.     

Analysis of growth and production coupling for batch cultures of Lactobacillus helveticus with the help of an unstructured model

An unstructured model has been developed by assuming a sigmoidal variation with time of specific growth rate and introducing an additional term in Luedeking and Piret production kinetics. The model fitted growth and production kinetic data. The model was helpful for examining the influence of initial yeast extract concentration on growth and production coupling. In richly supplemented media, the growth associated production mechanism prevailed, while in the case of poor nitrogen supplementation, most lactic acid production was non-growth-associated. Both contributions might be calculated from model parameters; this was especially interesting when the forms of growth and production kinetics did not allow a clear conclusion. The additional term in the production rate expression was a convenient way to characterize ‘usable nitrogen’ concentration in the medium. From the model a clear criterion has been derived for optimizing the nitrogen supplementation from an economic point of view.     

The origin of selection signatures on bovine chromosome 6

The extent and pattern of linkage disequilibrium (LD) between closely spaced markers contain information about population history, including past population size and selection history. Selection signatures can be identified by comparing the LD surrounding a putative selected allele at a locus to the putative non-selected allele. In livestock populations, locations of selection signatures identified in this way should be correlated with QTL affecting production traits, as the populations have been under strong artificial selection for these traits. We used a dense SNP map of bovine chromosome 6 to characterize the pattern of LD on this chromosome in Norwegian Red cattle, a breed which has been strongly selected for milk production. The pattern of LD was generally consistent with strong selection in regions containing QTL affecting milk production traits, including a strong selection signature in a region containing a mutation known to affect milk production. The results demonstrate that in livestock populations, the origin of selection signatures will often be QTL for livestock production traits, and illustrate the value of selection signatures in uncovering new mutations with potential effects on quantitative traits.     

Potential for Bioenergy Production from Sugarcane in China

There has been rapid economic development in China in recent decades, and demand for energy has consequently been increasing rapidly. Development and utilisation of clean and renewable energy has been promoted by the Chinese government to help sustain long-term and stable development. Sugarcane is being increasingly used in several countries as feedstock for renewable energy products, and is a major and expanding crop in southern China. In this paper, we discuss the potential of sugarcane as a feedstock for bioenergy production in China. It includes a review of (1) the existing sugarcane industry in China and key bio-physical factors affecting the extent to which sugarcane-based industries could supply feedstock for renewable energy production in China, (2) the economic and policy factors which are likely to affect production of bioenergy from sugarcane in China, and (3) recommendations on actions and policies that may assist with appropriate development of bioenergy production from sugarcane in China. Existing and expected future economic conditions are unlikely to favour production of biofuel from the sugar component in cane. However, the fibre component of cane remains an under-utilised resource component. A conclusion is made that sugarcane fibre has potential to contribute towards renewable electricity production in China. However, at present, favourable incentives do not exist to encourage this production. It is suggested that policies to facilitate cost-effective production of renewable electricity by sugar mills, consistent with national objectives regarding production of renewable energy, be considered by governments. Priorities for future research are in improving biomass yields per unit area of land and technologies for low-cost conversion of lignocellulosic biomass into biofuel.     

Engineering Saccharomyces cerevisiae for isoprenol production

Isoprenol (3-methyl-3-butene-1-ol) is a valuable drop-in biofuel and an important precursor of several commodity chemicals. Synthetic microbial systems using the heterologous mevalonate pathway have recently been developed for the production of isoprenol in Escherichia coli, and a significant yield and titer improvement has been achieved through a decade of research. Saccharomyces cerevisiae has been widely used in the biotechnology industry for isoprenoid production, but there has been no good example of isoprenol production reported in this host. In this study, we engineered the budding yeast S. cerevisiae for improved biosynthesis of isoprenol. The strain engineered with the mevalonate pathway achieved isoprenol production at the titer of 36.02 ± 0.92 mg/L in the flask. The IPP (isopentenyl diphosphate)-bypass pathway, which has shown more efficient isoprenol production by avoiding the accumulation of the toxic intermediate in E. coli, was also constructed in S. cerevisiae and improved the isoprenol titer by 2-fold. We further engineered the strains by deleting a promiscuous endogenous kinase that could divert the pathway flux away from the isoprenol production and improved the titer to 130.52 ± 8.01 mg/L. Finally, we identified a pathway bottleneck using metabolomics analysis and overexpressed a promiscuous alkaline phosphatase to relieve this bottleneck. The combined efforts resulted in the titer improvement to 383.1 ± 31.62 mg/L in the flask. This is the highest isoprenol titer up to date in S. cerevisiae and this work provides the key strategies to engineer yeast as an industrial platform for isoprenol production.     

Global Potential of Rice Husk as a Renewable Feedstock for Ethanol Biofuel Production

The production of ethanol for the energy market has traditionally been from corn and sugar cane biomass. The use of such biomass as energy feedstocks has recently been criticised as ill-fated due to competitive threat against food supplies. At the same time, ethanol production from cellulosic biomass is becoming increasingly popular. In this paper, we analyse rice husk (RH) as a cellulosic feedstock for ethanol biofuel production on the ground of its abundance. The global potential production of bioethanol from RH is estimated herein and found to be in the order of 20.9 to 24.3 GL per annum, potentially satisfying around one fifth of the global ethanol biofuel demand for a 10% gasohol fuel blend. Furthermore, we show that this is especially advantageous for Asia, in particular, India and China, where economic growth and demand for energy are exploding.     

The plant vesicular transport engineering for production of useful recombinant proteins

The molecular breeding of plants that have been genetically engineered for improved disease resistance and stress tolerance has been undertaken with the goal of improving food production. More recently, it has been realized that transgenic plants can serve as bioreactors for the production of proteins or compounds with industrial or clinical uses. Several different recombinant enzymes and antibodies have been produced in this manner. To maximize the potential of industrial plants as a production system for proteins, efficient expression systems utilizing promoters that optimize transgene expression, 5′-untranslated region elements for efficient translation, and appropriate post-translational modifications and localization must be developed. This review summarizes successful examples of the production of recombinant enzymes, antibodies, and vaccines using signal peptides that direct vesicular localization in transgenic plants. We further discuss the modulation of recombinant protein localization to the endoplasmic reticulum, vacuolar system, or extracellular compartments by varying the signal peptide.     

Compactin—a review

Compactin, a hypocholesterolemic molecule, is a competitive inhibitor of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase, which is a regulatory enzyme for cholesterol biosynthesis. The structural similarity and high affinity of the acid form of compactin and HMG, the natural substrate of enzyme, results in specific and effective inhibition of this enzyme. Inhibition results in reduced levels of mevalonic acid in the body, leading to pleiotropic effects. Various fungi have been used for the commercial production of compactin. Using different strategies for improving production levels, yields have been increased to around 900 times of the amount originally produced. Recently, the gene sequence responsible for compactin production has been cloned and sequenced. This review deals with the chemistry, mode of action, pharmacology, biosynthesis, and production of compactin. A comparative study of various reports dealing with the production of compactin is also included.     

靛蓝及其同类色素的微生物生产与转化

靛蓝类色素广泛应用于印染、食品和医药工业, 其环境友好的合成或生产途径越来越受到人们的关注, 特别是微生物生物合成。已经鉴定和分离了能够合成靛蓝类色素的多种微生物, 并且明确了起催化作用的主要是单加氧酶和双加氧酶。已经克隆和利用了一些加氧酶的基因, 构建了工程菌, 优化了其发酵过程。同时, 微生物合成靛蓝的生物转化也已经起步。这些进展将带来环境友好的靛蓝类色素的合成与生产。     

Glucose and acetate influences on the behavior of the recombinant strainEscherichia coli HB 101 (GAPDH)

Summary This study highlights data about the production of a recombinant protein (glyceraldehyde-3-phosphate dehydrogenase) byE. coli HB 101 (GAPDH) during batch and fed-batch fermentations in a complex medium. From a small number of experiments, this strain has been characterized in terms of protein production performance and glucose and acetate influences on growth and recombinant protein production. The present results show that this strain is suitable for recombinant protein production, in fed-batch culture 55 g L^–1 of biomass and 6 g L^–1 of GAPDH are obtained. However this strain, and especially GAPDH overproduction is sensitive to glucose availability. During fermentations, maximum yields of GAPDH production have been obtained in batch experiments for glucose concentration of 10 g L^–1, and in fed-batch experiments for glucose availability of 10 g h^–1 (initial volume 1.5 L). The growth of the strain and GAPDH overproduction are also inhibited by acetate. Moreover acetate has been noted as an activator of its own formation.     

Sustainable production of eicosapentaenoic acid-rich oil from microalgae: Towards an algal biorefinery

Utilization of sustainable natural resources such as microalgae has been considered for the production of biofuels, aquaculture feed, high-value bioactives such as omega-3 fatty acids, carotenoids, etc. Eicosapentaenoic acid (EPA) is an omega-3 fatty acid present in fish oil, which is of physiological importance to both humans and fish. Marine microalgae are sustainable sources of lipid rich in EPA, and different species have been explored for the production of EPA as a single product. There has been a rising interest in the concept of a multi-product biorefinery, focusing on the maximum valorization of the algal biomass. Targeting one or more value-added compounds in a biorefinery scenario can improve the commercial viability of low-value products such as triglycerides for biofuel. This approach has been viewed by technologists and experts as a sustainable and economically feasible possibility for the large-scale production of microalgae for its potential applications in biodiesel and jet fuel production, nutraceuticals, animal and aquaculture feeds, etc. In this review paper, we describe the recent developments in the production of high-value EPA-rich oil from microalgae, emphasizing the upstream and downstream bioprocess techniques, and the advantages of considering an EPA-rich oil-based biorefinery.