Recent advances on conversion and co-production of acetone-butanol-ethanol into high value-added bioproducts

Butanol is an important bulk chemical and has been regarded as an advanced biofuel. Large-scale production of butanol has been applied for more than 100 years, but its production through acetone–butanol–ethanol (ABE) fermentation process by solventogenic Clostridium species is still not economically viable due to the low butanol titer and yield caused by the toxicity of butanol and a by-product, such as acetone. Renewed interest in biobutanol as a biofuel has spurred technological advances to strain modification and fermentation process design. Especially, with the development of interdisciplinary processes, the sole product or even the mixture of ABE produced through ABE fermentation process can be further used as platform chemicals for high value added product production through enzymatic or chemical catalysis. This review aims to comprehensively summarize the most recent advances on the conversion of acetone, butanol and ABE mixture into various products, such as isopropanol, butyl-butyrate and higher-molecular mass alkanes. Additionally, co-production of other value added products with ABE was also discussed.     

Bioproduction of butanol from biomass: from genes to bioreactors

Butanol is produced chemically using either the oxo process starting from propylene (with H2 and CO over a rhodium catalyst) or the aldol process starting from acetaldehyde. The key problems associated with the bioproduction of butanol are the cost of substrate and butanol toxicity/inhibition of the fermenting microorganisms, resulting in a low butanol titer in the fermentation broth. Recent interest in the production of biobutanol from biomass has led to the re-examination of acetone-butanol-ethanol (ABE) fermentation, including strategies for reducing or eliminating butanol toxicity to the culture and for manipulating the culture to achieve better product specificity and yield. Advances in integrated fermentation and in situ product removal processes have resulted in a dramatic reduction of process streams, reduced butanol toxicity to the fermenting microorganisms, improved substrate utilization, and overall improved bioreactor performance.     

Integration of biocatalyst and process engineering for sustainable and efficient n‐butanol production

Recent environmental economic developments generate a need for sustainable and cost‐effective (microbial) processes for the production of high‐volume, low‐priced bulk chemicals. As an example, n‐butanol has, as a second‐generation biofuel, beneficial characteristics compared to ethanol in liquid transportation fuel applications. The industrial revival of the classic n‐butanol (ABE) fermentation requires process and strain engineering solutions for overcoming the main process limitations: product toxicity and low space–time yield. Reaction intensification on the biocatalyst, fermentation, and bioprocess level can be based on economic and ecologic evaluations using quantifiable constraints. This review describes the means of process intensification for biotechnological processes. A quantitative approach is then used for the comparison of the massive literature on n‐butanol fermentation. A comprehensive literature study—including key fermentation performance parameters—is presented and the results are visualized using the window of operation methodology. The comparison allowed the identification of the key constraints, high cell densities, high strain stability, high specific production rate, cheap in situ product removal, high n‐butanol tolerance, to operate in situ product removal efficiently, and cheap carbon source. It can thus be used as a guideline for the bioengineer during the combined biocatalyst, fermentation, and bioprocess development and intensification.     

Performance comparison of ethanol and butanol production in a continuous and closed-circulating fermentation system with membrane bioreactor

Since both ethanol and butanol fermentations are urgently developed processes with the biofuel-demand increasing, performance comparison of aerobic ethanol fermentation and anerobic butanol fermentation in a continuous and closed-circulating fermentation (CCCF) system was necessary to achieve their fermentation characteristics and further optimize the fermentation process. Fermentation and pervaporation parameters including the average cell concentration, glucose consumption rate, cumulated production concentration, product flux, and separation factor of ethanol fermentation were 11.45?g/L, 3.70?g/L/h, 655.83?g/L, 378.5?g/m²/h, and 4.83, respectively, the corresponding parameters of butanol fermentation were 2.19?g/L, 0.61?g/L/h, 28.03?g/L, 58.56?g/m²/h, and 10.62, respectively. Profiles of fermentation and pervaporation parameters indicated that the intensity and efficiency of ethanol fermentation was higher than butanol fermentation, but the stability of butanol fermentation was superior to ethanol fermentation. Although the two fermentation processes had different features, the performance indicated the application prospect of both ethanol and butanol production by the CCCF system.     

Fermentative butanol production by Clostridia

Butanol is an aliphatic saturated alcohol having the molecular formula of C(4)H(9)OH. Butanol can be used as an intermediate in chemical synthesis and as a solvent for a wide variety of chemical and textile industry applications. Moreover, butanol has been considered as a potential fuel or fuel additive. Biological production of butanol (with acetone and ethanol) was one of the largest industrial fermentation processes early in the 20th century. However, fermentative production of butanol had lost its competitiveness by 1960s due to increasing substrate costs and the advent of more efficient petrochemical processes. Recently, increasing demand for the use of renewable resources as feedstock for the production of chemicals combined with advances in biotechnology through omics, systems biology, metabolic engineering and innovative process developments is generating a renewed interest in fermentative butanol production. This article reviews biotechnological production of butanol by clostridia and some relevant fermentation and downstream processes. The strategies for strain improvement by metabolic engineering and further requirements to make fermentative butanol production a successful industrial process are also discussed.     

Integrated butanol recovery for an advanced biofuel: current state and prospects

Butanol has recently gained increasing interest due to escalating prices in petroleum fuels and concerns on the energy crisis. However, the butanol production cost with conventional acetone–butanol–ethanol fermentation by Clostridium spp. was higher than that of petrochemical processes due to the low butanol titer, yield, and productivity in bioprocesses. In particular, a low butanol titer usually leads to an extremely high recovery cost. Conventional biobutanol recovery by distillation is an energy-intensive process, which has largely restricted the economic production of biobutanol. This article thus reviews the latest studies on butanol recovery techniques including gas stripping, liquid–liquid extraction, adsorption, and membrane-based techniques, which can be used for in situ recovery of inhibitory products to enhance butanol production. The productivity of the fermentation system is improved efficiently using the in situ recovery technology; however, the recovered butanol titer remains low due to the limitations from each one of these recovery technologies, especially when the feed butanol concentration is lower than 1 % (w/v). Therefore, several innovative multi-stage hybrid processes have been proposed and are discussed in this review. These hybrid processes including two-stage gas stripping and multi-stage pervaporation have high butanol selectivity, considerably higher energy and production efficiency, and should outperform the conventional processes using single separation step or method. The development of these new integrated processes will give a momentum for the sustainable production of industrial biobutanol.     

Recent advances and state-of-the-art strategies in strain and process engineering for biobutanol production by Clostridium acetobutylicum

Butanol as an advanced biofuel has gained great attention due to its environmental benefits and superior properties compared to ethanol. However, the cost of biobutanol production via conventional acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum is not economically competitive, which has hampered its industrial application. The strain performance and downstream process greatly impact the economics of biobutanol production. Although various engineered strains with carefully orchestrated metabolic and sporulation-specific pathways have been developed, none of them is ideal for industrial biobutanol production. For further strain improvement, it is necessary to develop advanced genome editing tools and a deep understanding of cellular functioning of genes in metabolic and regulatory pathways. Processes with integrated product recovery can increase fermentation productivity by continuously removing inhibitory products while generating butanol (ABE) in a concentrated solution. In this review, we provide an overview of recent advances in C. acetobutylicum strain engineering and process development focusing on in situ product recovery. With deep understanding of systematic cellular bioinformatics, the exploration of state-of-the-art genome editing tools such as CRISPR-Cas for targeted gene knock-out and knock-in would play a vital role in Clostridium cell engineering for biobutanol production. Developing advanced hybrid separation processes for in situ butanol recovery, which will be discussed with a detailed comparison of advantages and disadvantages of various recovery techniques, is also imperative to the economical development of biobutanol.     

Biobutanol production in a Clostridium acetobutylicum biofilm reactor integrated with simultaneous product recovery by adsorption

Background

Clostridium acetobutylicum can propagate on fibrous matrices and form biofilms that have improved butanol tolerance and a high fermentation rate and can be repeatedly used. Previously, a novel macroporous resin, KA-I, was synthesized in our laboratory and was demonstrated to be a good adsorbent with high selectivity and capacity for butanol recovery from a model solution. Based on these results, we aimed to develop a process integrating a biofilm reactor with simultaneous product recovery using the KA-I resin to maximize the production efficiency of biobutanol.

Results

KA-I showed great affinity for butanol and butyrate and could selectively enhance acetoin production at the expense of acetone during the fermentation. The biofilm reactor exhibited high productivity with considerably low broth turbidity during repeated batch fermentations. By maintaining the butanol level above 6.5 g/L in the biofilm reactor, butyrate adsorption by the KA-I resin was effectively reduced. Co-adsorption of acetone by the resin improved the fermentation performance. By redox modulation with methyl viologen (MV), the butanol-acetone ratio and the total product yield increased. An equivalent solvent titer of 96.5 to 130.7 g/L was achieved with a productivity of 1.0 to 1.5 g?·?L^-1?·?h^-1. The solvent concentration and productivity increased by 4 to 6-fold and 3 to 5-fold, respectively, compared to traditional batch fermentation using planktonic culture.

Conclusions

Compared to the conventional process, the integrated process dramatically improved the productivity and reduced the energy consumption as well as water usage in biobutanol production. While genetic engineering focuses on strain improvement to enhance butanol production, process development can fully exploit the productivity of a strain and maximize the production efficiency.     

Improvements in Biobutanol Fermentation and Their Impacts on Distillation Energy Consumption and Wastewater Generation

In the conventional fermentation process to obtain butanol (a novel biofuel), product-induced toxicity results in a product stream with low concentration of butanol (～13 g/L) and limits the concentration of the sugar solution to less than 60 g/L. As a result, steam-consuming operations such as mash sterilization, downstream product recovery (distillation), and wastewater treatment are energy-intensive and important economic drawbacks. Based on the correlation between energy consumption of the distillation unit and butanol concentration in the fermentation beer, the present research points out that improvements in biobutanol processing intended to increase the concentration of butanol in the beer should have a minimum target of 36 g/L. Moreover, due to the dramatic effect of butanol concentration on the wastewater footprint, the volume of the effluent stream can be reduced by 60% (from 72 to 29 L stillage/L butanol) if the minimum concentration target is reached instead of the usual butanol titer of 13 g/L. These correlations were used as the basis to discuss the impacts of today’s research works (genetic strain improvement, utilization of lignocellulosic biomass feedstock, and development of new process technologies) on the energy consumption for complete dehydration of butanol and on wastewater generation.     

Production of acetone butanol ethanol (ABE) by a hyper-producing mutant strain of Clostridium beijerinckii BA101 and recovery by pervaporation.

A silicone membrane was used to study butanol separation from model butanol solutions and fermentation broth. Depending upon the butanol feed concentration in the model solution and pervaporation conditions, butanol selectivities of 20.88-68.32 and flux values of 158.7-215.4 g m(-)(2) h(-)(1) were achieved. Higher flux values (400 g m(-)(2) h(-)(1)) were obtained at higher butanol concentrations using air as sweep gas. In an integrated process of butanol fermentation-recovery, solvent productivities were improved to 200% of the control batch fermentation productivities. In a batch reactor the hyper-butanol-producing mutant strain C. beijerinckii BA101 utilized 57.3 g/L glucose and produced 24.2 g/L total solvents, while in the integrated process it produced 51.5 g/L (culture volume) total solvents. Concentrated glucose medium was also fermented. The C. beijerinckii BA101 mutant strain was not negatively affected by the pervaporative conditions. In the integrated experiment, acids were not produced. With the active fermentation broth, butanol selectivity was reduced by a factor of 2-3. However, the membrane flux was not affected by the active fermentation broth. The butanol permeate concentration ranged from 26.4 to 95.4 g/L, depending upon butanol concentration in the fermentation broth. Since the permeate of most membranes contains acetone, butanol, and ethanol (and small concentrations of acids), it is suggested that distillation be used for further purification.     

Ionic liquid-based in situ product removal design exemplified for an acetone–butanol–ethanol fermentation

Selecting an appropriate separation technique is essential for the application of in situ product removal (ISPR) technology in biological processes. In this work, a three-stage systematic design method is proposed as a guide to integrate ionic liquid (IL)-based separation techniques into ISPR. This design method combines the selection of a suitable ISPR processing scheme, the optimal design of an IL-based liquid–liquid extraction (LLE) system followed by process simulation and evaluation. As a proof of concept, results for a conventional acetone–butanol–ethanol fermentation are presented (40,000 ton/year butanol production). In this application, ILs tetradecyl(trihexyl)phosphonium tetracyanoborate ([TDPh][TCB]) and tetraoctylammonium 2-methyl-1-naphthoate ([TOA] [MNaph]) are identified as the optimal solvents from computer-aided IL design (CAILD) method and reported experimental data, respectively. The dynamic simulation results for the fermentation process show that, the productivity of IL-based in situ (fed-batch) process and in situ (batch) process is around 2.7 and 1.8fold that of base case. Additionally, the IL-based in situ (fed-batch) process and in situ (batch) process also have significant energy savings (79.6% and 77.6%) when compared to the base case.     

S-腺苷甲硫氨酸的研究进展

S-腺苷甲硫氨酸(SAM)是甲硫氨酸和三磷酸腺苷相结合的代谢物,广泛存在于动植物和微生物体内,参与40多种生化反应,主要作为三种代谢途径(转甲基、转硫基、转氨丙基)的前体,临床上被广泛用于治疗肝病、抑郁症、关节炎等。SAM的制备方法主要有化学合成法、酶促合成法、发酵法三种。化学合成的SAM是消旋体,需进行光学拆分,且存在产率低、原料L-高半胱氨酸价格昂贵和环境污染等问题。酶促合成法合成的SAM纯度高,但原料ATP成本太高。发酵法已成为目前生产SAM最常用的方法,欧洲利用发酵法生产SAM已实现了产业化,但国内的起步较晚,目前还处于实验室研究阶段。因此,应加强发酵法生产SAM的产业化关键技术研究。     

Fermentative production of butanol--the industrial perspective

A sustainable bacterial fermentation route to produce biobutanol is poised for re-commercialization. Today, biobutanol can compete with synthetic butanol in the chemical market. Biobutanol is also a superior biofuel and, in longer term, can make an important contribution towards the demand for next generation biofuels. There is scope to improve the conventional fermentation process with solventogenic clostridia and drive down the production cost of 1-butanol by deploying recent advances in biotechnology and engineering. This review describes re-commercialization efforts and highlights developments in feedstock utilization, microbial strain development and fermentation process development, all of which significantly impact production costs.     

Progress and perspectives on improving butanol tolerance

Fermentative production of butanol for use as a biofuel or chemical feedstock is regarded as a promising renewable technology that reduces greenhouse gas emissions and has the potential to become a substitute for non-sustainable chemical production route. However, butanol toxicity to the producing microbes remains a barrier to achieving sufficiently high titers for cost-effective butanol fermentation and recovery. Investigations of the external stress of high butanol concentration on butanol-producing microbial strains will aid in developing improved microbes with increased tolerance to butanol. With currently available molecular tool boxes, researchers have aimed to address and understand how butanol affects different microbes. This review will cover the individual organism’s inherent responses to surrounding butanol levels, and the collective efforts by researchers to improve production and tolerance. The specific microorganisms discussed here include the native butanol producer Clostridium species, the fermentation industrial model Saccharomyces cerevisiae and the photosynthetic cyanobacteria, the genetic engineering workhorse Escherichia coli, and also the butanol-tolerant lactic acid bacteria that utilize diverse substrates. The discussion will help to understand the physiology of butanol resistance and to identify specific butanol tolerance genes that will lead to informed genetic engineering strategies for new strain development.     

Use of in situ solid-phase adsorption in microbial natural product fermentation development

It has been half a century since investigators first began experimenting with adding ion exchange resins during the fermentation of microbial natural products. With the development of nonionic polymeric adsorbents in the 1970s, the application of in situ product adsorption in bioprocessing has grown slowly, but steadily. To date, in situ product adsorption strategies have been used in biotransformations, plant cell culture, the production of biofuels, and selected bulk chemicals, such as butanol and lactic acid, as well as in more traditional natural product fermentation within the pharmaceutical industry. Apart from the operational gains in efficiency from the integration of fermentation and primary recovery, the addition of adsorbents during fermentation has repeatedly demonstrated the capacity to significantly increase titers by sequestering the product and preventing or mitigating degradation, feedback inhibition and/or cytotoxic effects. Adoption of in situ product adsorption has been particularly valuable in the early stages of natural product-based drug discovery programs, where quickly and cost-effectively generating multigram quantities of a lead compound can be challenging when using a wild-type strain and fermentation conditions that have not been optimized. While much of the literature involving in situ adsorption describes its application early in the drug development process, this does not imply that the potential for scale-up is limited. To date, commercial-scale processes utilizing in situ product adsorption have reached batch sizes of at least 30,000 l. Here we present examples where in situ product adsorption has been used to improve product titers or alter the ratios among biosynthetically related natural products, examine some of the relevant variables to consider, and discuss the mechanisms by which in situ adsorption may impact the biosynthesis of microbial natural products.     

In situ extractive fermentation of acetone and butanol

The productivity of the acetone-butanol fermentation was increased by continuously removing acetone and butanol from the fermentation broth during fed-batch culture. Whole broth containing viable cells of Clostridium acetobutylicum was cycled to a Karr reciprocating plate extraction column in which acetone and butanol were extracted into oleyl alcohol flowing counter-currently through the column. By continuously removing these toxic metabolites from the broth, end product inhibition was reduced, and a concentrated feed solution containing 300 g/L glucose was fermented at an overall butanol productivity of 1.0 g/L h, 70% higher than the productivity of normal batch fermentation. The continuous extraction process provides flexible operation and lends itself to process scale-up.     

New approach for selective separation of dilute products from simulated clostridial fermentation broths using cyclodextrins

The ability of cyclodextrins (CD) to form crystalline insoluble complexes with organics was explored in this study in view of a selective separation of dilute products obtainable from three clostridial fermentation systems. To this purpose, a product or a product mixture at a concentration of 0.150 mM each were treated with alpha-CD or beta-CD (0.150 mM) in aqueous solutions as well as in a nutrient broth as a simulated fermentation medium. In the acetone-butanol-ethanol system, and in the butanol-iso-propanol system, alpha-CD was found to precipitate selectively 48% and 46% butanol after 1 h agitation at 30 degrees C. However, beta-CD was found to be superior for the butyric acid-acetic acid system because it selectively precipitated 100% butyric acid under the same conditions. Cooling the three product system with alpha-CD to 4 degrees C for 24 h increased significantly the crop of precipitates but decreased the selectivity for either butanol or butyric acid. Cyclodextrins were thus shown to offer potentially a new exciting possibility for downstream processing of low-concentration fermentation products.     

原位分离耦合技术制备生物丁醇的研究进展

笔者对吸附法、液液萃取法、气提法、渗透汽化法等提取技术原位分离耦合丁醇进行了综述,并对其分离特性与效果进行了比较。针对目前原位分离耦合发酵制备生物丁醇的应用现状和面临的挑战,并结合本课题组已取得的成果,对原位分离耦合发酵制备生物丁醇的前景进行了展望。     

Screening and characterization of butanol‐tolerant micro‐organisms

Aims: Poor butanol tolerance of solventogenic stains directly limits their butanol production during industrial‐scale fermentation process. This study was performed to search for micro‐organisms possessing elevated tolerance to butanol. Methods and Results: Two strains, which displayed higher butanol tolerance compared to commonly used solventogenic Clostridium acetobutylicum, were isolated by evolution and screening strategies. Both strains were identified as lactic acid bacteria (LAB). On this basis, a LAB culture collection was tested for butanol tolerance, and 60% of the strains could grow at a butanol concentration of 2·5% (v/v). In addition, an isolated strain with superior butanol tolerance was transformed using a certain plasmid. Conclusions: The results indicate that many strains of LAB possessed inherent tolerance of butanol. Significance and Impact of the Study: This study suggests that LAB strains may be capable of producing butanol to elevated levels following suitable genetic manipulation.     

Butanol recovery from fermentations by liquid-liquid extraction and membrane solvent extraction

Extraction can successfully be used for in-situ alcohol recovery in butanol fermentations to increase the substrate conversion. An advantage of extraction over other recovery methods may be the high capacity of the solvent and the high selectivity of the alcohol/water separation. Extraction, however, is a comprehensive operation, and the design of an extraction apparatus can be complex. The aim of this study is to assess the practical applicability of liquid-liquid extraction and membrane solvent extraction in butanol fermentations. In this view various aspects of extraction processes were investigated.Thirty-six chemicals were tested for the distribution coefficient for butanol, the selectivity of alcohol/water separation and the toxicity towards Clostridia. Convenient extractants were found in the group of esters with high molar mass.Liquid-liquid extraction was carried out in a stirred fermenter and a spray column. The formation of emulsions and the fouling of the solvent in a fermentation broth causes problems with the operation of this type of equipment. With membrane solvent extraction, in which the solvent is separated from the broth by a membrane, a dispersion-free extraction is possible, leading to an easy operation of the equipment. In this case the mass transfer in the membrane becomes important.With membrane solvent extraction the development of a process is emphasized in which the extraction characteristics of the solvent are combined with the property of silicone rubber membranes to separate butanol from water. In the case of apolar solvents with a high molar mass, the characteristics of the membrane process are determined completely by the solvent. In the case of polar solvents (e.g. ethylene glycol), the permselectivity of the membrane can profitably be used. This concept leads to a novel type of extraction process in which alcohol is extracted with a water-soluble solvent via a hydrophobic semipermeable membrane. This extraction process has been investigated for the recovery of butanol and ethanol from water. A major drawback in all processes with membrane solvent extraction was the permeation of part of the solvent to the aqueous phase.The extraction processes were coupled to batch, fed batch and continuous butanol fermentations to affirm the applicability of the recovery techniques in the actual process. In the batch and fed batch fermentations a three-fold increase in the substrate consumption could be achieved, in the continuous fermentation about 30% increase.