Isolation and characterization of novel 1,3-propanediol-producing Lactobacillus panis PM1 from bioethanol thin stillage

Conversion of glycerol to 1,3-propanediol (1,3-PDO) is an attractive option to increase the economic efficiency of the biofuel industry. A bacterial strain that produced 1,3-PDO in the presence of glycerol was isolated from thin stillage, the fermentation residue of bioethanol production. This 1,3-PDO-producing organism was identified as Lactobacillus panis through biochemical characteristics and by 16S rRNA sequencing. Characterization of the L. panis strain hereafter designated as PM1 revealed it was an aerotolerant acidophilic anaerobe able to grow over a wide range of temperatures; tolerant to high concentrations of sodium chloride, ethanol, acetic acid, and lactic acid; and resistant to many common antibiotics. L. panis PM1 could utilize glucose, lactose, galactose, maltose, xylose, and arabinose, but could not grow on sucrose or fructose. Production of 1,3-PDO by L. panis PM1 occurred only when glucose was available as the carbon source in the absence of oxygen. These metabolic characteristics strongly suggested NADH recycling for glucose metabolism is achieved through 1,3-PDO production by this strain. These characteristics classified L. panis PM1 within the group III heterofermentative lactic acid bacteria, which includes the well-characterized 1,3-PDO-producing strain, Lactobacillus reuteri. Metabolite production profiles showed that L. panis PM1 produced considerable amounts of succinic acid (~11–12 mM) from normal MRS medium, which distinguishes this strain from L. reuteri strains.     

Converting crude glycerol to 1,3-propandiol using resting and immobilized Klebsiella sp. HE-2 cells

In this study, 1,3-propanediol (1,3-PDO) was produced from crude glycerol through the fermentation of resting and immobilized cells of a Klebsieblla sp. HE-2 strain isolated from a hydrogen producing anaerobic sludge collected in Southern Taiwan. The Klebsieblla sp. HE-2 cells were first grown on a fermentation medium (FM medium). The medium was then switched to resting-cell medium (RC medium) tailored to improve the production of 1,3-PDO. Using a glycerol-amended FM medium, the soluble metabolites consisted of 1,3-PDO, 2,3-butanediol, and ethanol and byproducts (such as acetic acid and lactic acid) at a content of 18, 28, 49, and 5% (of total soluble metabolites), respectively. When the culture was transferred from the FM medium to the RC medium, the concentration of 1,3-PDO was doubled from 5 g/L to 10 g/L. Using immobilized cells of Klebsieblla sp. HE-2 greatly improved the operational stability and reusability of the cells, as the immobilized cells could be used for 6 cycles without significant activity loss. The immobilized cells were able to directly utilize non-pretreated crude glycerol obtained from a local biodiesel manufacturing plant for 1,3-PDO production with an efficiency comparable to that obtained from using pure glycerol.     

Alkaline conditions stimulate the production of 1,3-propanediol in Lactobacillus panis PM1 through shifting metabolic pathways

A novel Lactobacillus panis PM1 isolate was found to be capable of converting glycerol to 1,3-propanediol (1,3-PDO), an increasingly valuable commodity chemical. In this study the effects of various process parameters, including glucose and glycerol concentrations, inoculum size, temperature, aeration, pH, and carbon source were examined to determine the optimal conditions for the production of 1,3-PDO using a culture method simulating late log to early stationary phases. Inoculum size did not influence the production of 1,3-PDO, and temperature variance showed similar 1,3-PDO production between 25 and 37 °C under the examined conditions. Glycerol concentration and pH played a primary role in the final concentration of 1,3-PDO. The highest production occurred at 150–250 mM glycerol when 50 mM glucose was available. Alkaline initial conditions (pH 9–10) stimulated the production of 1,3-PDO which concurrently occurred with increased acetic acid production. Under these conditions, 213.6 mM of 1,3-PDO were produced from 300 mM glycerol (conversion efficiency was 71 %). These observations indicated that the production of 1,3-PDO was associated with the shift of the metabolic end-product ethanol to acetic acid, and that this shift resulted in an excess concentration of NADH available for the processing of glycerol to 1,3-PDO.     

Efficient production of 1,3-propanediol from fermentation of crude glycerol with mixed cultures in a simple medium

The objective of this study was to examine the applicability of mixed cultures for 1,3-propanediol (1,3-PDO) production from crude glycerol. Three different sources of mixed cultures were tested, where the mixed culture from a municipal wastewater treatment plant showed the best results. 1,3-PDO can be produced as the main product in this mixed culture with typical organic acids like acetic and butyric acids as by-products. The yield was in the range of 0.56–0.76 mol 1,3-PDO per mol glycerol consumed depending on the glycerol concentration. A final product concentration as high as 70 g/L was obtained in fed-batch cultivation with a productivity of 2.6 g/L h. 1,3-PDO can be kept in the culture several days after termination of the fermentation without being degraded. Degradation tests showed that 1,3-PDO is degraded much slower than other compounds in the fermentation broth. In comparison to 1,3-PDO production in typical pure cultures, the process developed in this work with a mixed culture achieved the same levels of product titer, yield and productivity, but has the decisive advantage of operation under complete non-sterile conditions. Moreover, a defined fermentation medium without yeast extract can be used and nitrogen gassing can be omitted during cultivation, leading to a strong reduction of investment and production costs.     

A microfiltration bioreactor to achieve high cell density in Sulfolobus solfataricus fermentation

A novel technique is proposed to achieve higher cell yield in extremophile fermentation. Because the accumulation of toxic compounds is thought to be responsible for low biomass yields, a bioreactor has been designed based on a microfiltration hollow-fiber module located inside the traditional fermentation vessel. Using the cul-tivation of the thermoacidophilic archeon Sulfolobus solfataricus Gı as a model, a biomass of 35 g l⁻¹ dry weight was obtained which proved greater than that of 2 g l⁻¹ obtained in batch fermentation. The bioreactor was characterized by running several fermentation experiments to check the high stability of the membrane module to sterilization cycles, high temperatures, and acidic pHs, even for prolonged periods of time. It was shown that the exhaust medium is unable to sustain growth for the presence of toxic compounds, and ultrafiltration and ion-exchange techniques were used in all the attempts to regenerate it. The results demonstrated the ability of the method to lower inhibitor concentrations and prolong the growth phase, thus achieving high cell density. Furthermore, they indicated that the toxic compounds are ionic species of less than 1kDa. Received: December 23, 1998 / Accepted: March 18, 1999     

Bioconversion of glycerol to 1,3-propanediol in thin stillage-based media by engineered Lactobacillus panis PM1

Thin stillage (TS) is a waste residue that remains after bioethanol production, and its disposal reflects the high costs of bioethanol production. Thus, the development of cost-effective ways to process TS is a pending issue in bioethanol plants. The aim of this study was to evaluate the utilization of TS for the production of the valuable chemical, 1,3-propanediol (1,3-PDO), by Lactobacillus panis PM1. Different fermentation parameters, including temperature, pH and strains [wild-type and a recombinant strain expressing a NADPH-dependent aldehyde reductase (YqhD) gene] were tested in batch and fed-batch cultivations. The highest 1,3-PDO concentration (12.85 g/L) and yield (0.84 g/g) were achieved by batch fermentation at pH-4.5/30 °C by the YqhD recombinant strain. Furthermore, pH-controlled batch fermentation reduced the total fermentation period, resulting in the maximal 1,3-PDO concentration of 16.23 g/L and yield of 0.72 g/g in TS without an expensive nutrient or nitrogen (e.g., yeast extract, beef extract, and peptone) supplementation. The addition of two trace elements, Mg²⁺ and Mn²⁺, in TS increased 1,3-PDO yield (0.74 g/g) without 3-hydroxypropionaldehyde production, the only intermediate of 1,3-PDO biosynthetic pathway in L. panis PM1. Our results suggest that L. panis PM1 can offer a cost-effective process that utilizes the TS to produce a value-added chemical, 1,3-PDO.     

Systems metabolic engineering of Corynebacterium glutamicum for high-level production of 1,3-propanediol from glucose and xylose

Corynebacterium glutamicum is a versatile chassis which has been widely used to produce various amino acids and organic acids. In this study, we report the development of an efficient C. glutamicum strain to produce 1,3-propanediol (1,3-PDO) from glucose and xylose by systems metabolic engineering approaches, including (1) construction and optimization of two different glycerol synthesis modules; (2) combining glycerol and 1,3-PDO synthesis modules; (3) reducing 3-hydroxypropionate accumulation by clarifying a mechanism involving 1,3-PDO re-consumption; (4) reducing the accumulation of toxic 3-hydroxypropionaldehyde by pathway engineering; (5) engineering NADPH generation pathway and anaplerotic pathway. The final engineered strain can efficiently produce 1,3-PDO from glucose with a titer of 110.4 g/L, a yield of 0.42 g/g glucose, and a productivity of 2.30 g/L/h in fed-batch fermentation. By further introducing an optimized xylose metabolism module, the engineered strain can simultaneously utilize glucose and xylose to produce 1,3-PDO with a titer of 98.2 g/L and a yield of 0.38 g/g sugars. This result demonstrates that C. glutamicum is a potential chassis for the industrial production of 1,3-PDO from abundant lignocellulosic feedstocks.     

Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol by a recombinant strain of Klebsiella pneumoniae

In this study, an aldehyde dehydrogenase (ALDH) was over-expressed in Klebsiella pneumoniae for simultaneous production of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO). Various genes encoding ALDH were cloned and expressed in K. pneumoniae, and expression of Escherichia colialdH resulted in the highest 3-HP titer in anaerobic cultures in shake flasks. Anaerobic fed-batch culture of this recombinant strain was further performed in a 5-L reactor. The 3-HP concentration and yield reached 24.4 g/L and 0.18 mol/mol glycerol, respectively, and at the same time 1,3-PDO achieved 49.3 g/L with a yield of 0.43 mol/mol in 24 h. The overall yield of 3-HP plus 1,3-PDO was 0.61 mol/mol. Over-expression of the E. coli AldH also reduced the yields of by-products except for lactate. This study demonstrated the possibility of simultaneous production of 3-HP and 1,3-PDO by K. pneumoniae under anaerobic conditions without supply of vitamin B₁₂.     

Construction of glycerol synthesis pathway in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> for bioconversion of glucose into 1,3-propanediol

1,3-Propanediol (1,3-PDO) is an important three-carbon compound widely used in new polyester polymer materials. Natural organisms that can produce 1,3-PDO from glycerol were well studied. However, no natural microorganisms found could directly convert glucose to 1,3-PDO due to its insufficient glycerol synthesis pathway. In this study, two essential glycerol synthesis genes, CgGPD gene (encoding glycerol-3-phosphate dehydrogenase from Candida glycerinogenes) and ScGPP2 gene (encoding glycerol-3-phosphatase from Saccharomyces cerevisiae), were expressed in wild-type Klebsiella pneumoniae, a natural 1,3-PDO producers with reduction pathway for 1,3-PDO synthesis from glycerol. The results of fermentation, key enzyme activities, and metabolites analysis confirmed that recombinant K. pneumoniae now possessed a metabolic pathway capable of converting glucose to 1,3-PDO. The strain could produce 1,3-PDO from glucose with a final titer of 17.27 g/L with 40 g/L glucose in the medium, showing a 1.26-fold increase compared with 30 g/L glucose. Also, adding certain concentrations of glycerol could quickly initiate the 1,3-PDO synthetic pathway and promote the accumulation of 1,3-PDO, which could shorten the fermentation cycle. These results have important implications for further studies involving the use of one strain for bioconversion of glucose to 1,3-PDO.     

Adaptation dynamics of Clostridium butyricum in high 1,3-propanediol content media

Aim of the present study was to evaluate the effect of exogenous additions of 1,3-propanediol (1,3-PDO) on microbial growth and metabolites production of Clostridium butyricum VPI 1718 strain, during crude glycerol fermentation. Preliminary batch cultures in anaerobic Duran bottles revealed that early addition of 1,3-PDO caused growth cessation in rather low quantities (15?g/L), while 1,3-PDO additions during the middle exponential growth phase up to 70?g/L resulted in an almost linear decrease of the specific growth rate (μ), accompanied by reduced glycerol assimilation, with substrate consumption being used mainly for energy of maintenance requirements. During batch trials in a 3-L bioreactor, the strain proved able to withstand more than 70?g/L of both biologically produced and externally added 1,3-PDO, whereas glycerol assimilation and metabolite production were carried on at a lower rate. Adaptation of the strain in high 1,3-PDO concentration environments was validated during its continuous cultivation with pulses of 1,3-PDO in concentrations of 31 and 46?g/L, where no washout phenomena were noticed. As far as C. butyricum cellular lipids were concerned, during batch bioreactor cultivations, 1,3-PDO addition was found to favor the biosynthesis of unsaturated fatty acids. Also, fatty acid composition was studied during continuous cultures, in which additions of 1,3-PDO were performed at steady states. Lipids were globally more saturated compared to batch cultures, while by monitoring of the transitory phases, it was noticed that the gradual diol washout had an evident impact in the alteration of the fatty acid composition, by rendering them more unsaturated.     

肺炎克雷伯氏菌利用甘油生产1,3-PDO发酵优化的研究进展

发展可再生能源,尤其是生物能源,具有显著的能量收益和碳减排效益。随着石油等不可再生资源的减少,许多大宗传统石油化工产品正不断被使用可再生原料的生物制造产品替代。生物发酵法生产1,3-丙二醇(1,3-PDO)顺应了这一潮流,具有广阔的发展前景。提高微生物发酵竞争力,优化发酵法生产1,3-PDO水平,势必增加1,3-PDO的生产效益。对肺炎克雷伯氏菌(Klebsiella pneumoniae)发酵法进行1,3-PDO生产的代谢机理、菌株筛选和利用、发酵参数的选择和优化以及发酵工程策略的设计和监测等进行综述,为利用生物柴油副产物甘油生产有重要工业价值的1,3-PDO产品提供参考。     

Cell immobilization for microbial production of 1,3-propanediol

Cell and enzyme immobilization are often used for industrial production of high-value products. In recent years, immobilization techniques have been applied to the production of value-added chemicals such as 1,3-Propanediol (1,3-PDO). Biotechnological fermentation is an attractive alternative to current 1,3-PDO production methods, which are primarily thermochemical processes, as it generates high volumetric yields of 1,3-PDO, is a much less energy intensive process, and generates lower amounts of environmental organic pollutants. Although several approaches including: batch, fed-batch, continuous-feed and two-step continuous-feed were tested in suspended systems, it has been well demonstrated that cell immobilization techniques can significantly enhance 1,3-PDO production and allow robust continuous production in smaller bioreactors. This review covers various immobilization methods and their application for 1,3-PDO production.     

Development of a novel recombinant strain Zygosacharomyces rouxii JL2011 for 1,3-propanediol production from glucose

1,3-propanediol (1,3-PDO) is one of the most important industrial chemicals due to its highly desired properties and its wide applications as a key component of the emerging polymer industry. Biotechnology route has been one of the most interesting methods for 1,3-PDO production, whereas, the dha genes were essential to 1,3-PDO biosynthesis. In this study, we cloned and placed the dha cassettes under the control of a glyceraldehyde 3-phosphate dehydrogenase gene promoter pGAP and homologous ZrFPS1 gene promoter pZrfps1; these two promoters were further integrated into the chromosome of Z. rouxii JL2011 to generate recombinant strain JL2011-GZ and JL2011-ZZ, respectively. The results showed that the two strains could produce 1,3-PDO from glucose with a final yield of 6.9 and 10.3 g/l, respectively. The engineered strain JL2011-ZZ showed a 2.3- and 1.5-fold increase in the specific activities and final concentration of 1,3-PDO, respectively, with respect to JL2011-GZ. Batch fermentation with aerobic/micro-aerobic combined strategy of JL2011-ZZ resulted a titer of 17.1 g/l and a yield from glucose of 8.6 %. These results demonstrated that JL2011-ZZ would be a potential strain for 1,3-PDO production from glucose.     

Development of Klebsiella pneumoniae J2B as microbial cell factory for the production of 1,3-propanediol from glucose

1,3-Propanediol (1,3-PDO) is an important platform chemical which has a wide application in food, cosmetics, pharmaceutical and textile industries. Its biological production using recombinant Escherichia coli with glucose as carbon source has been commercialized by DuPont, but E. coli cannot synthesize coenzyme B₁₂ which is an essential and expensive cofactor of glycerol dehydratase, a core enzyme in 1,3-PDO biosynthesis. This study aims to develop a more economical microbial cell factory using Klebsiella pneumoniae J2B which can naturally synthesize coenzyme B₁₂. To this end, the heterologous pathway for the production of glycerol from dihydroxyacetone-3-phosphate (DHAP), a glycolytic intermediate, was introduced to J2B and, afterwards, the strain was extensively modified for carbon and energy metabolisms including: (i) removal of carbon catabolite repression, (ii) blockage of glycerol export across the cell membrane, (iii) improvement of NADH regeneration/availability, (iv) modification of TCA cycle and electron transport chain, (v) overexpression of 1,3-PDO module enzyme, and (vi) overexpression of glucose transporter. A total of 33 genes were modified and/or overexpressed, and one resulting strain could produce 814 mM (62 g/L) of 1,3-PDO with the yield of 1.27 mol/mol glucose in fed-batch bioreactor culture with a limited supplementation of coenzyme B₁₂ at 4 μM, which is ~10 fold less than that employed by DuPont. This study highlights the importance of balanced use of glucose in the production of carbon backbone of the target chemical (1,3-PDO) and regeneration of reducing power (NADH). This study also suggests that K. pneumoniae J2B is a promising host for the production of 1,3-PDO from glucose.     

A robust soft sensor to monitor 1,3-propanediol fermentation process by Clostridium butyricum based on artificial neural network

With the aggravation of environmental pollution and energy crisis, the sustainable microbial fermentation process of converting glycerol to 1,3-propanediol (1,3-PDO) has become an attractive alternative. However, the difficulty in the online measurement of glycerol and 1,3-PDO creates a barrier to the fermentation process and then leads to the residual glycerol and therefore, its wastage. Thus, in the present study, the four-input artificial neural network (ANN) model was developed successfully to predict the concentration of glycerol, 1,3-PDO, and biomass with high accuracy. Moreover, an ANN model combined with a kinetic model was also successfully developed to simulate the fed-batch fermentation process accurately. Hence, a soft sensor from the ANN model based on NaOH-related parameters has been successfully developed which cannot only be applied in software to solve the difficulty of glycerol and 1,3-PDO online measurement during the industrialization process, but also offer insight and reference for similar fermentation processes.     

Glycerol assimilation and production of 1,3-propanediol by Citrobacter amalonaticus Y19

Citrobacter amalonaticus Y19 (Y19) was isolated because of its ability for carbon monoxide-dependent hydrogen production (water–gas shift reaction). This paper reports the assimilation of glycerol and the production of 1,3-propanediol (1,3-PDO) by Y19. Genome sequencing revealed that Y19 contained the genes for the utilization of glycerol and 1,2-propanediol (pdu operon) along with those for the synthesis of coenzyme B₁₂ (cob operon). On the other hand, it did not possess the genes for the fermentative metabolism of glycerol of Klebsiella pneumoniae, which consists of both the oxidative (dhaD and dhaK) and reductive (dhaB and dhaT) pathways. In shake-flask cultivation under aerobic conditions, Y19 could grow well with glycerol as the sole carbon source and produced 1,3-PDO. The level of 1,3-PDO production was improved when vitamin B₁₂ was added to the culture medium under aerobic conditions. Under anaerobic conditions, cell growth and 1,3-PDO production on glycerol was also possible, but only when an exogenous electron acceptor, such as nitrate or fumarate, was added. This is the first report of the glycerol metabolism and 1,3-PDO production by C. amalonaticus Y19.     

A new model for the anaerobic fermentation of glycerol in enteric bacteria: trunk and auxiliary pathways in Escherichia coli

Anaerobic fermentation of glycerol in the Enterobacteriaceae family has long been considered a unique property of species that synthesize 1,3-propanediol (1,3-PDO). However, we have discovered that Escherichia coli can ferment glycerol in a 1,3-PDO-independent manner. We identified 1,2-propanediol (1,2-PDO) as a fermentation product and established the pathway that mediates its synthesis as well as its role in the metabolism of glycerol. We also showed that the trunk pathway responsible for the conversion of glycerol into glycolytic intermediates is composed of two enzymes: a type II glycerol dehydrogenase (glyDH-II) and a dihydroxyacetone kinase (DHAK), the former of previously unknown physiological role. Based on our findings, we propose a new model for glycerol fermentation in enteric bacteria in which: (i) the production of 1,2-PDO provides a means to consume reducing equivalents generated in the synthesis of cell mass, thus facilitating redox balance, and (ii) the conversion of glycerol to ethanol, through a redox-balanced pathway, fulfills energy requirements by generating ATP via substrate-level phosphorylation. The activity of the formate hydrogen-lyase and F(0)F(1)-ATPase systems were also found to facilitate the fermentative metabolism of glycerol, and along with the ethanol and 1,2-PDO pathways, were considered auxiliary or enabling. We demonstrated that glycerol fermentation in E. coli was not previously observed due to the use of medium formulations and culture conditions that impair the aforementioned pathways. These include high concentrations of potassium and phosphate, low concentrations of glycerol, alkaline pH, and closed cultivation systems that promote the accumulation of hydrogen gas.     

Continuous production of 1,3-propanediol using raw glycerol with immobilized Clostridium beijerinckii NRRL B-593 in comparison to suspended culture

The continuous production of 1,3-propanediol (1,3-PDO) was investigated with Clostridium beijerinckii NRRL B-593 using raw glycerol without purification obtained from a biodiesel production process. Ceramic rings and pumice stones were used for cell immobilization in a packed-bed bioreactor. For comparison purpose, a control bioreactor with suspended culture was also run. The effect of hydraulic retention time (HRT) on the production of 1,3-PDO in both immobilized and suspended bioreactors were also investigated. The study revealed that HRT is an important factor for both immobilized and suspended systems and a HRT of 2 h is the best one in terms of volumetric production rate (g 1,3-PDO/L/h). Furthermore, cell immobilization had also obvious benefits especially for the robustness and the reliability of the production. The results indicated that cell immobilization achieved a 2.5-fold higher productivity in comparison to suspended cell system. Based on our results, continuous production of 1,3-PDO with immobilized cells is an efficient method, and raw glycerol can be utilized without any pretreatment.     

Production of tomato flavor volatiles from a crude enzyme preparation using a hollow-fiber reactor

In recent years there has been an increase in the interest in the production of compounds by isolation from natural sources or through processes that can be deemed "natural". This is of particular interest in the food and beverage industry for flavors and aromas. Hexanal, organoleptically known to possess "green character", is of considerable commercial interest. The objective of this study was to determine if the enzyme template known to be responsible for the synthesis of hexanal from linoleic acid (18:2) in tomato fruits could be harnessed using a hollow-fiber reactor. A hollow-fiber reactor system was set up and consisted of a XAMPLER ultrafiltration module coupled to a reservoir. The enzyme template was extracted from ripe tomato fruits and processed through an ultrafiltration unit (NMWC of 100 kDa) to produce a retentate enriched in soluble and membrane-associated lipoxygenase (LOX) and hydroperoxide lyase (HPL). This extract was recirculated through the lumen of the hollow-fiber ultrafiltration unit with the addition of substrate in the form of linoleic acid, with buffer addition to the reaction flask to maintain a constant retentate volume. Product formation was measured in the permeate using solid phase microextraction (SPME) developed for this system. At exogenous substrate concentrations of 16 mM and a transmembrane pressure of 70 kPa, hexanal production rates are in the order of 5.1 microg/min. Addition of Triton X-100 resulted in membrane fouling and reduced flux. The reactor system has been run for periods of up to 1 week and has been shown to be stable over this period.