High production of 1,3-propanediol from industrial glycerol by a newly isolated Clostridium butyricum strain

Batch and continuous cultures of a newly isolated Clostridium butyricum strain were carried out on industrial glycerol, the major by-product of the bio-diesel production process. For both types of cultures, the conversion yield obtained was around 0.55 g of 1,3-propanediol formed per 1 g of glycerol consumed whereas the highest 1,3-propanediol concentration, achieved during the single-stage continuous cultures was 35-48 g l-1. Moreover, the strain presented a strong tolerance at the inhibitory effect of the 1,3-propanediol, even at high concentrations of this substance at the chemostat (e.g. 80 g l-1). 1,3-Propanediol was associated with cell growth whereas acetate and butyrate seemed non growth-associated products. At low and medium dilution rates (until 0.1 h-1), butyrate production was favoured, whereas at higher rates acetate production increased. The maximum 1,3-propanediol volumetric productivity obtained was 5.5 g l-1 h-1. A two-stage continuous fermentation was also carried out. The first stage presented high 1,3-propanediol volumetric productivity, whereas the second stage (with a lower dilution rate) served to further increase the final product concentration. High 1,3-propanediol concentrations were achieved (41-46 g l-1), with a maximum volumetric productivity of 3.4 g l-1 h-1. A cell concentration decrease was reported between the second and the first fermentor.     

Conversion of glycerol to 1,3-propanediol by a newly isolated thermophilic strain

Of 60 different thermophilic enrichment cultures, 16 converted glycerol anaerobically to 1,3-propanediol. Two PD-forming strains were further enriched, isolated, and characterised. For the most active strain, AT1, the optimal cultivation parameters for pH and temperature were determined as 5.8 to 6.0 and 58°C, respectively. In batch-fermentations with AT1, 6.4 g propanediol per litre was formed with a productivity of 0.17 g l^–1 h^–1.     

Ethanol production from biodiesel-derived crude glycerol by newly isolated Kluyvera cryocrescens

The rapidly expanding market for biodiesel has increased the supply and reduced the cost of glycerol, making it an attractive sustainable feed stock for the fuel and chemical industry. Glycerol-based biorefinery is the microbial fermentation of crude glycerol to produce fuels and chemicals. A major challenge is to obtain microbes tolerant to inhibitors such as salts and organic solvents present in crude glycerol. Microbial screening was attempted to isolate novel strain capable of growing on crude glycerol as a sole carbon source. The newly isolated bacteria, identified as nonpathogenic Kluyvera cryocrescens S26 could convert biodiesel-derived crude glycerol to ethanol with high yield and productivity. The supplementation of nutrients such as yeast extract resulted in distinguished enhancement in cell growth as well as ethanol productivity under anaerobic condition. When glycerol fermentation is performed under microaerobic condition, there is also a remarkable improvement in cell growth, ethanol productivity and yield, compared with those under strict anaerobic condition. In batch fermentation under microaerobic condition, K. cryocrescens S26 produced 27 g/l of ethanol from crude glycerol with high molar yield of 80% and productivity of 0.61 g/l/h.     

Zobellella denitrificans strain MW1, a newly isolated bacterium suitable for poly(3-hydroxybutyrate) production from glycerol

Aims: To search for new bacteria for efficient production of polyhydroxyalkanoates (PHAs) from glycerol. Methods and Results: Samples were taken from different environments in Germany and Egypt, and bacteria capable of growing in mineral salts medium with glycerol as sole carbon source were enriched. From a wastewater sediment sample in Egypt, a Gram‐negative bacterium (strain MW1) was isolated that exhibited good growth and that accumulated considerable amounts of polyhydroxybutyrate (PHB) from glycerol and also from other carbon sources. The 16S rRNA gene sequence of this isolate exhibited 98·5% and 96·2% similarity to Zobellella denitrificans strain ZD1 and to Zobellella taiwanensis strain ZT1 respectively. The isolate was therefore affiliated as strain MW1 of Z. denitrificans. Strain MW1 grows optimally on glycerol at 41°C and pH 7·3 and accumulated PHB up to 80·4% (w/w) of cell dry weight. PHB accumulation was growth‐associated. Although it was not an absolute requirement, 20 g l^?1 sodium chloride enhanced both growth (5 g cell dry weight per litre) and PHB content (87%, w/w). Zobellella denitrificans strain MW1 is also capable to accumulate the poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) copolymer if sodium propionate was used as cosubstrate in addition to glycerol. Conclusions: A new PHB‐accumulating strain was isolated and identified. This strain is able to utilize glycerol for growth and PHB accumulation to high content especially in the presence of NaCl that will enable the utilization of waste glycerol from biodiesel industry. Significance and Impact of the Study: This study is the first report on accumulation of PHA in a member of the new genus Zobellella. Furthermore, utilization of glycerol as the sole carbon source for fast growth and PHB biosynthesis, growth in the presence of NaCl and high PHB contents of the cells will make this newly isolated bacterium a potent candidate for industrial production of PHB from crude glycerol occurring as byproduct during biodiesel production.     

A newly isolated fungal strain used as whole‐cell biocatalyst for biodiesel production from palm oil

A strain of Aspergillus niger isolated from atmospherically exposed bread and Jatropha curcas seed was utilized as a whole‐cell biocatalyst for palm oil methanolysis to produce fatty acid methyl esters (FAME), or biodiesel. The A. niger strain had a lipase activity of 212.58 mU mL^?1 after 144 h incubation at 25 °C with an initial pH value of 6.5, using 7% polypeptone (w/w on basal medium) as the nitrogen source and 3% olive oil (w/w on basal medium) as a carbon source. The A. niger cells spontaneously immobilized within polyurethane biomass support particles (BSPs) during submerged fermentation. Thereafter, the methanolysis of palm oil was achieved via a three‐step addition of methanol in the presence of BSPs‐immobilized with A. niger cells. The influence of water content, reaction temperature and enzyme concentration on reaction rate was investigated. An 8% water content and a temperature of 40 °C in the presence of 30 immobilized BSPs, resulted in an 87% FAME yield after 72 h.     

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.     

Tetramethylpyrazine production from glucose by a newly isolated Bacillus mutant

2,3,5,6-Tetramethylpyrazine (TTMP) was produced using a newly isolated Bacillus mutant. Culture medium optimization studies showed that soytone, an enzyme-hydrolysate of soybean meal, with the supplementation of vitamins, can fully replace yeast extract plus peptone in supporting TTMP production from glucose. In a 5-l fermenter, using the optimized medium which contained 20% glucose, 5% soytone, 3% (NH₄)₂HPO₄, and vitamin supplements, fermentations were carried out with stirring at 700 rpm, air flow at 1.0 vvm, controlled pH at 7.0, and temperature at 37 °C. TTMP reached 4.33 g l⁻¹ after 64.6 h cultivation. A product recovery method was described, which involved evaporation, crystallization, and lyophilization. The product purity was 99.88%, determined by GC with the normalization method. The main impurities were 2,3,5-trimethylpyrazine (0.09%) and 2-ethyl-3,5,6-trimethylpyrazine (0.02%), which were identified by GC/MS. ¹³C NMR determination also gave a consistent result. Natural and high purity of the product and the utilization of cheap green renewable materials make this process promising to compete with TTMP chemical synthetic methods.     

丁酸梭杆菌发酵甘油制备1,3-丙二醇的研究

本研究采用丁酸梭芽孢杆菌(Clostridium butyricum)从甘油发酵制备1,3—丙二醇。该发酵需厌氧培养,发酵培养基为：甘油6％,葡萄糖1％,玉米浆2％,(NH4)2SO40．2％。发酵温度为34℃,pH为6．5～7。在最佳条件下,发酵50h可产1,3—丙二醇40．7g／L,甘油摩尔转化率达68％。     

Optimization and characterization of pullulan production by a newly isolated high-yielding strain Aureobasidium melanogenum

Abstract

Pullulan is an extracellular water-soluble polysaccharide with wide applications. In this study, we screened strains that could selectively produce high molecular weight pullulan for application in industrial pullulan production. A new fungus strain A4 was isolated from soil and identified as Aureobasidium melanogenum based on colony characteristics, morphology, and internally transcribed spacer analysis. Thin-layer chromatography, Fourier-transform infrared spectroscopy, and nuclear magnetic resonance analysis suggested that the dominant exopolysaccharide produced by this strain, which presented a molecular weight of 1.384?×?10⁶ Dalton in in-gel permeation chromatography, was pullulan. The culture conditions for A. melanogenum A4 were optimized at 30?°C and 180?rpm: carbon source, 50?g/L maltose; initial pH 7; and 8?g/L Tween 80. Subsequently, batch fermentation was performed under the optimized conditions in a 5-L stirred-tank fermentor with a working volume of 3?L. The fermentation broth contained 303?g/L maltose, which produced 122.34?g/L pullulan with an average productivity of 1.0195?g/L/h and 82.32?g/L dry biomass within 120?h. The conversion efficiency of maltose to pullulan (Y%) and specific production rate (g/h/g dry cells) (Qs) reached 40.3% and 0.0251?g/L/g dry cells, respectively. The results showed strain A4 could be a good candidate for industrial production.     

Optimisation of β-glucuronidase production from a newly isolated Ganoderma applanatum

Media optimisation was attempted for β-glucuronidase production from a newly and locally isolated (Oxfordshire, UK) fungal strain of Ganoderma applanatum. Both fungal growth and β-glucuronidase activity were found to be greatly affected by varying the carbon or the nitrogen source with gum arabic and yeast extracts being the best carbon and nitrogen sources, respectively. Their concentrations were optimised at 8 g L⁻¹ for the former and 2 g L⁻¹ for the latter.

Work then proceeded to enhance the yield of β-glucuronidase in a controlled environment. Control, batch and fed-batch cultivations were performed in 2-L bioreactors using the optimised medium supplemented with cellobiuronic acid as inducer. Time profiles of biomass dry weight, carbohydrate consumption and β-glucuronidase production were obtained and the results showed that production of β-glucuronidase was noticeably increased by the addition of cellobiuronic acid in both batch and fed-batch fermentations. Although the addition did not produce a variation in the pattern of growth seen between control, and induced fermenters, higher levels of the enzyme were attained when adopting a fed-batch process with 1.09 U mL⁻¹ of culture, corresponding to a 5-fold enhancement in β-glucuronidase production rate compared with batch fermentation.     

Enhanced ethanol production from sugarcane juice by galactose adaptation of a newly isolated thermotolerant strain of Pichia kudriavzevii

The thermotolerant yeast strain isolated from sugarcane juice through enrichment technique was identified as a strain of Pichiakudriavzevii (Issatchenkiaorientalis) through molecular characterization. The P. kudriavzevii cells adapted to galactose medium produced about 30% more ethanol from sugarcane juice than the non-adapted cells. The recycled cells could be used for four successive cycles without a significant drop in ethanol production. Fermentation in a laboratory fermenter with galactose adapted P. kudriavzevii cells at 40 °C resulted in an ethanol concentration and productivity of 71.9 g L⁻¹ and 4.0 g L⁻¹ h⁻¹, respectively from sugarcane juice composed of about 14% (w/v) sucrose, 2% (w/v) glucose and 1% (w/v) fructose. In addition to ethanol, 3.30 g L⁻¹ arabitol and 4.19 g L⁻¹ glycerol were also produced, whereas sorbitol and xylitol were not formed during fermentation. Use of galactose adapted P. kudriavzevii cells for ethanol production from sugarcane juice holds potential for scale-up studies.     

Succinic acid production by a newly isolated bacterium

A new bacterial strain producing succinic acid was enriched from bovine rumen content. It is facultatively anaerobic, belongs to the family Pasteurellaceae and has similarity to the genus Mannheimia. In batch cultivations with D-glucose or sucrose the strain produced up to 5.8 g succinic acid l⁻¹ with a productivity and a yield of up to 1.5 g l⁻¹ h⁻¹ and 0.6 g g⁻¹, respectively. With crude glycerol up to 8.4 g l⁻¹, 0.9 g l⁻¹ h⁻¹ and 1.2 g g⁻¹ were obtained. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Efficient production of ethanol from crude glycerol by a Klebsiella pneumoniae mutant strain

A mutant strain of Klebsiella pneumoniae, termed GEM167, was obtained by γ irradiation, in which glycerol metabolism was dramatically affected on exposure to γ rays. Levels of metabolites of the glycerol reductive pathway, 1,3-propanediol (1,3-PD) and 3-hydroxypropionic acid (3-HP), were decreased in the GEM167 strain compared to a control strain, whereas the levels of metabolites derived from the oxidative pathway, 2,3-butanediol (2,3-BD), ethanol, lactate, and succinate, were increased. Notably, ethanol production from glycerol was greatly enhanced upon fermentation by the mutant strain, to a maximum production level of 21.5 g/l, with a productivity of 0.93 g/l/h. Ethanol production level was further improved to 25.0 g/l upon overexpression of Zymomonas mobilispdc and adhII genes encoding pyruvate decarboxylase (Pdc) and aldehyde dehydrogenase (Adh), respectively in the mutant strain GEM167.     

Biosynthesis and hyper production of pullulan by a newly isolated strain of Aspergillus japonicus-VIT-SB1

The main focus of this study was to screen and characterize novel microbial strains isolated from culinary leaf samples, capable of producing high concentrations of pullulan. Hundred isolates were screened from the phylloplane of different plants. The results revealed that eight strains had the capability to produce exopolysaccharide (EPS) and only one potential strain (designated as VIT-SB1) could produce the significant amount of EPS (3.9 ± 0.02 %) on the 6th day of the fermentation without optimisation. The EPS synthesized by VIT-SB1 strain was confirmed to be pullulan on the basis of the results of FT-IR, HPLC and the enzymatic (Pullulanase) analysis. More than 91 % hydrolysis of pullulan by pullulanase enzyme also indicated the presence of α (1 → 6) glycosidic linkages of α (1 → 4) linked maltotriose units. This VIT-SB1 strain was identified as Aspergillus japonicus based on the nucleotide sequence of the D1/D2 domain of Large-Subunit rRNA gene. The sequence was submitted to the GenBank Nucleotide sequence database with Accession No: KC128815. This study has confirmed that pullulan production capacity of this novel strain and Aureobasidium pullulans are comparable. Hence Aspergillus japonicus-VIT-SB1 strain can be commercially exploited as a potential pullulan producing strain.     

Ethanol production from galactose by a newly isolated Saccharomyces cerevisiae KL17

A wild-type yeast strain with a good galactose-utilization efficiency was newly isolated from the soil, and identified and named Saccharomyces cerevisiae KL17 by 18s RNA sequencing. Its performance of producing ethanol from galactose was investigated in flask cultures with media containing various combination and concentrations of galactose and glucose. When the initial galactose concentration was 20 g/L, it showed 2.2 g/L/h of substrate consumption rate and 0.63 g/L/h of ethanol productivity. Although they were about 70 % of those with glucose, such performance of S. cerevisiae KL17 with galactose was considered to be quite high compared with other strains reported to date. Its additional merit was that its galactose metabolism was not repressed by the existence of glucose. Its capability of ethanol production under a high ethanol concentration was demonstrated by fed-batch fermentation in a bioreactor. A high ethanol productivity of 3.03 g/L/h was obtained with an ethanol concentration and yield of 95 and 0.39 g/L, respectively, when the cells were pre-cultured on glucose. When the cells were pre-cultured on galactose instead of glucose, fermentation time could be reduced significantly, resulting in an improved ethanol productivity of 3.46 g/L/h. The inhibitory effects of two major impurities in a crude galactose solution obtained from acid hydrolysis of galactan were assessed. Only 5-Hydroxymethylfurfural (5-HMF) significantly inhibited ethanol fermentation, while levulinic acid (LA) was benign in the range up to 10 g/L.     

Enhanced hydrocarbon biodegradation by a newly isolated Bacillus subtilis strain

The relation between hydrocarbon degradation and biosurfactant (rhamnolipid) production by a new Bacillus subtilis 22BN strain was investigated. The strain was isolated for its capacity to utilize n-hexadecane and naphthalene and at the same time to produce surface-active compound at high concentrations (1.5 - 2.0 g l(-1)). Biosurfactant production was detected by surface tension lowering and emulsifying activity. The strain is a good degrader of both hydrocarbons used with degradability of 98.3 +/- 1% and 75 +/- 2% for n-hexadecane and naphthalene, respectively. Measurement of cell hydrophobicity showed that the combination of slightly soluble substrate and rhamnolipid developed higher hydrophobicity correlated with increased utilization of both hydrocarbon substrates. To our knowledge, this is the first report of Bacillus subtilis strain that degrades hydrophobic compounds and at the same time produces rhamnolipid biosurfactant.     

Fermentation strategies for 1,3-propanediol production from glycerol using a genetically engineered Klebsiella pneumoniae strain to eliminate by-product formation

We generated a genetically engineered Klebsiella pneumoniae strain (AK-VOT) to eliminate by-product formation during production of 1,3-propanediol (1,3-PD) from glycerol. In the present study, the glycerol-metabolizing properties of the recombinant strain were examined during fermentation in a 5 L bioreactor. As expected, by-product formation was completely absent (except for acetate) when the AK-VOT strain fermented glycerol. However, 1,3-PD productivity was severely reduced owing to a delay in cell growth attributable to a low rate of glycerol consumption. This problem was solved by establishing a two-stage process separating cell growth from 1,3-PD production. In addition, nutrient co-supplementation, especially with starch, significantly increased 1,3-PD production from glycerol during fed-batch fermentation by AK-VOT in the absence of by-product formation.     

Isolation and basic characterization of a β‐glucosidase from a strain of Lactobacillus brevis isolated from a malolactic starter culture

Aims: To study glycosidase activities of a Lactobacillus brevis strain and to isolate an intracellular β‐glucosidase from this strain. Methods and Results: Lactic acid bacteria (LAB) isolated from a commercially available starter culture preparation for malolactic fermentation were tested for β‐glycosidase activities. A strain of Lact. brevis showing high intracellular β‐d ‐glucosidase, β‐d ‐xylosidase and α‐l ‐arabinosidase activities was selected for purification and characterization of its β‐glucosidase. The pure glucosidase from Lact. brevis has also side activities of xylosidase, arabinosidase and cellobiosidase. It is a homotetramer of 330 kDa and has an isoelectric point at pH 3·5. The K_m for p‐nitrophenyl‐β‐d ‐glucopyranoside and p‐nitrophenyl‐β‐d ‐xylopyranoside is 0·22 and 1·14 mmol l^?1, respectively. The β‐glucosidase activity was strongly inhibited by gluconic acid δ‐lactone, partially by glucose and gluconate, but not by fructose. Ethanol and methanol were found to increase the activity up to twofold. The free enzyme was stable at pH 7·0 (t_1/2 = 50 day) but not at pH 4·0 (t_1/2 = 4 days). Conclusions: The β‐glucosidase from Lact. brevis is widely different to that characterized from Lactobacillus casei ( Coulon et al. 1998 ) and Lactobacillus plantarum ( Sestelo et al. 2004 ). The high tolerance to fructose and ethanol, the low inhibitory effect of glucose on the enzyme activity and the good long‐term stability could be of great interest for the release of aroma compounds during winemaking. Significance and Impact of the study: Although the release of aroma compounds by LAB has been demonstrated by several authors, little information exists on the responsible enzymes. This study contains the first characterization of an intracellular β‐glucosidase isolated from a wine‐related strain of Lact. brevis.