A novel downstream process for 1,3-propanediol from glycerol-based fermentation

In this paper, a downstream process for purification of 1,3-propanediol from glycerol-based fermentation broth was investigated. The purification of 1,3-propanediol from fermentation broth was achieved by a process combining microfiltration, charcoal treatment, vacuum distillation, and silica gel chromatography. The broth was first filtered through hollow fiber cartridge, wherein 98.7% of biomass was removed. Soluble proteins and other color impurities in the broth were removed by the use of activated charcoal at optimal concentration of 30 g l⁻¹ where the soluble proteins in the broth decreased to 0.1 g l⁻¹ (96.0% protein loss). The obtained broth when concentrated by vacuum distillation resulted in the crystallization of inorganic salts. Subsequently, 1,3-propanediol was purified by gradient chromatography using silica gel as a stationary phase and mixture of chloroform and methanol as a mobile phase. Finally, with the optimal flow rate of 10 ml min⁻¹ and loading amount of 80 ml, the yield of 1,3-propanediol achieved was 89%. The overall yield of 1,3-propanediol using the proposed procedure was 75.47%. The developed method was found to be a simple, rapid, and efficient procedure for the purification of 1,3-propanediol from fermentation broth.     

Use of activated charcoal for the removal of patulin from cider.

Penicillium urticae (NRRL 2159A) was grown in culture broth containing 1 muCi of [1-14C-A1acetate to produce [14C]patulin. [14C]patulin was purified from the broth and added to apple cider. After the patulin concentration of the cider was adjusted to 30 mug/ml with unlabeled patulin, the cider was subjected to various charcoal treatments. [14C]patulin was completely removed by shaking the cider with 20 mg of activated charcoal per ml and by eluting the cider through a 40- to 60-mesh charcoal column. Activated charcola at 5 mg/ml reduced patulin in naturally contaminated cider to nondetectable levels.     

Effect of activated charcoal, autoclaving and culture media on sucrose hydrolysis

The effect of activated charcoal, autoclaving and culture media on sucrose hydrolysis in tissue culture media was investigated. Activated charcoal acidified an aqueous sucrose (5%) solution and culture media by about 1 to 2 units after autoclaving. Sucrose hydrolysis in tissue culture media and/or aqueous sucrose (5%) solutions containing activated charcoal (buffered to pH 5.8) was dependent on both the hydrogen ion concentration (pH) and autoclaving. After autoclaving, 70%, 56% and 53% sucrose hydrolysis were respectively recorded in a 5% sucrose solution, Murashige and Skoog (MS) and Gamborg B5 (B5) liquid media in the presence of 1% activated charcoal, added before autoclaving. In the absence of activated charcoal, autoclaving resulted in about 20% of the sucrose being hydrolyzed.     

Overcoming inhibitors in a hemicellulosic hydrolysate: improving fermentability by feedstock detoxification and adaptation of <Emphasis Type="Italic">Pichia stipitis</Emphasis>

In order to improve the fermentative efficiency of sugar maple hemicellulosic hydrolysates for fuel ethanol production, various methods to mitigate the effects of inhibitory compounds were employed. These methods included detoxification treatments utilizing activated charcoal, anion exchange resin, overliming, and ethyl acetate extraction. Results demonstrated the greatest fermentative improvement of 50% wood hydrolysate (v/v) by Pichia stipitis with activated charcoal treatment. Another method employed to reduce inhibition was an adaptation procedure to produce P. stipitis stains more tolerant of inhibitory compounds. This adaptation resulted in yeast variants capable of improved fermentation of 75% untreated wood hydrolysate (v/v), one of which produced 9.8 g/l ± 0.6 ethanol, whereas the parent strain produced 0.0 g/l ± 0.0 within the first 24 h. Adapted strains RS01, RS02, and RS03 were analyzed for glucose and xylose utilization and results demonstrated increased glucose and decreased xylose utilization rates in comparison to the wild type. These changes in carbohydrate utilization may be indicative of detoxification or tolerance activities related to proteins involved in glucose and xylose metabolism.     

Optimal production of polyhydroxyalkanoates (PHA) in activated sludge fed by volatile fatty acids (VFAs) generated from alkaline excess sludge fermentation

To reduce the production cost of polyhydroxyalkanoates (PHA) and disposal amount of excess sludge simultaneously, the feasibility of using fermentative volatile fatty acids (VFAs) as carbon sources to synthesize PHA by activated sludge was examined. At pH 11.0, 60 degrees C and fermentative time of 7d, the VFAs yield was 258.65 mgTOC/gVSS. To restrain cell growth during PHA production, the released phosphorus and residual ammonium in the fermentative VFAs was recovered by the formation of struvite precipitation. Acetic acid was the predominant composition of the fermentative VFAs. PHA accumulation in excess sludge occurred feeding by fermentative VFAs with aerobic dynamic feeding process. The maximum PHA content accounted for 56.5% of the dry cell. It can be concluded from this study that the VFAs generated from excess sludge fermentation were a suitable carbon source for PHA production by activated sludge.     

Continuous production of high-content fructooligosaccharides by a complex cell system

A complex biocatalyst system with a bioreactor equipped with a microfiltration (MF) module was employed to produce high-content fructooligosaccharides (FOS) in a continuous process initiated by a batch process. The system used mycelia of Aspergillus japonicus CCRC 93007 or Aureobasidium pullulans ATCC 9348 with beta-fructofuranosidase activity and Gluconobacter oxydans ATCC 23771 with glucose dehydrogenase activity. Calcium carbonate slurry was used to control pH to 5.5, and gluconic acid in the reaction mixture was precipitated as calcium gluconate. Sucrose solution with an optimum concentration of 30% (w/v) was employed as feed for the complex cell system, and high-content FOS was discharged continuously from a MF module. The complex cell system was run at 30 degrees C with an aeration rate of 5 vvm and produced more than 80% FOS with the remainder being 5-7% glucose and 8-10% sucrose on a dry weight basis, plus a small amount of calcium gluconate. The system worked for a 7-day continuous production process with a dilution rate of 0.04 h(-1), and the volumetric productivity for total FOS was more than 160 g L(-1) h(-1).     

A novel downstream process for highly pure 1,3‐propanediol from an efficient fed‐batch fermentation of raw glycerol by Clostridium pasteurianum

An efficient downstream process without prior desalination was developed for recovering 1,3‐propanediol (1,3‐PDO) with high purity and yield from broth of a highly productive fed‐batch fermentation of raw glycerol by Clostridium pasteurianum. After removal of biomass and proteins by ultrafiltration, and concentration by water evaporation, 1,3‐PDO was directly recovered from the broth by vacuum distillation with continuous addition and regeneration of glycerol as a supporting agent. Inorganic salts in the fermentation broth were crystallized but well suspended by a continuous flow of glycerol during the distillation process, which prevented salt precipitation and decline of heat transfer. On the other hand, ammonium salt of organic acids were liberated as ammonia gas and free organic acids under vacuum heating. The latter ones formed four types of 1,3‐PDO esters of acetic acid and butyric acid, which resulted in yield losses and low purity of 1,3‐PDO (< 80%). In order to improve the efficiency of final 1,3‐PDO rectification, we examined alkaline hydrolysis to eliminate the ester impurities. By the use of 20% (w/w) water and 2% (w/w) sodium hydroxide, > 99% reduction of 1,3‐PDO esters was achieved. This step conveniently provided free 1,3‐PDO and the sodium salt of organic acids from the corresponding esters, which increased the 1,3‐PDO yield by 7% and prevented a renewed formation of esters. After a single stage distillation from the hydrolyzed broth and a followed active carbon treatment, 1,3‐PDO with a purity of 99.63% and an overall recovery yield of 76% was obtained. No wastewater with high‐salt content was produced during the whole downstream process. The results demonstrated that the monitoring and complete elimination of 1,3‐PDO esters are crucial for the efficient separation of highly pure 1,3‐PDO with acceptable yield from fermentation broth of raw glycerol.     

Immobilization of fructosyltransferase by chitosan and alginate for efficient production of fructooligosaccharides

The effective system of reusing mycelial fructosyltransferase (FTase) immobilized with two polymers, chitosan and alginate were evaluated for continuous production of fructooligosaccharides (FOS). The alginate beads were successfully developed by maintaining spherical conformation of using 0.3% (w/v) sodium alginate with 0.1% (w/v) of CaCl₂ solution for highest transfructosylating activity. The characteristics of free and immobilized FTase were investigated and results showed that optimum pH and temperature of FTase activity were altered by immobilized materials. A successive production of FOS by FTase entrapped alginate beads was observed at an average of 62.96% (w/w) up to 7 days without much losing its activity. The data revealed by HPLC analysis culminate 67.75% (w/w) of FOS formation by FTase entrapped alginate beads and 42.79% (w/w) by chitosan beads in 36 h of enzyme substrate reaction.     

Ethanol production from starch in a pervaporation membrane bioreactor using Clostridium thermohydrosulfuricum

To attain both high productivity and efficient recovery of ethanol from broth, a membrane bioreactor consisting of a jar fermentor and a pervaporation system was applied to the direct production of ethanol from uncooked starch with a thermophilic anaerobic bacterium, Clostridium thermohydrosulfuricum. From four types of ethanol-selective membranes tested, microporous polytetrafluoroethylene (PTFE) membrane, the pores of which are impregnated with silicone rubber, was chosen for its large flux, high ethanol selectivity, and high stability. During fed-batch fermentation with pervaporation in the membrane bioreactor, ethanol was continuously extracted and concentrated in two traps with concentrations at 5.6%-6.2% (w/w) in trap 1 (20 degrees C) and 27%-32% (w/w) in trap 2 (liquid N(2)), while the ethanol concentration in the broth was maintained at 0.85-0.9% (w/w). Due to the low ethanol concentration in the broth, and the immobilization of bacterial cells by the membrane, the number of viable cells, and, eventually, the ethanol productivity, increased in the membrane bioreactor.     

Production of Nucleic Acid-Related Substances by Fermentative Processes

During the course of the investigation on the production of nucleotide by fermentative processes, it was found that a large amount of ATP and ADP or GTP and GDP, in addition to a smaller amount of AMP or GMP, accumulated in the culture broth when Brevibacterium ammoniagenes ATCC 6872 was incubated in a medium containing adenine or guanine.

After treatment of the culture filtrate with charcoal, the nucleotides were isolated by ion-exchange chromatography on Dowex-1 × 2 (Cl^?-form). They were identified by paper-chromatography, ultraviolet absorption spectra and analyses of base, ribose and phosphate. The ATP preparation from the broth had the same activity with that of authentic sample in the β-aspartokinase system from Corynebacterium glutamicum.     

蒺藜内生真菌发酵液抗菌及抗肿瘤活性初步研究

【目的】筛选出具有抗菌和抗肿瘤活性的蒺藜内生真菌。【方法】采用牛津杯法、稻瘟菌模型及肿瘤细胞模型评价蒺藜内生真菌PDB和察氏培养基发酵产物的抗菌活性和肿瘤细胞毒性。【结果】PDB培养基发酵液和察氏培养基发酵液抑菌圈直径大于10 mm的菌株分别占总菌株数的19.05%和23.81%。PDB培养基发酵液和察氏培养基发酵液对稻瘟菌的最小抑制浓度(MIC)低于10%的菌株分别占总菌株数的19.05%和47.61%。对肿瘤细胞抑制率高于50%的PDB发酵产物占PDB发酵产物总数的52.38%, 而对肿瘤细胞抑制率高于50%的察氏发酵产物占察氏发酵产物总数的28.57%。【结论】部分蒺藜内生真菌的发酵产物具有抗菌和抗肿瘤活性。     

Mechanical properties of chitosan/bamboo charcoal composite films made with normal and surface oxidized charcoal

Chitosan/bamboo charcoal composite films were prepared by blending chitosan with either virgin bamboo charcoal or bamboo charcoal modified by nitric acid oxidation to provide more hydrophilic regions on the bamboo charcoal surface. Investigation of the physical properties of these composite films revealed that the tensile strength and Young’s modulus of the chitosan films were enhanced in a dose-dependent manner by the inclusion of modified bamboo charcoal at up to 1% (w/w), whilst the elongation at break was increased by inclusion of modified bamboo charcoal at up to 0.5% (w/w). In contrast, chitosan composites with virgin bamboo charcoal at up to 0.5% or 1.0% (w/w) showed no enhancement of the tensile strength or Young’s modulus, respectively, and both parameters were reduced with higher levels of virgin bamboo charcoal. Oil, and especially water, absorption of the composite films displayed a marked and dose-dependent increase compared to those of the pure chitosan film.     

Production of ethanol from thin stillage by metabolically engineered Escherichia coli

Thin stillage is a by-product generated in large amounts during the production of ethanol that is rich in carbon sources like glycerol, glucose and maltose. Unfortunately, the fermentation of thin stillage results in a mixture of organic acids and ethanol and minimum utilization of glycerol, the latter a compound that can represent up to 80% of the available substrates in this stream. We report here the efficient production of ethanol from thin stillage by a metabolically engineered strain of Escherichia coli. Simultaneous utilization of glycerol and sugars was achieved by overexpressing either the fermentative or the respiratory glycerol-utilization pathway. However, amplification of the fermentative pathway (encoded by gldA and dhaKLM) led to more efficient consumption of glycerol and promoted the synthesis of reduced products, including ethanol. A previously constructed strain, EH05, containing mutations that prevented the accumulation of competing by-products (i.e. lactate, acetate, and succinate) and overexpressing the fermentative pathway for glycerol utilization [i.e. strain EH05 (pZSKLMgldA)], efficiently converted thin stillage supplemented with only mineral salts to ethanol at yields close to 85% of the theoretical maximum. Ethanol accounted for about 90% (w/w) of the product mixture. These results, along with the comparable performance of strain EH05 (pZSKLMgldA) in 0.5 and 5 l fermenters, indicate a great potential for the adoption of this process by the biofuels industry.     

Microflora of the aerobic preservation of directly brined green olives from Hojiblanca cultivar

New procedures for the preservation stage of ripe olives from Hojiblanca cultivar were studied. An aerobic fermentative process was used with initial pH correction (0.3% acetic acid) and various NaCl concentrations: 6, 3 and 0% (w/v) in tap water. Treatments were carried out at industrial level and the spontaneous changes monitored. At initial salt concentrations of 6 and 3% (w/v) NaCl, pH rose progressively, reaching 4.3 at equilibrium maintaining during this period a constant free lactic acidity of around 0.4% (w/v). When the initial solution was tap waste, however, the pH decreased rapidly to stabilize at about 3.7, and lactic acidity increased continuously to reach values over 1% (w/v) at the end of the preservation process. In all treatments aeration effectively purged the carbon dioxide from the preservation brines, preventing shrivelling of olives. The microbial growth was strongly influenced by the initial NaCl concentration. At 6 and 3%, only yeasts grew, the most abundant being Pichia membranaefaciens, P. vini, P. fermentans and Hansenula polymorpha. However, when there was no NaCl, lactic acid bacteria colonized the solution. Lactobacillus plantarum and Pediococcus inopinatus were the only species found. In this case there was a co-existence between yeasts and lactic acid bacteria. As the treatment that supported lactic acid bacteria achieved the best final pH and acidity for olive stability, it may help to overcome the obstacles to a lactic fermentative process during the preservation stage of ripe olives from the Hojiblanca cultivar.     

Charcoal and activated carbon as adsorbate of phytotoxic compounds – a comparative study

This study compares the potential of natural charcoal from Scots pine ( Pinus sylvestris L.) and activated carbon to improve germination under the hypothesis that natural charcoal adsorbs phytotoxins produced by dwarf-shrubs, but due to it's chemical properties to a lesser extent than activated carbon. Activated carbon has been used in many bioassays as an adsorbate to clean aqueous solutions.
We used aqueous extracts from young leaves of Calluna vulgaris (L.) Hull and Vaccinium myrtillus (L.) as phytotoxin sources in two different concentrations (10 and 14 gr. of dried leaves in 100 ml distilled water). Germination of pine seeds was prevented by the higher concentration of both species, while the lower ones did not show significantly reduced germination. Both ericaceous species showed a very similar potential to prevent germination of Scots pine seeds.
Supplemented carbon (activated carbon, powdered or granulated pine charcoal) restored germination in strong extracts. Adding activated carbon resulted in germination of almost 100%. With pine charcoals added, lower germination percentages were observed. The charcoal powder was more effective (60% for C. vulgaris ; 28% for V. myrtillus ) than the charcoal granulate (30% and 16%, respectively) in restoring germination.
Chemical and surface analysis of the three carbon supplements revealed that activated carbon had by far the biggest active surface area (641 m² g⁻¹), and thus many more cavities to bind phytotoxins than natural charcoal (total surface area of 142 m² g⁻¹).
We conclude, that charcoal produced by forest fires can have a positive effect on seed germination, but to a much lesser extent than activated carbon. Previous studies, which used activated carbon as an equivalent for charcoal, overestimated the effect of charcoal on germination.     

Sugarcane molasses and yeast powder used in the Fructooligosaccharides production by Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611

Different concentrations of sucrose (3–25% w/v) and peptone (2–5% w/v) were studied in the formulation of media during the cultivation of Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611. Moreover, cane molasses (3.5–17.5% w/v total sugar) and yeast powder (1.5–5% w/v) were used as alternative nutrients for both strains’ cultivation. These media were formulated for analysis of cellular growth, β-Fructosyltransferase and Fructooligosaccharides (FOS) production. Transfructosylating activity (U _t ) and FOS production were analyzed by HPLC. The highest enzyme production by both the strains was 3% (w/v) sucrose and 3% (w/v) peptone, or 3.5% (w/v) total sugars present in cane molasses and 1.5% (w/v) yeast powder. Cane molasses and yeast powder were as good as sucrose and peptone in the enzyme and FOS (around 60% w/w) production by studied strains.     

Highly pure 1,3‐propanediol: Separation and purification from crude glycerol‐based fermentation

A new separation and purification process was developed for recovering 1,3‐propanediol (1,3‐PD) from crude glycerol‐based fermentation broth with high purity. The downstream process integrated chitosan flocculation, activated carbon decolorization, fixed bed cation exchange resin adsorption, and vacuum distillation. Breakthrough curves were measured considering the effect of sample concentration, flow rate, temperature, and resin stack height. Yoon–Nelson model was proposed to fit the fixed bed adsorption. The characteristic column parameters were calculated. Optimal condition for adsorption was 1,3‐PD, 30.0 g/L; flow rate, 1.00 mL/min; stacking height, 30.0 cm; and temperature, 298 K. Ethanol‐water (75%, 1 mL/min) was used as eluent to separate 1,3‐PD and glycerol with 95.3% 1,3‐PD elution rate. After vacuum distillation, the overall purity and yield of 1,3‐PD were 99.2% and 80.8% in the purification process, respectively. This is a simple and efficient downstream strategy for 1,3‐PD purification.     

Biotechnological production and application of fructooligosaccharides

Currently, prebiotics are all carbohydrates of relatively short chain length. One important group is the fructooligosaccharides (FOS), a special kind of prebiotic associated to the selective stimulation of the activity of certain groups of colonic bacteria. They have a positive and beneficial effect on intestinal microbiota, reducing the incidence of gastrointestinal infections and also possessing a recognized bifidogenic effect. Traditionally, these prebiotic compounds have been obtained through extraction processes from some plants, as well as through enzymatic hydrolysis of sucrose. However, different fermentative methods have also been proposed for the production of FOS, such as solid-state fermentations utilizing various agro-industrial by-products. By optimizing the culture parameters, FOS yields and productivity can be improved. The use of immobilized enzymes and cells has also been proposed as being an effective and economic method for large-scale production of FOS. This article is an overview of the results considering recent studies on FOS biosynthesis, physicochemical properties, sources, biotechnological production and applications.     

Structural analysis and optimised production of fructo- oligosaccharides by levansucrase from Acetobacter diazotrophicus SRT4

Major fructo-oligosaccharides (FOS) produced by levansucrase (EC 2.4.1.10) from Acetobacter diazotrophicus SRT4 were characterised as 1-kestose and nystose by acid hydrolysis and 13C-NMR spectroscopy. The highest yields of 1-kestose (481 mM; 241 g/l) and nystose (81 mM; 54 g/l) were achieved at initial sucrose concentration of 1754 mM (600 g/l), pH 5.5 and 40°C. The synthesized FOS reached 50% (w/w) of total sugars in the reaction mixture, with a conversion efficiency over 70% (w/w) based on the amount of sucrose converted to 1-kestose.     

Fed-batch production and simple isolation of succinoglycan from Agrobacterium tumefaciens

Succinoglycan production by Agrobacterium tumefaciens was increased from 9.5 g/l to 14 g/l when the microorganism was cultivated under fed-batch condition, as compared to batch fermentation. Cells and broth impurities were removed from the fermentation broth with activated charcoal at a temperature of 90°C, yielding a clear and odorless solution of the polymer. Viscosity mesurement, NMR spectroscopy and Mw determination proved that the polymer retains its main features, practically unaffected by the heat treatment.