Filtered Fermentation Broth Research Articles

Spectrophotometric analysis of bioactive metabolites and fermentation optimisation of Streptomyces sp. HU2014 with antifungal potential against Rhizoctonia solani

Streptomyces spp. produce a range of secondary metabolites that can inhibit phytopathogen growth. This study aimed to optimise the environmental and nutritional parameters for improving the production of antifungal components by Streptomyces sp. HU2014 through statistical approaches. Spectrophotometry and bioassay revealed that two fractions, F6 and F8, from the cell-free filtrates of HU2014 culture broth were rich in antifungal compounds. Subsequently, single-factor experiments were conducted using glucose-peptone-yeast extract medium as basal medium, and the effects of different parameters on the production of bioactive metabolites by Streptomyces sp. HU2014 were evaluated and optimised using shake-flask experiments as follows: incubation time, 10 days; temperature, 25 °C; agitation speed, 150 rpm; inoculum size, 5% (v/v); initial pH, 7.0; and medium composition, dextrin, yeast extract and KNO3. The central composite face-centred design from response surface methodology predicted 39.6 g/L dextrin, 8.5 g/L yeast extract and 2.5 g/L KNO3 as the optimum medium composition. The validation experiment confirmed that a 50-fold dilution of the optimised filtered fermentation broth of the strain culture showed an increase of 14.9% in the inhibition of Rhizoctonia solani YL-3 when compared with the basal medium. A good correlation between measured and predicted values of the model validated both the responses. Thus, the present study used statistical optimisation to improve the bioactive metabolites productivity in Streptomyces sp. HU2014.

Production and extraction of medium chain carboxylic acids at a semi-pilot scale

Abstract Microbial chain elongation enables the conversion of organic wastes and agroindustrial sidestreams into medium chain carboxylic acids (MCCAs), with application in the production of plasticizers, personal care products, functional feed additives, and fuels. This work describes the up-scaling of the production of MCCAs from thin stillage, and their separation into an oil at kilogram scale with a MCCA content of more than 72%. The chain elongation occurred at a maximum rate of 1.7 ± 0.6 g MCCA L−1 d−1 without the use of ethanol as electron donor, even when it was supplied. Two in-line extraction trains are compared: (1) solvent extraction based on pertraction followed by electrochemical phase separation of the acids through a 2-compartment membrane electrolysis (ME) cell, and (2) direct electrochemical extraction from the fermentation broth using a 3-compartment ME cell. Using the first treatment train, n-caproic acid was extracted at an efficiency of 73%. The decrease in the fermentation pH from 5.5 to 5.0 enabled doubling the MCCA and oil production rate without a concomitant increase in NaOH dosage for pH stabilization. The selectivity brought about by pertraction and its capacity to up-concentrate MCCAs in an alkaline-pH extract enabled a more efficient use of the electricity for ME (as low as 6.2 kWh kg−1 oil) when compared to a period when ME was applied directly to the filtered fermentation broth (160 kWh kg−1 oil). The 3-compartment ME cell was able to phase separate oil extracting directly from the fermentation broth but it was not selective for longer-chain carboxylic acids due to co-extraction of short chain carboxylic acids and inorganic anions.

Factors Affecting Precipitation of Calcium Lactate from Fermentation Broths and from Aqueous Solution

Lactic acid is one of the most important organic acids which is being extensively used around the globe in a range of industrial and biotechnological applications. Lactic acid can be produced either by fermentation or by chemical synthesis but the biotechnological fermentation process has several advantages compared to the other one. However fermentation broth contains a number of impurities which must be removed from the broth in order to achieve more pure lactic acid. Efficiency of recovery is crucial to the economy of the whole process as well since the costs of separation and recovery are responsible for more than half of the entire cost of production. In the traditional procedure, the heated and filtered fermentation broth is concentrated to allow crystallization or precipitation of calcium lactate, followed by addition of sulphuric acid to remove the calcium in form of calcium sulphate. The disadvantage of this procedure is the relatively high solubility of calcium lactate which causes product loss in the crystallization step. Therefore we investigated the effects of four operating parameters of the crystallization/precipitation process from two different fermentation broths and from an aqueous solution. Thus we applied three central composite statistical designs, in which the examined parameters were the temperature of the solution, the duration of the process, the effect of ethanol addition to the solution as well as the effect of multi-cycle precipitation after separation of the precipitated calcium lactate. According to the results, more than 50 percent calcium lactate precipitation increase can be obtained from fermentation broth by adding sufficient amount of ethanol and adjusting the proper temperature of the process.

EXTRACTION OF ACETOIN FROM FERMENTATION BROTH USING AN ACETONE/PHOSPHATE AQUEOUS TWO-PHASE SYSTEM

An aqueous two-phase system (ATPS) consisting of acetone and phosphate was used to extract acetoin from fermentation broth. The influence of phase composition on partition of acetoin was investigated. When the filtered fermentation broth was used, relatively high partition coefficient (22.3) and recovery coefficient (96.4%) of acetoin were obtained by a system composed of 30% (w/w) acetone and 35% (w/w) dipotassium hydrogen phosphate. Then the system was applied to extract unfiltered fermentation broth directly, and the recovery coefficient of acetoin was 94.3%. Simultaneously, the byproduct 2,3-butanediol could also be extracted with the recovery coefficient of 93.5%. In addition, the removal of residual sucrose, cells, proteins, and prodigiosin from the fermentation broth was studied, and the removal ratios of these impurities were all above 85%. Ultimately, the recovery of phosphate in the bottom phase was explored, and the recovery coefficient could reach 93.7% through pH adjustment and dilution crystallization. The recovered phosphate also showed good ATPS extraction ability. This method provides a new possible way for the separation of acetoin from fermentation broth.

Systematic downstream process development for purification of baccatin III with key performance indicators

Performance and cost-efficiency of biotechnological processes largely depend on decisions made in early phases of process development. Experimental investigation of process alternatives, unit operations, mass separating agents and operating conditions should therefore follow a systematic approach. Results should be evaluated and alternatives benchmarked with the aim of designing the most cost-efficient total process. Key performance indicators can meet this demand prior to availability of complete process concepts or mass balances. In this paper utilization and benefit of purification performance index (PPI), purification fingerprints and separation cost indicator (SCI) are demonstrated for the example of an experimentally-based downstream process development. Adsorption is investigated as a first purification step to separate baccatin III, a precursor of the anti-cancer drug paclitaxel, from the supernatant of a plant cell culture of Taxus baccata. Nine sorbents and 11 solvents (desorbents) are screened at different phase ratios. Concentration changes of the target substance and 13 major contaminants are evaluated and rated by costs with key performance indicators to find an optimal process concept. As a result, 94% pure baccatin III can be obtained from filtered fermentation broth by a simple low-cost downstream process consisting of three steps only: adsorption, washing and desorption.

Adsorption equilibria of bio-based butanol solutions using zeolite

1-Butanol can be produced by clostridial fermentations with acetone and ethanol as by-products. The butanol can be present up to ∼20 g L −1 depending on process conditions and microbial strain. The high-silica zeolite CBV28014 has been proven to adsorb butanol selective over water, while showing higher affinity for butanol than for acetone and ethanol. Multi-component acetone–butanol–ethanol (ABE) adsorption on CBV28014 has been modeled using a single site extended Langmuir adsorption model and the ideal adsorbed solution (IAS) theory model. The IAS model describes multi-component adsorption of ABE in synthetic mixtures and ABE in filtered fermentation broth by CBV28014 more accurately than the single site extended Langmuir model.

A comparison of direct infusion MS and GC‐MS for metabolic footprinting of yeast mutants

Recent technical advances in mass spectrometry (MS) have propelled this technology to the forefront of methods employed in metabolome analysis. Here, we compare two distinct analytical approaches based on MS for their potential in revealing specific metabolic footprints of yeast single-deletion mutants. Filtered fermentation broth samples were analyzed by GC-MS and direct infusion ESI-MS. The potential of both methods in producing specific and, therefore, discriminant metabolite profiles was evaluated using samples from several yeast deletion mutants grown in batch-culture conditions with glucose as the carbon source. The mutants evaluated were cat8, gln3, ino2, opi1, and nil1, all with deletion of genes involved in nutrient sensing and regulation. From the analysis, we found that both methods can be used to classify mutants, but the classification depends on which metabolites are measured. Thus, the GC-MS method is good for classification of mutants with altered nitrogen regulation as it primarily measures amino acids, whereas this method cannot classify mutants involved in regulation of phospholipids metabolism as well as the direct infusion MS (DI-MS) method. From the analysis, we find that it is possible to discriminate the mutants in both the exponential and stationary growth phase, but the data from the exponential growth phase provide more physiological relevant information. Based on the data, we identified metabolites that are primarily involved in discrimination of the different mutants, and hereby providing a link between high-throughput metabolome analysis, strain classification, and physiology.

Selective Reverse Micellar Extraction of Three Proteins from Filtered Fermentation Broth Using Response Surface Methodology

The influence of three system parameters [bis(2-ethylhexyl)sulfosuccinate (AOT) concentration, pH, and temperature] on the selective separation of cytochrome c, lysozyme, and ribonuclease A from buffer solution and a filtered fermentation broth was examined. It was found that a minimal AOT concentration exists for ≥90% extraction from a buffer solution, and this concentration depends on the pH of solution and hydrophobicity of the protein. Extraction from filtered broth resulted in a reduction of the minimal concentration for both cytochrome c and lysozyme, while for ribonuclease A it was unchanged. It appears that certain broth constituents act as cosurfactants and reduce the charge repulsion between the surfactant headgroups leading to smaller micelles. This is turn led to a reduction in the pH range, although extraction yields were unaltered. The water content (W 0) was measured in a reverse micellar phase in contact with buffer, filtered fermentation broth, and molecular weight fractionated or extracted broth. This data led to the conclusion that the broth constituents responsible for changes in the minimal AOT concentration and W 0 were low molecular weight positively charged amino acids and phospholipids. Finally, response surface methodology was used to optimize key system parameters in order to maximize protein extraction, and this technique minimizes the experiments required. By using these optimized conditions, a mixture of the three proteins could be recovered from a filtered broth with high yields (70–97%) and high purity. Furthermore, the initial protein concentration was found not to influence protein recovery.

Determination of carbon sources in fermentation media using high-performance anion-exchange liquid chromatography and pulsed amperometric detection

The technique of high pH anion-exchange chromatography method with pulsed amperometric detection (HPAEC-PAD) has been adapted for the rapid determination of common carbohydrates present in fermentation broths. Simple water dilution of filtered fermentation broth samples was the sole sample preparation step required. The samples were analyzed using a Dionex CarboPac PA1 column with 150 m M NaOH as the mobile phase at a flow-rate of 1 ml/min and a total run time of 20 min. A gradient method was also developed to resolve species which exhibited similar t R in the isocratic procedure. Among the analytes examined in this study were ethanol, glycerol, galactose, glucose, mannose, fructose, raffinose, ribose and lactose. Examples from several microbial fermentations using chemically defined or complex medium are presented.

Evidence for a Novel Insecticidally Active Exotoxin Produced By the HD 116 Strain of Bacillus thuringiensis var. morrisoni

Liquid chromatographic and bioassay data provide evidence for a novel exotoxin produced by the HD 116 strain of Bacillus thuringiensis var. morrisoni. The exotoxin from HD 116 differs from the well known beta-exotoxin produced by the HD 59 strain of Bacillus thuringiensis var. thuringiensis. Chromatograms of aliquots of filtered fermentation broth taken before and after inoculation with the organisms HD 116 and HD 59 were compared. Based on retention times of the obtained peaks, chromatographic fractions were obtained and activity against first instar larvae of the Colorado potato beetle (Coleoptera: Chrysomelidae) was determined. Fractions based upon similar retention times that were active from var. morrisoni were not active from var. thuringiensis and vice versa. Our conclusion that the new exotoxin may be a diphosphate analog of thuringiensin is based on a comparison of relative retention times among several adenine nucleotides. We have tentatively named this new extracellular exotoxin sigma-exotoxin.

A35512, a complex of new antibacterial antibiotics produced by Streptomyces candidus. I. Isolation and characterization.

The new antibiotic complex A35512 produced by Streptomyces candidus was isolated from the filtered fermentation broth. The individual factors A, B, C, E, and H were separated and purified by column chromatography. A35512B, the major factor, was isolated as the dihydrochloride salt, a white crystalline compound with an approximate empirical formula of C90H101 N8O39Cl.2HCl. The A35512 antibiotics belong to the glycopeptide class of antibiotics and possess high in vitro and in vivo activity against Gram-positive bacteria.