Continuous Asymmetric Reduction Of 4-Oxoisophorone By Thermophilic Bacteria Using A Hollow Fiber Reactor

Continuous asymmetric reduction of 4-oxoisophorone by the thermophilic bacterium Thermomonospora curvata JTS321 was examined using three reactor systems: packed bed, fluidized bed and hollow fiber. T. curvata was immobilized in polyacrylamide-hydrazide gels when used in the packed and fluidized bed reactors. Of the three reactor systems, the highest productivity (964 mg.1^-1.h^-1) was observed in the fluidized bed reactor. However, many cells grew outside of the gel matrix, causing product contamination. The productivity of the hollow fiber reactor was 504 mg.1^-1.h^-1; the problem of cell contamination of the product was avoided, as the molecular cut-off of the hollow fibers (400 000) was of an appropriate size to prevent cell leakage to the product stream. We therefore consider that the hollow fiber reactor is most suitable for continuous microbial conversions.     

Bioreactor for solid-state cultivation of <Emphasis Type="Italic">Phlebiopsis gigantea</Emphasis>

Phlebiopsis gigantea fungus used in biological control of root rot is currently cultivated commercially in disposable, sterilizable plastic bags. A novel packed bed bioreactor was designed for cultivating P. gigantea and compared to the plastic bag method and to a tray bioreactor. The spore viability of 5.4 × 10⁶ c.f.u./g obtained with the packed bed bioreactor was of the same order of magnitude as the viabilities obtained with the other cultivation methods. Furthermore, the packed bed bioreactor was less time and space consuming and easier to operate than the tray bioreactor.     

Molecular Monitoring of Microbial Population Dynamics During Operational Periods of Anaerobic Hybrid Reactor Treating Cassava Starch Wastewater

This study characterized the microbial community and population dynamics in an anaerobic hybrid reactor (AHR) treating cassava starch wastewater. Methanogens and nonmethanogens were followed during the start-up and operation of the reactor, and linked to operational and performance data. Biomass samples taken from the sludge bed and packed bed zones of the AHR at intervals throughout the operational period were examined by 16S rRNA fluorescence in situ hybridization (FISH). The start-up seed and the reactor biomass were sampled during the feeding of the wastewater with a chemical oxygen demand (COD) value of 8 g L⁻¹ and a hydraulic retention time (HRT) of 8 days. These samples were characterized by the predominance of cells with long-rod morphology similar to Methanosaeta spp. Following a sharp operational change, accomplished by increasing the COD concentration of the organic influent from 8 to 10 g L⁻¹ and reducing the HRT from 8 to 5 days, there was a doubling of the organic loading rate, a reduction of the COD removal efficiency, as well as decreased methane content in the biogas and an accumulation of total volatile acids in the reactor. Moreover, this operational change resulted in a significant population shift from long-rod Methanosaeta-like cells to tetrad-forming Methanosarcina-like cells. The distributions of microbial populations involved in different zones of the AHR were determined. The results showed that nonmethanogens became the predominant population in both sludge and the packed bed zone. However, the percentage of methanogens in the packed bed zone was higher than that in the sludge bed zone. This higher percentage of methanogens was likely caused by the fact that the packed bed zone provided a suitable environmental condition with an appropriate nutrient availability for methanogen growth.     

Isolation and identification of a new metabolite of microbial conversion of upgraded neutral fraction of Polish tall oil by means of four strains of Mycobacterium sp.

Summary A new metabolite, 5-alpha-androstane-3,6,17-trione, was isolated as a product of microbial conversion of upgraded neutral fraction of the Polish tall oil byMycobacterium NRRL B-3683, NRRL B-3805, MB 3683, and MB 3805.     

A new method of whole microbial cell immobilization

Summary A simple method for producing gelatin-immobilized microbial cells is described. The microorganism used as an example was baker's yeast (Saccharomyces cerevisiae). The gel particles containing these cells were utilized as an immobilized enzyme (invertase) both in stirred batch and packed bed systems.     

Continuous Asymmetric Reduction Of 4-Oxoisophorone By Thermophilic Bacteria Using A Hollow Fiber Reactor

Continuous asymmetric reduction of 4-oxoisophorone by the thermophilic bacterium Thermomonospora curvata JTS321 was examined using three reactor systems: packed bed, fluidized bed and hollow fiber. T. curvata was immobilized in polyacrylamide-hydrazide gels when used in the packed and fluidized bed reactors. Of the three reactor systems, the highest productivity (964 mg.1^-1.h^-1) was observed in the fluidized bed reactor. However, many cells grew outside of the gel matrix, causing product contamination. The productivity of the hollow fiber reactor was 504 mg.1^-1.h^-1; the problem of cell contamination of the product was avoided, as the molecular cut-off of the hollow fibers (400 000) was of an appropriate size to prevent cell leakage to the product stream. We therefore consider that the hollow fiber reactor is most suitable for continuous microbial conversions.     

Nutrient and Microbial Dynamics in Biosolids Amended Soils Following Rainfall Simulation

Municipal waste treatment plants are mandated by U.S. EPA to treat domestic wastewater prior to releasing it to receiving streams. The dewatering and high temperature drying processes at the plant are considered effective in reducing microbial contaminants in the waste. The resulting solid material (biosolid) is rich in nutrients that may serve as a value-added product for plant growth. In this study, we examined the nutrient value of biosolids, their potential biological and chemical risks that could result from surface application to two Mid-Atlantic soils: Bojac (coarse-loamy, mixed, thermic Typic Hapludult) and Cullen (clayey, mixed, thermic Typic Hapludult). Soils were placed on tilt beds and packed to their respective bulk density. Biosolids were added at a rate of 2.24 Mg/ha equivalent and mixed with the top 5 cm of the soil bed. Simulated rain was applied at a rate of 65 mm h^?1 for 45 minutes. Surface runoff and percolation water were collected and analyzed for elemental content, Escherichia coli (E. coli) and total coliform bacteria. Among the nutrient elements of concern (P, Zn, Mn, and Cu) in biosolids, none were found to be higher than the specified EPA limits. The concentration of P was highest in runoff and percolation water from beds packed with Bojac and biosolids. The combined effects of high clay (35%), Al (1.14%), and Fe (5.11%) in Cullen increased its P-adsorbing capacity. Low levels of E. coli and other coliform bacteria were present in samples from biosolids-treated beds packed with Cullen. Microbial counts in runoff and percolation samples varied with soil type; in some instances they were ten-fold higher in Bojac than in Cullen. The results obtained in this study suggest that surface runoff from land applications of biosolids might contribute to microbial contamination of receiving waters near agricultural fields.     

Identification of mathematical models of complex cultures of microorganisms in continuous cultivation—Numerical approach

A numerical procedure, suitable for parameter estimation in complex continuous cultivation was designed and is described here. We successfully tested the proposed algorithm that was based on a combination of direct and random search minimization of squares of residuals for multistage cultivation systems. As examples, the production of neutral solvents byClostridium acetobutylicum and chlortetracycline production byStreptomyces aureofaciens were selected.     

Partial purification properties of human epidermal growth factor from recombinant Escherichia coli by expanded bed adsorption

Human epidermal growth factor is a polypeptide hormone having many diverse biological functions. This paper first presents the recovery results of human epidermal growth factor (hEGF) immediately from the fermentation broth of recombinant Escherichia coli by using an expanded bed system (a couple of STREAMLINE25 and ÄKTA explorer 100). The influences of operational conditions such as linear flow rate, gradient length of NaCl concentration, pH and sample concentration on the purification performances of hEGF in expanded and packed bed modes with STREAMLINE DEAE resin were systematically evaluated. After optimization, the practical recovery procedure in the expanded bed mode was carried out on a scaled-up system under the conditions of linear flow rates of 183 cm/h (upward) and 37 cm/h (downward), sample volume of 300 ml and column bed height of 13.8 cm which yielded a primary product of hEGF from the cell-free supernatant containing hEGF after centrifugation at 4000 rev/min for 15 min. As a result, the hEGF concentration in the product was higher than 20% (w/v), the concentration factor was greater than 4.3 and the total yield was higher than 80%, respectively. At the same time, the results of hEGF recovery by using expanded bed adsorption (EBA), packed bed chromatography (PBC) and salting out were compared. The results show that the procedure of hEGF recovery in expanded bed adsorption has some advantages over the other two procedures, because of its higher concentration factor, recovery yield, productivity, hEGF concentration in the primary product and shorter duration of purification run.     

Production of ℓ-malic acid by Paecilomyces varioti

Summary Preliminary data for production of -malic acid from calcium acetate byPaecilomyces varioti is presented. Shake flask cultures with free cells and with cells immobilised in calcium alginate beads gave comparable results, acid concentrations of approximately 8 g/l being produced after 5 days from a medium containing 4% w/w of calcium acetate. A packed bed reactor, operated as an extended batch with product recycle, produced maximum acid concentrations of 32.6 g/l, equivalent to 73% of the maximum theoretical yield, after 3 days. Evidence obtained indicated that spores were more active than mycelia in the production of malic acid.     

Extractive fermentation of lactic acid by immobilizedLactobacillus casei using ion—exchange resin

Summary A novel method of lactic acid fermentation byLactobacillus casei immobilized in Ca—alginate gels is described, in which an ion—exchange resin packed column is attached to a fermentor for separation of lactic acid from fermentative broth. The technique successfully alleviated the restriction imposed by lactic acid on bacterial growth and product formation. As compared to the conventional batch fermentation, the new fermentation technique enhanced the lactic acid productivity and sugar conversion rate from 0.328g/L·h and 88. 2% to 0.482g/L·h and 98.6%, respectively.     

Changes in microbial biomass,respiration and nutrient status of beech (Fagus sylvatica) leaf litter processed by millipedes (Glomeris marginata)

The effect of processing of beech leaf litter (Fagus sylvatica L.) of different ages by the diplopodGlomeris marginata (Villers) on status and turnover of microorganisms was investigated in the laboratory. Microbial biomass, basal respiration and metabolic quotient of litter-material from three different beechwood sites of a basalt hill forming a gradient from basalt (upper part of the hill) to limestone (lower part of the hill) were determined each season (February, May, August and November). The same microbial parameters were also measured after these litter materials had been processed byG. marginata (faecal pellets of an average age of 4 days). Short-term changes in microbial biomass and respiration in leaf material and faecal pellets from February and August were investigated after 1, 2, 5, 10, 20 and 40 days of incubation. The ergosterol content of August samples was determined. Processing of beech leaf litter byG. marginata increased microbial biomass in February and May but reduced microbial biomass in August and November. It was concluded that processing of litter materials in February and May increased accessibility of carbon resources to microorganisms by fragmentation. In contrast, in litter materials from August and November carbon resources were depleted and fragmentation by diplopods did not increase availability of carbon resources. Addition of carbon (glucose) and nutrients (nitrogen and phosphorus) to litter and faecal pellets indicated that processing of beech litter reduced nutrient deficiency of the microflora. Ergosterol content in faecal pellets was reduced strongly after beech leaf litter processing byG. marginata, indicating a decrease in fungal biomass. Presumably, in faecal pellets bacteria flourished at the expense of fungi.     

Comparison of batch, packed bed and expanded bed purification of A. niger cellulase using cellulose beads

Rigid macroporous cross-linked cellulose beads were prepared and used as a useful affinity medium for purification of A. niger cellulase from commercial preparation, in batch; packed bed and expanded bed modes. The beads bound 99% activity in both packed bed and expanded bed modes and upto 91% activity could be recovered by washing the adsorbent with 1 M phosphate buffer, pH 7.0. While batch adsorption and elution gave only 4-fold purification, packed bed operation gave 14-fold purification and expanded bed, the highest, 36-fold purification.     

Prediction of the course of continuous fermentation on the basis of analysis of the batch process

A way of predicting the conditions of product formation in a two-state continuous cultivation on the basis of analyzing the batch process is described. The prediction applies to processes where the product formation is proportional to the growth rate and where the relationship between culture activity and the external conditions is linear. Using the example of glucose oxidase production byAspergillus niger the prediction is borne out. The difference between the predicted and found values does not exceed 15%. Dedicated to Academician Ivan Málek on the occasion of his 60th birthday     

Identification of sources of microbial pathogens on cantaloupe rinds from pre-harvest to post-harvest operations

Foodborne illness outbreaks involving cantaloupes have increased dramatically in the past 15 years in the United States and other countries. The need for the identification of the microbial sources that contaminate cantaloupe rinds has been raised by various investigators. This study was undertaken to identify the agricultural, industrial and human sources of microbial contamination from the pre- to post-harvest operations of cantaloupes grown at ten different farms in southern Texas. Results indicate that irrigation water contained a wide range of microorganisms that could cause human illness and were able to survive on the rind of cantaloupes before, at, and after harvesting. Fungi, total aerobic bacteria and total coliform bacteria were not completely eliminated by chlorinated water in the disinfection tanks of the six packinghouses under investigation. There were significant (P < 0.05) reductions on rind populations of fungi and total aerobic bacteria as well as drastic reductions in total coliform bacteria on the rinds after the disinfection and rinsing steps in all packing facilities. There was no evidence of the presence of Escherichia coli O157:H7 on packed cantaloupes across packinghouses. Less than a geometric mean of 1 c.f.u. cm⁻² of salmonellae were detected on the surface of packed cantaloupes in two of the packinghouses, and approximately ten times more salmonellae were found on the packed fruit processed in the remaining packinghouses. A similar trend was observed with listeriae. Results suggested that microbial loads originating from river water may survive on the rind or may re-infest cantaloupes after the disinfection and rinsing process at the packinghouses. Disinfection techniques and aseptic handling of cantaloupes at the packing facilities need a closer evaluation to ensure a safe product.     

Yeast cells entrapped in low-gelling temperature agarose for the continuous production of ethanol

Summary Continuous ethanol production byS. uvarum immobilized in a low-gelling temperature agarose namely SeaPlaque agarose was studied in a packed bed reactor at 30°C using sugarcane molasses containing 13.5% fermentable sugars as feed. The productivity at 95% conversion was 23 g/l.h (on reactor volume basis). The bioreactor was run continuously at a fixed dilution rate and it retained 60% of its initial activity upto 80 days.     

Effects of External Mass Transfer and Product Inhibition on a Simulated Immobilized Enzyme‐Catalyzed Reactor for Lactose Hydrolysis

A mathematical model has been developed for predicting the performance and simulation of a packed bed immobilized enzyme reactor performing lactose hydrolysis, which follows Michaelis‐Menten kinetics with competitive product (galactose) inhibition. The performance characteristics of a packed bed immobilized enzyme reactor have been analyzed taking into account the effects of various diffusional phenomena like axial dispersion and external mass transfer limitations. The model design equations are then solved by Galerkin's method and orthogonal collocation on finite elements. The effects of external mass transfer and axial dispersion have been studied and their effects were shown to reduce the external effectiveness factor. The effects of product inhibition have been investigated at different operating conditions correlated at different regimes using dimensionless moduli (St, γ, θ, Da)¹⁾. The product inhibition was shown to reduce the substrate conversion, and, additionally, to decrease the effectiveness factor when Da > Da_xo, however, it increases the effectiveness factor when Da < Da_xo. The effectiveness factor is found to be independent of the product inhibition at a crossover point at which Da_xo is defined. Effects of St and Pe have been investigated at different kinetic regimes and the results show that their effects have a strong dependency on the kinetic parameters θ, γ (i.e., K_m/K_p), and Da_xo.     

Isolation of a thermotolerant bacterium producing medium-chain-length polyhydroxyalkanoate

Aims:  The aim of this study was to isolate a thermotolerant micro‐organism that produces polyhydroxyalkanoates (PHAs) composed of medium‐chain‐length (mcl) HA units from a biodiesel fuel (BDF) by‐product as a carbon source. Methods and Results:  We successfully isolated a thermotolerant micro‐organism, strain SG4502, capable to accumulate mcl‐PHA from a BDF by‐product as a carbon source at a cultivation temperature of 45°C. The strain could also produce mcl‐PHA from acetate, octanoate and dodecanoate as sole carbon sources at cultivation temperatures up to 55°C. Taxonomic studies and 16S rRNA gene sequence analysis revealed that strain SG4502 was phylogenetically affiliated with species of the genus Pseudomonas. This study is the first report of PHA synthesis by a thermotolerant Pseudomonas. Conclusions:  A novel thermotolerant bacterium capable to accumulate mcl‐PHA from a BDF by‐product was successfully isolated. Significance and Impact of the Study:  A major issue regarding industrial production of microbial PHAs is their much higher production cost compared with conventional petrochemical‐based plastic materials. Especially significant are the cost of a fermentative substrate and the running cost to maintain a temperature suitable for microbial growth. Thus, strain SG4502, isolated in this study, which assimilates BDF by‐product and produces PHA at high temperature, would be very useful for practical application in industry.     

Packing of large‐scale chromatography columns with irregularly shaped glass based resins using a stop‐flow method

Rigid chromatography resins, such as controlled pore glass based adsorbents, offer the advantage of high permeability and a linear pressure‐flow relationship irrespective of column diameter which improves process time and maximizes productivity. However, the rigidity and irregularly shaped nature of these resins often present challenges in achieving consistent and uniform packed beds as formation of bridges between resin particles can hinder bed consolidation. The standard flow‐pack method when applied to irregularly shaped particles does not yield well‐consolidated packed beds, resulting in formation of a head space and increased band broadening during operation. Vibration packing methods requiring the use of pneumatically driven vibrators are recommended to achieve full packed bed consolidation but limitations in manufacturing facilities and equipment may prevent the implementation of such devices. The stop‐flow packing method was developed as an improvement over the flow‐pack method to overcome these limitations and to improve bed consolidation without the use of vibrating devices. Transition analysis of large‐scale columns packed using the stop‐flow method over multiple cycles has shown a two‐ to three‐fold reduction of change in bed integrity values as compared to a flow‐packed bed demonstrating an improvement in packed bed stability in terms of the height equivalent to a theoretical plate (HETP) and peak asymmetry (A_s). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1319–1325, 2014     

A structured approach to design and operation of biotransformation processes

A structured approach to design and operation of biotransformation (bioconversion) processes, based on previous case studies, has been developed. This requires knowledge of the key characteristics of a biotransformation which determine the constraints on process selection. The approach is illustrated for five biotransformations, two enzymic and three microbial. Some generic problems such as low water solubility and volatility of reactants, reactant and product toxicity have been identified. The microblal oxidations of toluene and fluorobenzene to toluenecis-glycol and fluorocatechol respectively byPseudomonas putida have been used to illustrate how these constraints may be overcome by addition of tetradecane as a second liquid phase, use of a membrane oxygenator and introduction ofin situ product removal.