Disruption of baker's yeast by a new bead mill

Summary A continuous high-speed bead mill of novel design (Sulzer Annu Mill 01) was tested for cell disruption of baker's yeast as a model system. The efficiency of cell disruption was evaluated for the relative amount of released protein. The effects of rotation speed, cell concentration and flow rate of cell suspension on the cell disruption were investigated. The maximum yield of released protein was found to be 2.62 kg protein/L.h. This novel design appears to be more effective than existing commercially available mills.Notations Cs cell concentration (g packed yeast/L) - F flow rate of suspension, mL/min - F_R cumulative residence time distribution - N rotation speed of the rotor (rpm) - P number of passes of suspension through mill - R amount of protein released from cell, mg/g packed yeast - R_m maximum amount of protein released, mg/g packed yeast - t time, s - mean residence time, s     

A kinetic analysis of cell disruption by bead mill. The influence of bead loading, bead size and agitator speed.

The influence of operating parameters such as bead loading, peripheral velocity and bead size on the kinetic behavior of cell disruption in a bead mill was investigated. The bead mill was equipped with a single rotating disc and operated batchwise. Analysis of the data showed that the frequency of bead collision may be correlated to the observed first-order process, applying a new concept called effective disruption volume. It was found that the first-order rate constant was proportional to the square of bead loading within the other experimental conditions examined and increased with the decrease in bead diameter. A new disruption kinetics was proposed, explaining all the observed data in terms of the frequency of bead collision and the concept of effective disruption volume. Although other types of microorganisms were not examined, the concept may well be extended to various kinds of cells.     

Characterization of E. coli cell disintegrates from a bead mill and high pressure homogenizers

The protein releases, the particle size distribution and the viscosity of disrupted E. coli suspensions from Dyno Mill KDL, Manton Gaulin 15 M-8TA and Microfluidizer M-110 were determined. The effects of these parameters on separation of the cell debris from the protein solution by centrifugation and by filtration were also examined. All three disintegration methods investigated give approximately the same protein and enzyme releases but considerably different physical properties of the cell disintegrates which influences centrifugation and filtration. The separation degree of biomass during centrifugation is only slightly affected by increasing degree of disruption (increasing protein releases) in the bead mill, while an increase in the degree of disruption in the two high pressure homogenizers drastically reduces the centrifugal degree of separation. However, increasing degrees of disruption result in shorter filtration times during filtration for all three disintegration methods. The results show further that the cell concentration only has a minor influence on protein releases in the Microfluidizer high-pressure homogenizer, while an increase in the biomass content reduces the separability of the cell disintegrate both in filtration and in centrifugation.     

Performance and microbial diversity of palm oil mill effluent microbial fuel cell

Aim: To evaluate the bioenergy generation and the microbial community structure from palm oil mill effluent using microbial fuel cell. Methods and Results: Microbial fuel cells enriched with palm oil mill effluent (POME) were employed to harvest bioenergy from both artificial wastewater containing acetate and complex POME. The microbial fuel cell (MFC) showed maximum power density of 3004 mW m^?2 after continuous feeding with artificial wastewater containing acetate substrate. Subsequent replacement of the acetate substrate with complex substrate of POME recorded maximum power density of 622 mW m^?2. Based on 16S rDNA analyses, relatively higher abundance of Deltaproteobacteria (88·5%) was detected in the MFCs fed with acetate artificial wastewater as compared to POME. Meanwhile, members of Gammaproteobacteria, Epsilonproteobacteria and Betaproteobacteria codominated the microbial consortium of the MFC fed with POME with 21, 20 and 18·5% abundances, respectively. Conclusions: Enriched electrochemically active bacteria originated from POME demonstrated potential to generate bioenergy from both acetate and complex POME substrates. Further improvements including the development of MFC systems that are able to utilize both fermentative and nonfermentative substrates in POME are needed to maximize the bioenergy generation. Significance and Impact of the Study: A better understanding of microbial structure is critical for bioenergy generation from POME using MFC. Data obtained in this study improve our understanding of microbial community structure in conversion of POME to electricity.     

Experiences with a 20 litre industrial bead mill for the disruption of microorganisms

Suspensions of several yeast strains and bacterial species were disrupted in a continuously operating industrial agitator mill of 22.7 litre internal working volume. The influence of agitator speed, flow rate, concentration of microorganisms in the slurry, packing density of glass beads and bead diameter on the disruption process was studied using baker's yeast (Saccharomyces cerevisiae). Cell disintegration was followed by assaying the appearance of protein and the activities of d-glucose-6-phosphate dehydrogenase [d-glucose-6-phosphate:NADP⁺ oxidoreductase, EC 1.1.1.49] and α-d-glucosidase [α-d-glucoside glucohydrolase, EC 3.2.1.20] in the soluble fraction. The best operating conditions for the disintegration of baker's yeast with respect to activity yield appeared to be at a rotational speed of 1100 rev/min, a flow rate of 100 litre h^?1 and a cell concentration of 40% (w/v). The location of the desired enzyme in the cell is of importance for the choice of bead diameter and packing density of the glass beads. Temperature increase and power consumption during disintegration are also strongly influenced by the bead loading in the mill. With optimized parameters, 200 kg baker's yeast can be processed per hour with a degree of disintegration >85%. The disruption process in the mill was found to be very effective for several yeast species tested, e.g. Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Candida boidinii. The usefulness of the Netzsch LME 20-mill for the disruption of bacteria species was demonstrated with Escherichia coli, Brevibacterium ammoniagenes, Bacillus sphaericus and Lactobacillus confusus. As expected, the mill capacity for bacterial disruption was significantly smaller than for the yeast. Between 10 and 20 kg per h bacteria may be processed, depending on the organism.     

Cellulose bead entrapped microbial cells for biotechnical applications

Immobilization of intact or pretreated microbial cells instead of partially purified enzymes offers several advantages. A novel method has been applied to entrap Actinoplanes missouriensis, Bacillus coagulans, Kluyveromyces fragilis, K. lactis, Saccharomyces cerevisiae, Serratia sp., and Streptomyces albus cells within α-cellulose beads, with activity recoveries of about 22 to 85%. The best results were obtained with S. albus for glucose isomerase and with S. cerevisiae for invertase. The application of entrapped glucose isomerase-active A. missouriensis cells to increase the sweetness of β-galactosidase-hydrolysed dairy products was investigated in detail. Pressure drop across the column reactor bed was negligible; the stability of the entrapped enzyme was highest at pH 7.5 in the presence of both Mg²⁺and Co²⁺, although Co²⁺could be omitted with little effect on performance; and the activity was drastically affected by Ca²⁺content of substrate due to competition with Mg²⁺. The reactor was successfully operated for more than 5 weeks for the isomerization of demineralized concentrated whey hydrolysate of 8% lactose, 23% galactose, and up to 36% added glucose to obtain a syrup of sweetness approaching that of sucrose.     

Evaluation of the potential of a bead mill for the release of intracellular bacterial enzymes

The suitability of bead mills for the release of intracellular bacterial enzymes has been studied using the Dyno-Mill Model KDL. The effect of cell concentration, bead size and agitation speed on the release of beta-lactamase from Enterobacter cloacae P99 was examined. Scale-up considerations included, the best operational values for these parameters were 1 g cell paste suspended in 2.5 ml buffer, 0.25 mm diameter glass beads and 15 ms ⁻¹ agitation speed. These conditions proved suitable for the release of enzymes from other Gram-negative bacteria in both batch and continuous processes.     

Kinetics of enzyme release from breadmaking yeast cells in a bead mill

Summary Four intracellular enzymes from two species of breadmaking yeasts- S. cerevisiae and C. boidinii- have been measured as a function of time during its disruption using a bead mill in batch operation. The amount and rate of enzyme released was dependent on its location inside the cell as well as on the kind of yeast. The maximum amount of invertase, a-D-glucosidase, alcohol dehydrogenase and fumarase was obtained at 2,5,10,15 min. respectively for S. cerevisiae. C. boidinii did not show either invertase nor a-D glucosidase activity and the maximum amount of alcohol dehydrogenase and fumarase were reached at 5 and 20 min. respectively.     

Synthesis of a mesoporous functional copolymer bead carrier and its properties for glucoamylase immobilization

A series of mesoporous and hydrophilic novel bead carriers containing epoxy groups were synthesized by modified inverse suspension polymerization. Glycidyl methacrylate and acryloyloxyethyl trimethyl ammonium chloride were used as the monomers, and divinyl benzene, allyl methacrylate, and ethylene glycol dimethacrylate as crosslinking agents, respectively. The resulting carriers were employed in the immobilization of glucoamylase (Glu) with covalent bond between epoxy groups and enzymes. The activity recovery of the three series of immobilized Glus could reach 76%, 79%, and 86%, respectively. The immobilized Glus exhibit excellent stability and reusability than that of the free ones.     

Changes in microbial and soil properties following amendment with treated and untreated olive mill wastewater

We investigated the effect of untreated and biologically treated olive mill wastewater (OMW) spreading on the soil characteristics and the microbial communities. The water holding capacity, the salinity and the content of total organic carbon, humus, total nitrogen, phosphate and potassium increased when the spread amounts of the treated or untreated OMW increased. The OMW treated soil exhibited significantly higher respiration compared to the control soil. However, the C-CO2/C(tot) ratio decreased from 1.7 in the control soil to 0.5 in the soil amended with 100 m3 ha(-1) of untreated OMW. However, it slightly decreased to 1.15 in the soil amended with 400 m3 ha(-1) of treated OMW. The treated OMW increased the total mesophylic number while the number of fungi and nitrifiers decreased. Actinomycetes and spore-forming bacteria were neither sensitive to treated nor to untreated OMW. The total coliforms increased with higher doses of treated and untreated OMW. A toxic effect of the untreated OMW appeared from 100 m3 ha(-1). This toxicity was more significant with 200 m3 ha(-1), where microflora of total mesophilic, yeasts and moulds, actinomycetes, and nitrifiers were seriously inhibited except for total coliforms and spore-forming bacteria.     

Parallelized disruption of prokaryotic and eukaryotic cells via miniaturized and automated bead mill

The application of integrated microbioreactor systems is rapidly becoming of more interest to accelerate strain characterization and bioprocess development. However, available high‐throughput screening capabilities are often limited to target extracellular compounds only. Consequently, there is a great demand for automated technologies allowing for miniaturized and parallel cell disruption providing access to intracellular measurements. In this study, a fully automated bead mill workflow was developed and validated for four different industrial platform organisms: Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Aspergillus niger. The workflow enables up to 48 parallel cell disruptions in microtiter plates and is applicable at‐line to running lab‐scale cultivations. The resulting cell extracts form the basis for quantitative omics studies where no rapid metabolic quenching is required (e.g., genomics and proteomics).     

Chemical and toxic evaluation of a biological treatment for olive-oil mill wastewater using commercial microbial formulations

Olive-oil-mill wastewater (OMW) has significant polluting properties due to its high levels of chemical oxygen demand (COD), biochemical oxygen demand (BOD), and phenols. In the present study, different commercial bacterial formulations were used in the biological treatment of OMW. COD and toxicity testing using primary consumers of the aquatic food chain (the rotifer Brachionus calyciflorus and the crustacean Daphnia magna) were employed to evaluate abatement of the organic load and reduction of the toxic potential. In addition, the four most active formulations were tested mixed pair-wise on the basis of their unique characteristics in order to evaluate the improvement of treatment. The effect of treatment was assessed by measuring COD removal, reduction of total phenols, and decreased toxicity. The results obtained with the mixed formulations showed that the maximum removal of the organic load was about 85%, whereas phenols were reduced by about 67%. The toxicity for rotifers decreased by 43% and for crustaceans by about 83%.     

微生物絮凝剂在甘蔗糖厂中的应用研究

经过初筛、复筛,从活性污泥中分离出一株富产絮凝剂并适用于甘蔗蔗汁絮凝澄清的微生物菌株A。根据生化和生理特征及光学显微镜和电镜扫描的形态观察,初步鉴定该菌株为轮枝孢属(Verticilliumsp.)。同时对菌株进行培养条件的初步实验,获得该菌株产絮凝剂的最佳培养条件为:pH6.0,28℃,140r/min,培养4d。最佳碳氮源分别为葡萄糖和牛肉膏。微生物发酵液在蔗汁沉降试验中的絮凝率为69.2%,清汁色值IU为87.4。     

The effect of freeze-thawing on the release of intracellular proteins from Escherichia coli by means of a bead mill

World Journal of Microbiology and Biotechnology -     

Scale-up impacts on mass transfer and bioremediation of suspended naphthalene particles in bead mill bioreactors

Scale-up effects on mass transfer and bioremediation of suspended naphthalene particles have been studied in 20 and 58L bead mill bioreactors and compared to data generated earlier with a laboratory scaled bioreactor. The bead mill bioreactor performance with respect to naphthalene mass transfer rate was dependent on the size and loading of the inert particles, as well as the rotational speed of the roller apparatus. The optimum operating conditions were found to be 15mm glass beads at a loading of 50% (total volume of particles/working volume of bioreactor: v/v%) and a bioreactor rotational speed of 50rpm. The highest naphthalene mass transfer coefficients obtained in the large scale system under these optimum conditions (19.6 and 22.4h(-1) for 20 and 58L vessels, respectively) were higher than those determined previously in a 2.5L bead mill bioreactor (0.7h(-1)). The acute toxicity tests indicated that the bioreactor effluent was less toxic than the untreated naphthalene suspension. Biodegradation rates obtained in these large scale bead mill bioreactors under optimum conditions (36-37.4mgL(-1)h(-1)) were higher than those achieved in the control bioreactors of similar sizes (11.4 and 11.6mgL(-1)h(-1)) but were slower than those previously determined in a 2.5L bead mill bioreactor (59-61.5mgL(-1)h(-1)). The limitation of oxygen in the large scale systems and damage of the bacterial cells due to the crushing effects of the large beads are likely contributing factors in the lower observed biodegradation rates. The optimum conditions with respect to naphthalene mass transfer might not necessarily translate to optimum performance with regard to bioremediation.     

Development of a solar-powered microbial fuel cell

Aims: To understand factors that impact solar‐powered electricity generation by Rhodobacter sphaeroides in a single‐chamber microbial fuel cell (MFC). Methods and Results: The MFC used submerged platinum‐coated carbon paper anodes and cathodes of the same material, in contact with atmospheric oxygen. Power was measured by monitoring voltage drop across an external resistance. Biohydrogen production and in situ hydrogen oxidation were identified as the main mechanisms for electron transfer to the MFC circuit. The nitrogen source affected MFC performance, with glutamate and nitrate‐enhancing power production over ammonium. Conclusions: Power generation depended on the nature of the nitrogen source and on the availability of light. With light, the maximum point power density was 790 mW m^?2 (2·9 W m^?3). In the dark, power output was less than 0·5 mW m^?2 (0·008 W m^?3). Also, sustainable electrochemical activity was possible in cultures that did not receive a nitrogen source. Significance and Impact of the Study: We show conditions at which solar energy can serve as an alternative energy source for MFC operation. Power densities obtained with these one‐chamber solar‐driven MFC were comparable with densities reported in nonphotosynthetic MFC and sustainable for longer times than with previous work on two‐chamber systems using photosynthetic bacteria.     

Modelling of a microbial fuel cell process

Summary An electrochemical model for a microbial fuel cell process is proposed here. The model was set up on the basis of the experimental results and analysis of biochemical and electrochemical processes. Simulation of the process shows that the model describes the process reasonably well. The analysis of model simulation illustrates how the current output depends on the substrate concentration, mediator concentration and other main variables. The relationship between the current output and over-voltage is revealed from the modelling study.     

Metabolic shifts: a fitness perspective for microbial cell factories

Performance of industrial microorganisms as cell factories is limited by the capacity to channel nutrients to desired products, of which optimal production usually requires careful manipulation of process conditions, or strain improvement. The focus in process improvement is often on understanding and manipulating the regulation of metabolism. Nonetheless, one encounters situations where organisms are remarkably resilient to further optimization or their properties become unstable. Therefore it is important to understand the origin of these apparent limitations to find whether and how they can be improved. We argue that by considering fitness effects of regulation, a more generic explanation for certain behaviour can be obtained. In this view, apparent process limitations arise from trade-offs that cells faced as they evolved to improve fitness. A deeper understanding of such trade-offs using a systems biology approach can ultimately enhance performance of cell factories.