Comparison of Electrode Reduction Activities of Geobacter sulfurreducens and an Enriched Consortium in an Air-Cathode Microbial Fuel Cell

An electricity-generating bacterium, Geobacter sulfurreducens PCA, was inoculated into a single-chamber, air-cathode microbial fuel cell (MFC) in order to determine the maximum electron transfer rate from bacteria to the anode. To create anodic reaction-limiting conditions, where electron transfer from bacteria to the anode is the rate-limiting step, anodes with electrogenic biofilms were reduced in size and tests were conducted using anodes of six different sizes. The smallest anode (7 cm², or 1.5 times larger than the cathode) achieved an anodic reaction-limiting condition as a result of a limited mass of bacteria on the electrode. Under these conditions, the limiting current density reached a maximum of 1,530 mA/m², and power density reached a maximum of 461 mW/m². Per-biomass efficiency of the electron transfer rate was constant at 32 fmol cell⁻¹ day⁻¹ (178 μmol g of protein⁻¹ min⁻¹), a rate comparable to that with solid iron as the electron acceptor but lower than rates achieved with fumarate or soluble iron. In comparison, an enriched electricity-generating consortium reached 374 μmol g of protein⁻¹ min⁻¹ under the same conditions, suggesting that the consortium had a much greater capacity for electrode reduction. These results demonstrate that per-biomass electrode reduction rates (calculated by current density and biomass density on the anode) can be used to help make better comparisons of electrogenic activity in MFCs.     

Simultaneous wastewater treatment,electricity generation and biomass production by an immobilized photosynthetic algal microbial fuel cell

A photosynthetic algal microbial fuel cell (PAMFC) was constructed by the introduction of immobilized microalgae (Chlorella vulgaris) into the cathode chamber of microbial fuel cells to fulfill electricity generation, biomass production and wastewater treatment. The immobilization conditions, including the concentration of immobilized matrix, initial inoculation concentration and cross-linking time, were investigated both for the growth of C. vulgaris and power generation. It performed the best at 5 % sodium alginate and 2 % calcium chloride as immobilization matrix, initial inoculation concentration of 10⁶ cell/mL and cross-linking time of 4 h. Our findings indicated that C. vulgaris immobilization was an effective and promising approach to improve the performance of PAMFC, and after optimization the power density and Coulombic efficiency improved by 258 and 88.4 %, respectively. Important parameters such as temperature and light intensity were optimized on the performance. PAMFC could achieve a COD removal efficiency of 92.1 %, and simultaneously the maximum power density reached 2,572.8 mW/m³ and the Coulombic efficiency was 14.1 %, under the light intensity of 5,000 lux and temperature at 25 °C.     

A photochemical fuel cell system using Anabaena N-7363

Summary A blue-green algae, Anabaena N-7363, was immobilized in 2% agar gel. The hydrogen productivity of the immobilized algae was three times higher than that of free algae. The maximum hydrogen production rate by the immobilized blue-green algae was 0.52 moles h^–1 g^–1 (of wet gel) in the medium without nitrogen sources under illumination (10,000 lux). The oxygen evolved was then removed by a reactor containing aerobic bacteria. A photo-current of 15–20 mA was continuously produced for 7 days by the photochemical fuel cell system consisting of the immobilized Anabaena reactor, the oxygen-removing reactor and the hydrogen-oxygen fuel cell. The conversion ratio of hydrogen to current was from 80% to 100%.     

Studies on sugar isomerases of Lactobacillus sp.: Whole cell immobilization of d-glucose isomerase

A new soil isolate of Lactobacillus sp. grown in Yamanaka medium under submerged conditions showed the presence of d-glucose, d-xylose and d-ribose isomerases in washed cell suspension and cell free extracts. d-Xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5) and d-ribose isomerase (d-ribose ketol-isomerase, EC 5.3.1.20) activities reached a maximum in 48 h of growth and then declined. d-Glucose isomerase (d-glucose 6-phosphate isomerase, d-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) activity was maximum after 72 h and remained constant for ～120 h of growth. d-Glucose isomerase activity increased with the increase in number of generations of culture and reached a maximum in 5–6 generations, whereas d-xylose and d-ribose isomerase activities decreased. The washed and starved whole cells could be heat treated and immobilized on the rough surface of glass rods or glass slides using acetone treatment. The heat treated immobilized cells showed only the presence of d-glucose isomerase activity and showed no d-xylose and d-ribose isomerase activities. d-Glucose isomerase activity of heat treated immobilized cells was inhibited less by sorbitol, mannitol, sodium arsenate, cysteine and calcium ions than the free d-glucose isomerase activity in fresh untreated washed whole cells and cell free extracts. EDTA inhibition had the same effect for both forms. Ca²⁺inhibition could be reversed by adding Mg²⁺ions.     

Photochemical energy conversion using immobilized blue-green algae

A blue-green alga, Anabaena N-7363, was immobilized in 2% κ-carrageenan gel. The hydrogen productivity of the immobilized algae was 2.4 times higher than that of free algae, with a maximum rate of hydrogen production of 3.24 mmol h⁻¹ g⁻¹ dry gel, in a nitrogen free medium under illumination (6000 lux). The immobilized blue-green algae (39 kg wet gel) was employed for continuous production of hydrogen under illumination (6000 lux), producing 0.5–1.1 ml min⁻¹ for more than 8 days. The hydrogen produced was supplied to a phosphoric acid fuel cell, which generated an approximate 50 mW power output and a current of 300 mA over a period of 4 h.     

Microorganism-immobilized carbon nanoparticle anode for microbial fuel cells based on direct electron transfer

A fast and convenient bacterial immobilization method was proposed as an attempt to improve the anode efficiency of a microbial fuel cell, in which bacteria were entrapped into carbon nanoparticle matrix. The direct electron transfer from the entrapped bacterial cells to the anode was verified using cyclic voltammogram (CV). Using the immobilized bioanode, the start-up time of the MFC was greatly reduced. Meanwhile, the maximum power density of 1,947 mW m⁻² with the modified anode was much higher than that with the biofilm-based carbon cloth anode (1,479 mW m⁻²). Impedance measurements suggested that performance improvement resulted from the decrease in charge transfer and diffusion resistances. The results demonstrated that bacteria immobilization using carbon nanoparticle matrix was a simple and efficient approach for improving the anodes performances in MFCs.     

The effect of Mn2+ on ethanol production from lactose using Kluyveromyces marxianus IMB3 immobilized in magnetically responsive matrices

The thermotolerant, ethanol producing yeast strain, K. marxianus IMB3 was immobilized in calcium alginate containing magnetically responsive Fe₃O₄ particles. In these studies the β-galactosidase derived from K. marxianus IMB3 was immobilized onto the Fe₃O₄ particles prior to inclusion into the alginate matrix. Ethanol production by the immobilized microorganism in the presence of Fe₃O₄ reached a maximum of 16?g/L on 40?g/L lactose whereas prior immobilization of the enzyme to the particles and inclusion into the alginate matrix increased ethanol production to a maximum concentration of 18 g/L. When Mn²⁺ was incorporated into fermentations containing the immobilized enzyme in the alginate matrix, ethanol production increased further to a maximum concentration of 20?g/L. In addition, the behaviour of the magnetically responsive biocatalyst containing the co-immobilized enzyme was examined in a batch-fed system in the presence and absence of Mn²⁺.     

Continuous biodegradation of parathion by immobilized Sphingomonas sp. in magnetically fixed-bed bioreactors and evaluation of the enzyme stability of immobilized bacteria

Magnetically-modified Sphingomonas sp. was prepared using covalent binding of magnetic nanoparticles on to the cell surface. The magnetic modified bacteria were immobilized in the fixed-bed bioreactors (FBR) by internal and external magnetic fields for the biodetoxification of a model organophosphate, parathion: 93 % of substrate (50 mg parathion/l) was hydrolyzed at 0.5 ml/min in internal magnetic field fixed-bed bioreactor. The deactivation rate constants (at 1 ml/min) were 0.97 × 10^?3, 1.24 × 10^?3 and 4.17 × 10^?3 h^?1 for immobilized bacteria in external and internal magnetic field fixed-bed bioreactor and FBR, respectively. The deactivation rate constant for immobilized magnetically modified bacteria in external magnetic field fixed-bed bioreactor (EMFFBR) was 77 % lower than that of immobilized cells by entrapping method on porous basalt beads in FBR at 1 ml/min. Immobilized magnetic modified bacteria exhibited maximum enzyme stability in EMFFBR.     

Cell immobilization in composite agar layer microporous membrane structures: growth kinetics of gel-entrapped cultures and cell leakage limitation by a microporous membrane

Agar discs containing different amounts of viable Escherichia coli cells (from 10 to 10⁶ organisms·g^–1 agar) were incubated in a nutrient medium and the growth of agar-entrapped bacteria and free (released) cells was monitored. The study was repeated with composite immobilized-cell structures obtained by placing a microporous membrane filter between the gel matrix and the incubation medium. In both cases, immobilized cells grew exponentially and reached a peak concentration an order of magnitude higher than that of free (suspended) cell cultures. The maximum specific growth rates of entrapped bacteria, ranging between 0.0115 min^–1 and 0.0145 min^–1, i.e., slightly higher than that of control free cultures (0.011 min^–1), showed no clear dependence on the initial cell loading (ICL). The microporous filter proved efficient in limiting cell leakage since it noticeably lengthened the leakage time at a given ICL. This efficiency, however, decreased at high ICL and high growth rate of immobilized organisms. Correspondence to: G.-A. Junter     

Quantitative Microbiological Analysis of Bacterial Community Shifts in a High-Rate Anaerobic Bioreactor Treating Sulfite Evaporator Condensate

The bacterial population of a high-rate, anaerobic, fixed-bed loop reactor treating sulfite evaporator condensate from the pulp industry was studied over a 14-month period. This period was divided into seven cycles that included a startup at the beginning of each cycle. Some 82% of the total biomass was immobilized on and between the porous glass rings filling the reactor. The range of the total number of microorganisms in these biofilms was 2 × 10⁹ to 7 × 10⁹ cells per ml. Enumeration and characterization by microbiological methods and by phase-contrast, epifluorescence, and electron microscopy showed that the samples consisted mainly of the following methanogens: a Methanobacterium sp., a Methanosarcina sp., a Methanobrevibacter sp., and a Methanothrix sp., as well as furfural-degrading sulfate-reducing bacteria resembling Desulfovibrio furfuralis. Viable counts of hydrogenotrophic methanogens were relatively stable (mostly within the range of 3.2 × 10⁸ to 7.5 × 10⁸ cells per ml), but Methanobrevibacter cells increased from <5 to 30% of the total hydrogenotrophic count after transfer of the fixed bed into a second reactor vessel. Acetotrophic methanogens reached their highest numbers of 1.3 × 10⁸ to 2.6 × 10⁸ cells per ml in the last fermentation cycles. They showed a morphological shift from sarcinalike packets in early samples to single coccoid forms in later phases of the fermentation. Furfural-degrading sulfate reducers reached counts of 1 × 10⁷ to 5.8 × 10⁷ cells per ml. The distribution of the chief metabolic groups between free fluid and biofilms was analyzed in the fifth fermentation cycle: 4.5 times more furfural degraders were found in the free fluid than in the biofilms. In contrast, 5.8 times more acetotrophic and 16.6 times more hydrogenotrophic methanogens were found in the biofilms than in the free liquid. The data concerning time shifts of morphotypes among the trophic groups of methanogens corroborated the trends observed by using immunological assays on the same samples.     

Shewanella putrefaciens Adhesion and Biofilm Formation on Food Processing Surfaces

Laboratory model systems were developed for studying Shewanella putrefaciens adhesion and biofilm formation under batch and flow conditions. S. putrefaciens plays a major role in food spoilage and may cause microbially induced corrosion on steel surfaces. S. putrefaciens bacteria suspended in buffer adhered readily to stainless steel surfaces. Maximum numbers of adherent bacteria per square centimeter were reached in 8 h at 25°C and reflected the cell density in suspension. Numbers of adhering bacteria from a suspension containing 10⁸ CFU/ml were much lower in a laminar flow system (modified Robbins device) (reaching 10² CFU/cm²) than in a batch system (reaching 10⁷ CFU/cm²), and maximum numbers were reached after 24 h. When nutrients were supplied, S. putrefaciens grew in biofilms with layers of bacteria. The rate of biofilm formation and the thickness of the film were not dependent on the availability of carbohydrate (lactate or glucose) or on iron starvation. The number of S. putrefaciens bacteria on the surface was partly influenced by the presence of other bacteria (Pseudomonas fluorescens) which reduced the numbers of S. putrefaciens bacteria in the biofilm. Numbers of bacteria on the surface must be quantified to evaluate the influence of environmental factors on adhesion and biofilm formation. We used a combination of fluorescence microscopy (4′,6′-diamidino-2-phenylindole staining and in situ hybridization, for mixed-culture studies), ultrasonic removal of bacteria from surfaces, and indirect conductometry and found this combination sufficient to quantify bacteria on surfaces.     

Steroid transformation using magnetically immobilized Mycobacterium sp.

Mycobacterium sp. (NRRL-B 3683) has been immobilized by adhesion of magnetic materials of submicron size to the bacterial surface. Preparations based on laboratory-prepared magnetic oxide that had been derivatized with hydrophobic octyltrichlorosilane showed the best properties. The magnetically immobilized bacteria were used for side-chain degradation of cholesterol into androsta-1,4-diene-3,17-dione. The magnetic bacteria behaved as free cells in the transformation media and no mass transfer limitations were observed. The magnetic bacteria could be used repeatedly without any cell loss, the cells being retrieved at the end of each transformation cycle by a magnet.     

Continuous production of lignin peroxidase by immobilized Phanerochaete chrysosporium in a pilot scale bioreactor

Lignin peroxidase was continuously produced by nylon-web or polyurethane immobilized Phanerochaete chrysosporium ATCC 24725 in a modified Biostate E® bioreactor, agitated either with the air and oxygen flow alone or in combination with a mechanical stirrer. Lignin peroxidase production started rapidly, and activities as high as ∼600 U l⁻¹ were reached as early as in 3 days. At best a total activity yield of ∼10 000 U was obtained during one week's continuous production with the maximum activity of ∼750 U l⁻¹ with nylon-web immobilized fungus, veratryl alcohol as an activator, and 2,2-dimethylsuccinate as buffer, although the relatively inexpensive benzyl alcohol and sodium tartrate peformed satisfactorily as the activator and buffer, respectively.     

l-Glutamate production by protoplasts immobilized in carrageenan gel

Protoplastization of Brevibacterium flavum cultured in a medium containing 50 μg l⁻¹ and 5 units penicillin per ml was performed by lysozyme treatment. The protoplasts were immobilized in various polymer matrices, such as agar, polyacrylamide, calcium alginate, and κ-carrageenan and then used for l-glutamate production from glucose and urea in a batch system. The protoplasts immobilized in κ-carrageenan gels showed the highest productivity of l-glutamate being twice that of immobilized whole cells under optimum conditions. The maximum productivity reached 2.3 mg ml⁻¹ initially. The immobilized B. flavum protoplasts could be used 8 times (192 h) for l-glutamate production retaining about 22% of the initial productivity during the last reaction.     

Conversion of Glycerol to Dihydroxyacetone by Immobilized Whole Cells of Acetobacter xylinum

Enzymatic production of dihydroxyacetone (DHA) was studied by immobilization of the whole cells of acetic acid bacteria capable of oxidizing glycerol to DHA. Acetobacter xylinum A-9 cells immobilized in a polyacrylamide gel were selected as the most favorable enzyme preparation. The enzymatic properties of immobilized cells converting glycerol to DHA were investigated and compared with those of intact cells. The optimum pH for the immobilized cells was broad (4.0 to 5.5), whereas the intact cells had a narrow pH optimum at 5.5. The thermal stability of the immobilized cells was somewhat higher than that of the intact cells. Apparent K_m values for glycerol with both intact and immobilized cells were about equal, 6.3 × 10⁻² to 6.5 × 10⁻² M. The complete conversion of glycerol to DHA was achieved within 40 h under optimum conditions, and pure crystalline DHA was readily isolated from the reaction mixture with over 80% yield.     

Dehydrogenation of cortisol by Arthrobacter simplex immobilized as supported monolayer

Arthrobacter simplex has been successfully immobilized by adhesion on glass, either by coating the support with colloidal particles of hydrous alumina or by pretreating the cells with aluminium ions. The use of glass slides as a model support has shown that a single, dense and regular layer of immobilized cells is achieved. The quantity of immobilized cells is about 7 × 10⁷ cells cm^?2. Immobilization on glass beads or glass wool packed as a bed in a column has also been successful. Transformation of cortisol to prednisolone has been tested under no-growth conditions in the absence of nutrients. The specific activity of immobilized cells is not significantly different from that of free cells. The use of a microreactor with the immobilized bacteria as biocatalyst demonstrated the feasibility of repeated use of the microorganisms.     

Biochemical energy conversion using immobilized whole cells of Clostridium butyricum

Hydrogen producing bacteria, Clostridium butyricum, were immobilized in agar gel (2 per cent). The immobilized whole cells were employed for continuous production of hydrogen from alcohol factory waste waters. The hydrogen production rate became constant above BOD 1500 ppm when hydrogen production was performed with a batch system. The immobilized whole cells continuously produced hydrogen over a 20-day period. The amount of hydrogen produced was about 6 ml/min/kg wet gels. Hydrogen produced was supplied to the hydrogen-oxygen (air) fuel cells. The maximum cell voltage of cell I and II was about 0.55 and 0.66 V respectively when the flow rate of hydrogen was 6 ml/min. The limiting current density changed from 0.4 to 40 mA/cm2 as the resistance between the electrodes changed from 1 to 100 ohmz. The fuel cell was left on for 7 days and the current from 550 to 500 mA was obtained continuously over a 7 day period.     

Lipase-mediated Transformation of Vegetable Oils into Biodiesel using Propan-2-ol as Acyl Acceptor

Propan-2-ol was used as an acyl acceptor for immobilized lipase-catalyzed preparation of biodiesel. The optimum conditions for transesterification of crude jatropha (Jatropha curcas), karanj (Pongamia pinnata) and sunflower (Helianthus annuus) oils were 10% Novozym-435 (immobilized Candida antarctica lipase B) based on oil weight, alcohol to oil molar ratio of 4:1 at 50 °C for 8 h. The maximum conversions achieved using propan-2-ol were 92.8, 91.7 and 93.4% from crude jatropha, karanj and sunflower oils, respectively. Reusability of the lipase was maintained over 12 repeated cycles with propan-2-ol while it reached to zero by 7^th cycle when methanol was used as an acyl acceptor, under standard reaction conditions. Revisions requested 22 December 2005; Revisions received 26 January 2006     

Expression of cbsA encoding the collagen-binding S-protein of Lactobacillus crispatus JCM5810 in Lactobacillus casei ATCC 393(T)

The cbsA gene encoding the collagen-binding S-layer protein of Lactobacillus crispatus JCM5810 was expressed in L. casei ATCC 393^T. The S-protein was not retained on the surface of the recombinant bacteria but was secreted into the medium. By translational fusion of CbsA to the cell wall sorting signal of the proteinase, PrtP, of L. casei, CbsA was presented at the surface, rendering the transformants able to bind to immobilized collagens.     

Production of xylanase by immobilized Trichoderma reesei SAF3 in Ca-alginate beads

In the present study, the optimum conditions for the production of xylanase by immobilized spores of Trichoderma reesei SAF3 in calcium alginate beads were determined. The operational stability of the beads during xylanase production under semi-continuous fermentation was also studied. The influence of alginate concentration (1, 2, 3, and 4%) and initial cell loading (100, 200, 300, 400, and 500 beads per flask) on xylanase production was considered. The production of xylanase was found to increase significantly with increasing concentration of alginate and reached a maximum yield of 3.12 ± 0.18 U ml⁻¹ at 2% (w/v). The immobilized cells produced xylanase consistently up to 10 cycles and reached a maximum level at the forth cycle (3.36 ± 0.2 U ml⁻¹).