Plant cell immobilization in a dual hollow fiber bioreactor

Summary Lithospermum erythrorhizon was immobilized in a dual hollow fiber bioreactor (DHFBR) to maintain high cell density and to operate continuously. The cells grew well and its dry biomass density was 325 g/L of the void volume for the cell growth. Volumetric and specific productivities of phenolics were 221 mg/L.day and 0.68 mg/g.dry wt.day, respectively, which are 58 and 2 times of those of shake flask cultures.     

Hydrogen and methane production from desugared molasses using a two‐stage thermophilic anaerobic process

Hydrogen and methane production from desugared molasses by a two‐stage thermophilic anaerobic process was investigated in a series of two up‐flow anaerobic sludge blanket (UASB) reactors. The first reactor that was dominated with hydrogen‐producing bacteria of Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium aciditolerans could generate a high hydrogen production rate of 5600 mL H₂/day/L, corresponding to a yield of 132 mL H₂/g volatile solid (VS). The effluent from the hydrogen reactor was further converted to methane in the second reactor with the optimal production rate of 3380 mL CH₄/day/L, corresponding to a yield of 239 mL CH₄/g VS. Aceticlastic Methanosarcina mazei was the dominant methanogen in the methanogenesis stage. This work demonstrates that biohydrogen production can be very efficiently coupled with a subsequent step of methane production using desugared molasses. Furthermore, the mixed gas with a volumetric content of 16.5% H_2, 38.7% CO₂, and 44.8% CH₄, containing approximately 15% energy by hydrogen is viable to be bio‐hythane.     

Kinetics of CH4 production from H2 and CO2 in a hollow fiber reactor by plug flow reaction model

For microbial production of CH₄ from H₂ and CO₂, a hollow fiber reactor had been developed to increase an interfacial area between liquid and gas phases. The CH₄ production with the hollow fiber reactor was analyzed by applying a plug flow reaction model of a tubular reactor. It was possible to apply the model to the reaction of CH₄ production. The relationships between influent gas velocity, length of reactor and reaction yield were simulated by the reaction model. The plug flow reaction model was useful to design a hollow fiber bioreactor for the biomethanation of H₂ and CO₂.     

Lactic acid production by Lactobacillus delbreuckii in a hollow fiber fermenter

Summary Lactic acid was produced by viable Lactobacillus delbreuckii NRRL-B445 in a hollow fiber fermenter. Final cell densities in the fluid surrounding the fibers in the fermenter were apparently as high as 480 gms DW/L, and volumetric productivities reached 100 gms/L-hr lactic acid. The observed cell yields were appreciably lower than batch cell yields.     

Continuous ethanol production by immobilized yeast in a fluidized reactor

Summary In order to minimize the adverse effect of CO₂ gas in a packed bed immobilized yeast reactor, a fluidized bed reactor was used for the continuous production of ethanol from glucose. Immobilized yeast was prepared by entrapping whole cells of Saccharomyces cerevisiae within a Caalginate matrix. It was found that the efficiency of the ethanol production in a fluidized bed reactor was 100% better than that for a packed bed reactor system. The alcohol productivity obtained was 21 g/l/hr in a fluidized bed reactor at 94% of conversion level.     

Continuous production of acrylamide byBrevibacterium sp. immobilized in a dual hollow fiber bioreactor

Summary Acrylamide was continuously produced from acrylonitrile usingBrevibacterium sp. CHl grown and immobilized in a dual hollow fiber bioreactor of 8.0 cm³. The biomass reached as high as 200 gm/L of the space available for the cell growth. The volumetric productivity of the reactor was 88 gm/L. h and the conversion of acrylonitrile varied with acrylonitrile concentration, pH and feed rate.     

Methanogenesis in Arizona,USA dryland streams

Methanogenesis was studied in five streams of central and southern Arizona by examining the distribution of methane in interstitial water and evasion of methane in three subsystems (hyporheic, parafluvial and bank sediments). In Sycamore Creek, the primary study site (studied during summer and early autumn), methane content of interstitial water exhibited a distinct spatial pattern. In hyporheic (sediments beneath the wetted channel) and parfluvial zones (active channel sediments lateral to the wetted channel), which were well oxygenated due to high hydrologic exchange with the surface stream and had little particulate organic matter (POM), interstitial methane concentration averaged only 0.03 mgCH₄-C/L. Bank sediments (interface between the active channel and riparian zone), in contrast, which were typically vegetated, had high POM, low hydrologic exchange and concomitantly low dissolved oxygen levels, had interstitial concentration averaging 1.5 mgCH4-C/L. Methane emission from Sycamore Creek, similar to methane concentration, averaged only 3.7 mgCH₄-C·m⁻²·d⁻¹ from hyporheic and parafluvial zones as opposed to 170 mgCH₄-C·m⁻²·d⁻¹ from anoxic bank sediments. Methane in four additional streams sampled (one sampling date during late winter) was low and exhibited little spatial variation most likely due to cooler stream temperatures. Interstitial methane in parafluvial and bank sediments of all four streams ranged from only 0.005 to 0.1 mgCH₄-C/L. Similarly methane evasion was also low from these streams varying from 0 to 5.7 mgCH₄-C·m⁻²·d⁻¹. The effects of organic matter and temperature on methanogenesis were further examined by experimentally manipulating POM and temperature in stoppered flasks filled with hyporheic sediments and stream water. Methane production significantly increased with all independent variables. Methane production is greatest in bank sediments that are relatively isolated hydrologically and lowest in hyporheic and parafluvial sediments that are interactive with the surface stream.     

Bacterial Processes of the Methane Cycle in Bottom Sediments of Lake Baikal

The activity of methanogenic and methanotrophic bacteria was evaluated in bottom sediments of Lake Baikal. Methane concentration in Baikal bottom sediments varied from 0.0053 to 81.7 ml/dm³. Bacterial methane was produced at rates of 0.0004–534.7 l CH₄/(dm³ day) and oxidized at rates of 0.005–1180 l CH₄/(dm³ day). Peak methane production and oxidation were observed in Frolikha Bay near a methane vent. Methane was emitted into water at rates of 49.2–4340 l CH₄/(m² day). Rates of bacterial methane oxidation in near-bottom water layers ranged from 0.002 to 1.78 l/(l day). Methanogens and methanotrophs were found to play an important role in the carbon cycle through all layers of sediments, particularly in the areas of methane vent and gas-hydrate occurrence.     

Lysis of Micrococcus lysodeikticus cells by lysozyme covalently immobilized on the lumen of hollow fibers

Summary A lytic enzyme reactor for microbial cell lysis is described in which lysozyme is immobilized on the lumen of hemodialyzer hollow fibers using epichlorohydrin as a coupling agent. The cell suspension flows through the lumen without any hindrance where the cells are lyzed by the immobilized lysozyme efficiently. Micrococcus lysodeikticus cells at concentrations of 0.25 g/L and 5 g/L were successfully lyzed without clogging the hollow fiber. In comparison with lysozyme immobilized on submicron particles, the activity retention was at least 8 times higher.     

Effect of calcium chloride on the growth and ethanol production by free cells of Zymomonas mobilis

Summary The effect of calcium chloride concentration on the growth rate and ethanol production using free cells of Zymomonas mobilis was studied. There was no appreciable change in rates of cell mass production and ethanol formation in the medium containing upto 2g/L CaCl₂. On further increase in CaCl₂ concentration, the rates started decreasing. However, the ethanol yield decreased and biomass yield increased with increase in CaCl₂ concentration.     

Effect of enzyme additions on methane production and lignin degradation of landfilled sample of municipal solid waste

Operation of waste cells as landfill bioreactors with leachate recirculation is known to accelerate waste degradation and landfill gas generation. However, waste degradation rates in landfill bioreactors decrease with time, with the accumulation of difficult to degrade materials, such as lignin-rich waste. Although, potential exists to modify the leachate quality to promote further degradation of such waste, very little information is available in literature. The objective of this study was to determine the viability of augmenting leachate with enzymes to increase the rate of degradation of lignin-rich waste materials. Among the enzymes evaluated MnP enzyme showed the best performance in terms of methane yield and substrate (lignin) utilization. Methane production of 200 mL CH₄/g VS was observed for the MnP amended reactor as compared to 5.7 mL CH₄/g VS for the control reactor. The lignin reduction in the MnP amended reactor and control reactor was 68.4% and 6.2%, respectively.     

Biomethanation of H2 and CO2 by Methanobacterium thermoautotrophicum in membrane and ceramic bioreactors

Direct conversion of gaseous H₂ and CO₂ to CH₄ was achieved with Methanobacterium thermoautotrophicum ΔH (DSM 1053) cells fixed either on a cellulose acetate membrane or inside a porous silica-alumina ceramic support.In a membrane bioreactor with cellulose acetate (5 μmø), methane production rate increased in proportion to the contact area between the gases and the methanogen cells, giving a methane production rate of 0.75 ml CH₄/cm² contact area/h. The initial fixed-cell mass of 0.2 mg dry cell/cm² of contact area increased to 1 mg/cm² after 12 h of cultivation (steady state).In the ceramic bioreactor (cylindrical, 30 mmø × 70; av. pore size 100 μ, and porosity 79.7%), the methane production rate at steady state was 6 l CH₄/l ceramic/l. The methanogen cells grew homogeneously inside the ceramic up to 7 cm depth, and the cell density ranged from 20 to 30 mg dry cell/cm³ ceramic.     

Effect of CO2 enrichment and elevated temperature on methane emissions from rice, Oryza sativa

Methane emissions from rice grown within Temperature Gradient Greenhouse Tunnels under doubled CO₂ concentrations were 10–45 times less than emissions from control plants grown under ambient CO₂. For two cultivars of rice (cvs. Lemont and IR-72), methane emissions increased with a temperature increase of 2°, from outdoor ambient temperatures to the first cell of the ambient CO₂ tunnel (ambient temperature + 2 °C). Within both tunnels and for both cultivars methane emissions decreased with further temperature increases (from 2° to 5 °C above ambient). Carbon dioxide enrichment stimulated both above- and below-ground production. Our original hypothesis was that increased CO₂ would stimulate plant productivity and therefore stimulate methane emission, since direct linkages between these parameters have been observed. We hypothesize that CO₂ enrichment led to the attenuation of methane production due to increased delivery of oxygen to the rhizosphere because of increased root biomass and porosity. The increased root biomass due to elevated CO₂ may have more effectively aerated the soil, suppressing methane production. However, this study may be unique because the low organic content (< 1%) of the sandy soils in which the rice was grown created very little oxygen demand.     

Biomethanation: Anaerobic fermentation of CO2, H2 and CO to methane

Studies to examine the microbial fermentation of coal gasification products (CO₂, H₂ and CO) to methane have been done with a mixed culture of anaerobic bacteria selected from an anaerobic sewage digestor. The specific rate of methane production at 37°C reached 25 mmol/g cell hr. The stoichiometry for methane production was 4 mmol H₂/mol CO₂. Cell recycle was used to increase the cell concentration from 2.5 to 8.3 g/liter; the volumetric rate of methane production ran from 1.3 to 4 liter/liter hr. The biogasification was also examined at elevated pressure (450 psi) and temperature to facilitate interfacing with a coal gasifier. At 60°C, the specific rate of methane production reached 50 mmol/g cell hr. Carbon monoxide utilization by the mixed culture of anaerobes and by a Rhodopseudomonas species was examined. Both cultures are able to carry out the shift conversion of CO and water to CO₂ and hydrogen.     

Nitrogen removal from purified swine wastewater using biogas by semi-partitioned reactor

Nitrate and ammonium removal from purified swine wastewater using biogas and air was investigated in continuous reactor operation. A novel type of reactor, a semi-partitioned reactor (SPR), which enables a biological reaction using methane and oxygen in the water phase and discharges these unused gases separately, was operated with a varying gas supply rate. Successful removal of NO(3)(-) and NH(4)(+) was observed when biogas and air of 1L/min was supplied to an SPR of 9L water phase with a NO(2,3)(-)-N and NH(4)(+)-N removal rate of 0.10 g/L/day and 0.060 g/L/day, respectively. The original biogas contained an average of 77.2% methane, and the discharged biogas from the SPR contained an average of 76.9% of unused methane that was useable for energy like heat or electricity production. Methane was contained in the discharged air from the SPR at an average of 2.1%. When gas supply rates were raised to 2L/min and the nitrogen load was increased, NO(3)(-) concentration was decreased, but NO(2)(-) accumulated in the reactor and the NO(2,3)(-)-N and NH(4)(+)-N removal activity declined. To recover the activity, lowering of the nitrogen load and the gas supply rate was needed. This study shows that the SPR enables nitrogen removal from purified swine wastewater using biogas under limited gas supply condition.     

Use of sulfate reducing cell suspension bioreactors for the treatment of SO2 rich flue gases

This paper describes a novel bioscrubber concept for biological flue gas desulfurization, based on the recycling of a cell suspension of sulfite/sulfate reducing bacteria between a scrubber and a sulfite/sulfate reducing hydrogen fed bioreactor. Hydrogen metabolism in sulfite/sulfate reducing cell suspensions was investigated using batch activity tests and by operating a completely stirred tank reactor (CSTR). The maximum specific hydrogenotrophic sulfite/sulfate reduction rate increased with 10% and 300%, respectively, by crushing granular inoculum sludge and by cultivation of this sludge as cell suspension in a CSTR. Operation of a sulfite fed CSTR (hydraulic retention time 4 days; pH 7.0; sulfite loading rate 0.5–1.5 g SO ₃ ^2- l^-1 d^-1) with hydrogen as electron donor showed that high (up to 1.6 g l^-1) H₂S concentrations can be obtained within 10 days of operation. H₂S inhibition, however, limited the sulfite reducing capacity of the CSTR. Methane production by the cell suspension disappeared within 20 days reactor operation. The outcompetition of methanogens in excess of H₂ can be attributed to CO₂ limitation and/or to sulfite or sulfide toxicity. The use of cell suspensions opens perspectives for monolith or packed bed reactor configurations, which have a much lower pressure drop compared to air lift reactors, to supply H₂ to sulfite/sulfate reducing bioreactors.     

Electrolysis-enhanced anaerobic digestion of wastewater

This study demonstrates enhanced methane production from wastewater in laboratory-scale anaerobic reactors equipped with electrodes for water electrolysis. The electrodes were installed in the reactor sludge bed and a voltage of 2.8-3.5 V was applied resulting in a continuous supply of oxygen and hydrogen. The oxygen created micro-aerobic conditions, which facilitated hydrolysis of synthetic wastewater and reduced the release of hydrogen sulfide to the biogas. A portion of the hydrogen produced electrolytically escaped to the biogas improving its combustion properties, while another part was converted to methane by hydrogenotrophic methanogens, increasing the net methane production. The presence of oxygen in the biogas was minimized by limiting the applied voltage. At a volumetric energy consumption of 0.2-0.3 Wh/L_R, successful treatment of both low and high strength synthetic wastewaters was demonstrated. Methane production was increased by 10-25% and reactor stability was improved in comparison to a conventional anaerobic reactor.     

High density culture of hybridoma cells in a dual hollow fiber bioreactor

Summary Hybridoma cells were cultured for two months in the dual hollow fiber bioreactor (DHFBR) which had been successfully used for high cell density cultures of various microbial cells. In batch suspension culture the concentration of monoclonal antibody (Mab) against human Chorionic Gonadotropin (hCG) and the cell density of Alps 25-3 hybridoma cells were obtained in 30 μg/mL and 2.35×10⁶ cells/mL, respectively. The continuous culture with DHFBR produced Mab of 100–130 μg/mL for 30 days and the estimated cell density in the extracapillary space of DHFBR was 1.87×10⁸ cells/mL based on the antibody production rate. The productivity of Mab was 205 mg/day per litre of the total reactor volume while that of the batch suspension culture was only 10 mg/L day.     

Biogeochemical Processes of Methane Cycle in the Soils,Bogs, and Lakes of Western Siberia

The biogeochemical processes of methane production and oxidation were studied in the upper horizons of tundra and taiga soils and raised bogs and lake bottom sediments near the Tarko-Sale gas field in western Siberia. Both in dry and water-logged soils, the total methane concentration (in soil particles and gaseous phase) was an order of magnitude higher than in the soil gaseous phase alone (22 and 1.1 nl/cm³, respectively). In bogs and lake bottom sediments methane concentration was as high as 11 l/cm³. Acetate was the major precursor of the newly formed methane. The rate of aceticlastic methanogenesis reached 55 ng C/(cm³day), whereas that of autotrophic methanogenesis was an order of magnitude lower. The most active methane production and oxidation were observed in bogs and lake sediments, where the ¹³C values of CO₂were inversely related to the intensity of bacterial methane oxidation. Methane diffusing from bogs and lake bottom sediments showed ¹³C values ranging from –78 to –47, whereas the ¹³C value of carbon dioxide ranged from –18 to –1. In these ecosystems, methane emission comprised from 3 to 206 mg CH₄/(m²day). Conversely, the dry and water-logged soils of the tundra and taiga took up atmospheric methane at a rate varying from 0.3 to 5.3 mg CH₄/(m²day). Methane consumption in soils was of biological nature. This was confirmed by the radioisotopic method and chamber experiments, in which weighting of methane carbon was observed (the ¹³C value changed from –51 to –41).     

Methane production from dairy manure filtrate and supernatants

An anaerobic fixed-film reactor receiving screened dairy manure filtrate and supernatants was operated at 35°C and a hydraulic retention time (HRT) of 1 day. Methane production rates were very similar for both the screened slurry and supernatants. The results indicated that using supernatants from the sedimentation process could simplify the operational procedure in a methane production system. The utilization of a fixed-film reactor in methane production process could accommodate a hydraulically flushed dairy waste treatment system.