Flowcell culture of Porphyromonas gingivalis biofilms under anaerobic conditions

We have developed an anaerobic biofilm culture system. The system is inexpensive, simple to use and, unlike an anaerobic glovebox, requires no dedicated space. As a test of the system, Porphyromonas gingivalis was cultured under low oxygen (1–2 ppm) and under anaerobic conditions (≤0.1 ppm O₂). In the presence of small amounts of oxygen, the organism attached and formed an initial biofilm over the course of 4 h, but the biofilm was unable to maintain its growth and had lost biomass after 18 h. Also, ambiguous results were obtained when the biofilm was stained with a viability stain. Under anaerobic conditions, the biofilm was able to continue growth — biomass was greater after 18 h than after 4 h, and the anaerobic biofilm had a less ambiguous staining pattern than did the low-O₂-grown biofilm.     

Zn-Fe LDHs改性石英砂生物膜体系对BDE-47的去除效果与机理

文章以四溴联苯醚(BDE-47)为目标污染物, 利用共沉淀法制备Zn-Fe LDHs覆膜改性石英砂基质, 在好氧、厌氧及两者交替条件下, 研究腐败希瓦氏菌CN32(Shewanella putrefaciens CN32) 在LDHs改性基质上生物膜形成过程及其对培养液中BDE-47的去除效果; 通过监测反应体系中Fe2+和H₂O₂浓度变化探讨BDE-47的生物及非生物去除机制。结果表明, LDHs改性不影响石英砂基质表面生物膜的形成, 但在好氧条件下, Zn-Fe LDHs石英砂改性基质对CN32电子传递链活性存在一定抑制作用, 而在厌氧条件下, LDHs改性会影响基质生物膜胞外聚合物(EPS)组成特性, 使多糖占比升高。无论在好氧还是厌氧条件下, 基质生物膜反应体系中EPS总浓度均显著高于纯菌CN32体系; 且在好氧与交替条件下, 基质生物膜的形成均显著提高反应体系中BDE-47的去除效果(约25%)。在交替条件下, 前3次循环(72h内)BDE-47的去除以基质吸附为主; 72h后, 生物膜吸附与生物降解共同发挥作用, 且LDHs改性基质在后期上升潜力更大。研究报道了LDHs改性基质生物膜形成特性及其对水相中PBDEs去除的潜力, 为强化人工湿地中PBDEs生物降解提供新思路。     

Decolorization of reactive dyes by mixed cultures isolated from textile effluent under anaerobic conditions

In the present study mixed cultures that could grew in the molasses media were isolated from textile dye effluent and its decolorization activity was studied in a batch system under anaerobic conditions, in order to determine the optimal conditions required for the highest decolorization activity. The optimum pH value for decolorization was determined as 8 for all the dyes tested. In the experiment with pH 8 dye decolorizations by mixed cultures were investigated at about 96.2–1031.3 mg l⁻¹ initial dye concentrations. The highest dye removal rates of mixed cultures were 94.9% for Reactive Red RB, 91.0% for Reactive Black B and 63.6% for Remazol Blue at 953.2, 864.9 and 1031.3 mg l⁻¹ initial dye concentrations respectively within 24 h incubation period. When the Reactive Red RB was used, approximately 82–98% total color removal was obtained at between 96.2 and 953.2 mg l⁻¹ initial dye concentrations after 12 h of incubation at 35 °C. These results show that our enriched mixed cultures have the potential to serve as an excellent biomass for the use in reactive dye removal from wastewaters under anaerobic conditions.     

L(-)-carnitine production using a recombinant Escherichia coli strain

The L(-)-carnitine production by biotransformation using the recombinant strain Escherichia coli pT7-5KE32 has been studied and optimized with crotonobetaine and D(+)-carnitine as substrates. A resting rather than a growing cells system for L(-)-carnitine production was chosen, crotonobetaine being the best substrate. High biocatalytic activity was obtained after growing the cells under anaerobic conditions at 37°C and with crotonobetaine or L(-)-carnitine as inducer. The growth incubation temperature (37°C) was high enough as to activate the heat-inducible λp_L promoter inserted in the plasmid pGP1-2. The best biotransformation conditions were with resting cells, under aerobiosis, with 4 g l⁻¹ and 100 mM biomass and substrate concentrations respectively. Under these conditions the biotransformation time (1 h) was shorter and the L(-)-carnitine yield (70%) higher than previously reported. Consequently productivity value (11.3 g l⁻¹h⁻¹) was highly improved when comparing with other published works. The resting cells could be reused until eight times maintaining product yield levels well over 50% that meant to increase ten times the L(-)-carnitine obtained per gram of biomass.     

Growth and biochemical characterization of microalgal biomass produced in bubble column and airlift photobioreactors: studies in fed-batch culture

Relatively large (0.19 m column diameter, 2 m tall, 0.06 m³ working volume) outdoor bubble column and airlift bioreactors (a split-cylinder and a draft-tube airlift device) were compared for monoseptic fed-batch culture of the microalga Phaeodactylum tricornutum. The three photobioreactors produced similar biomass versus time profiles and final biomass concentration (4 kg m⁻³). The maximum specific growth rate observed within a daily illuminated period in the exponential growth phase, had a value of 0.08 h⁻¹ on the third day of culture. Because of night-time losses of biomass, the specific growth rate averaged over the 4-days of exponential phase was 0.021 h⁻¹ for the three reactors.

The biomass in the vertical column reactors did not experience photoinhibition under conditions (photosynthetically active daily averaged irradiance value of 1150±52 μE m⁻² s⁻¹) that are known to cause photoinhibition in conventional thin-tube horizontal loop reactors. Because of good gas-liquid mass transfer, the dissolved oxygen concentration in the reactors at peak photosynthesis remained <120% of air saturation; thus, oxygen inhibition of photosynthesis and photo-oxidation of the biomass did not occur. Carbohydrate accumulation (up to 13% w/w) by the biomass was favored during light-limited linear growth. A declining light intensity caused a more than five-fold increase in cellular carotenoids but the chlorophylls increased only by about 2.5-fold during the course of the culture. In the stationary phase, up to 2% of the biomass was chlorophylls and carotenoids constituted up to 0.5% of the biomass dry weight.     

Spent media from cultures of environmental isolates of Escherichia coli can suppress the deficiency of biofilm formation under anoxic conditions of laboratory E. coli strains

The prevailing lifestyle of bacteria is sessile and they attach to surfaces in structures known as biofilms. In Escherichia coli, as in many other bacteria, biofilms are formed at the air-liquid interface, suggesting that oxygen has a critical role in the biofilm formation process. It has been reported that anaerobically growing E. coli laboratory strains are unable to form biofilms even after 96 h of incubation on Luria Bertani (LB) medium. After analyzing 22,000 transposon-induced and 26,000 chemically-induced mutants we failed to isolate an E. coli laboratory strain with the ability to form biofilm under anaerobic growth conditions. Notably, seven strains from a collection of E. coli isolated from different hosts and the environment had the ability to form biofilm in the absence of oxygen. Interestingly, spent medium from cultures of one strain, Souza298, can promote biofilm formation of E. coli laboratory strains growing under anaerobic conditions. Our results led us to propose that laboratory E. coli strains do not release (or synthesize) a molecule needed for biofilm formation under anoxic conditions but that they bear all the required machinery needed for this process.     

Microbial degradation of hydrocarbons in soil during aerobic/anaerobic changes and under purely aerobic conditions

Microbial hydrocarbon degradation in soil was studied during periodical aerobic/anaerobic switching and under purely aerobic conditions by using a pilot-scale plant with diesel-fuel-contaminated sand. The system worked according to the percolation principle with controlled circulation of process water and aeration. Periodical switching between 4 h of aerobic and 2 h of anaerobic conditions was achieved by repeated saturation of the soil with water. Whatever the cultivation mode, less than 50% of the diesel was degraded after 650 h because the hydrocarbons were adsorbed. Contrary to expectations, aerobic/anaerobic changes neither accelerated the rate of degradation nor reduced the residual hydrocarbon content of the soil. Obviously the pollutant degradation rate was determined mainly by transport phenomena and less by the efficiency of microbial metabolism. The total mass of oxygen consumed and carbon dioxide produced was greater under aerobic/anaerobic changing than under aerobic conditions, although the mass of hydrocarbons degraded was nearly the same. As shown by an overall balance of microbial growth and by a carbon balance, the growth yield coefficient was smaller during aerobic/anaerobic changes than under aerobic conditions. Received: 25 November 1997 /  Received revision: 15 January 1998 / Accepted: 18 January 1998     

Effects of oxygen on the growth and metabolism of Actinomyces viscosus

Abstract Actinomyces viscosus is a predominant microorganism in dental plaque. It is, just as the oral Streptococcus spp., a saccharolytic and aero-tolerant organism. We have investigated the effects of oxygen on the growth and metabolism of A. viscosus . To this end A. viscosus Ut 2 was grown in a glucose limited chemostat culture on a chemically defined medium ( D = 0.2 h⁻¹) with exposure to variable amounts of oxygen. The Y_glucose increased from 62.5 g · mol⁻¹ under anaerobic conditions to 149 g · mol⁻¹ under aerobic conditions, while, concomitantly, the carbon recovery from acidic fermentation products decreased from 75% to 7%. Addition of [¹⁴C]glucose to the chemostat showed that the glucose, which was not converted to acidic fermentation products, was instead converted to carbon dioxide or used for the production of biomass. Under aerobic and anaerobic conditions identical cytochrome spectra, containing only two cytochrome b -type absorption bands, were found. It was concluded that electron transport phosphorylation probably occurs both under aerobic and anaerobic conditions. Anaerobically, fumarate served as the electron acceptor, while the high growth yields observed under aerobic conditions are likely to be explained by citric acid cycle activity coupled to electron transport phosphorylation.     

Factors affecting the production of hydrogen sulphide by Lactobacillus sake L13 growing on vacuum-packaged beef

Lactobacillus sake L13 produced hydrogen sulphide during growth at 0°C on vacuum-packaged beef of normal pH (5·6–5·8) when the packaging films used had oxygen permeabilities as high as 200 ml/m²/24 h/atm (measured at 25°C and 98% relative humidity. No hydrogen sulphide was detected when the film permeability was 300 ml/m²/24 h/atm. Sulphmyoglobin was formed whenever hydrogen sulphide was present except when the film permeability was very low (1 ml of oxygen/m²/24 h/atm). Lactobacillus sake L13 also produced hydrogen sulphide when grown on beef under anaerobic conditions at 5°C. When meat pH was high (6·4–6·6) hydrogen sulphide was first detected after incubation for 9 d. When 250 μg of glucose was added to each g of high pH meat, or when meat pH was normal (5·6–5·8), hydrogen sulphide was first detected after incubation for 18 d. The spoilage of beef by hydrogen sulphide-producing lactobacilli is more rapid when the pH of the meat is high because high-pH meat contains less glucose. Sulphmyoglobin formation and greening can be prevented by the use of packaging films of very low oxygen permeability.     

Effect of oxygen transfer rate on β-carotene production from synthetic medium by Blakeslea trispora in shake flask culture

The effect of oxygen transfer rate (OTR) on β-carotene production by Blakelsea trispora in shake flask culture was investigated. The results indicated that the concentration of β-carotene (704.1 mg/l) was the highest in culture grown at maximum OTR of 20.5 mmol/(l h). In this case, the percentage of zygospores was over 50.0% of the biomass dry weight. On the other hand, OTR level higher than 20.5 mmol/(l h) was found to be detrimental to cell growth and pigment formation. To elucidate the effect of oxidative stress on β-carotene synthesis, the accumulation of hydrogen peroxide during fermentation under different OTRs was determined. A linear response of β-carotene synthesis to the level of H₂O₂ was observed, indicating that β-carotene synthesis is stimulated by H₂O₂. However, there was an optimal concentration of H₂O₂ (2400 μM) in enhancing β-carotene synthesis. At a higher concentration of H₂O₂, β-carotene decreased significantly due to its toxicity.     

Formate oxidation by Wolinella recta ATCC 33238 with oxygen as electron acceptor

Abstract The formate oxidizing capacity of Wolinella recta ATCC 33238 was studied in relation to growth under anaerobic and microaerobic conditions. Three distinct activities could be recognized: (a) cyanide-insensitive H₂O₂-producing oxidation of formate; (b) peroxidation of formate (H₂O₂-consuming); (c) oxidation of formate via an electron transport chain with oxygen as the electron acceptor. The contribution of these different formate oxidizing components during the growth of W. recta was dependent on the extent of aeration. It is suggested that due to the relative increase in overall H₂O₂ formation at higher oxygen tensions growth of W. recta appears possible only under anaerobic and microaerobic conditions.     

Operation strategies for biohydrogen production with a high-rate anaerobic granular sludge bed bioreactor

Long-term operation for biohydrogen production with an efficient carrier-induced granular sludge bed (CIGSB) bioreactor had encountered problems with poor biomass retention at a low hydraulic retention (HRT) as well as poor mass-transfer efficiency at a high HRT or under a prolonged operation period. This work was undertaken to develop strategies enabling better biomass retention and mass-transfer efficiency of the CIGSB reactors. Supplementation of calcium ion was found to enhance mechanical strength of the granular sludge. Addition of 5.4–27.2 mg/l of Ca²⁺ also led to an over three-fold increase in biomass concentration and a nearly five-fold increase in the H₂ production rate (up to 5.1 l H₂/h/l). Two reflux strategies were utilized to enhance the mass-transfer efficiency of the CIGSB system. The liquid reflux (LR) strategy enhanced the H₂ production rate by 2.2-fold at an optimal liquid upflow velocity of 1.09 m/h, which also gave a maximal biomass concentration of ca. 22 g VSS/l. Similar optimal H₂ production rate was also obtained with the gas reflux (GR) strategy at a rate of 1.0–1.49 m/h, whereas the biomass concentration decreased to 2–7 g VSS/l and thereby the specific H₂ production rate was higher than that with LR. The operation strategies applied in this work were effective to allow stable and efficient H₂ production for nearly 100 days.     

Biotreatment of swine manure by intensive lagooning during winter

The intent of the proposed system was to develop an in situ biotreatment system for swine manure that should involved minimum handling by the farmer. A demonstration plant has been installed in the Paris region. The intensive lagooning system consists of algal ponds, daphnid ponds and a polishing fish pond working in series with a total area of 2100 m² and a total volume of 3600 m³.

The performance of the system was evaluated under winter conditions with raw decanted swine manure by measuring N-NH₄⁺ and P-PO₄³⁻ removal, pH, temperature, water oxygen, algal biomass and daphnid productions.

Results showed that low temperatures (<5°C) did not allow any significant biomass production (0·41–0·68 g dry mass/m² d). Ammonia-nitrogen, mostly stripped (98% lost by stripping), and phosphate removals were small. However, as soon as the temperature increased in spring (March), ammonia-nitrogen removal was improved with a large contribution (71%) due to stripping and the algal productivity increased to ≈ 3·5 g dry mass/m² d; CO₂ appeared to be a limiting factor.

Ammonia-nitrogen, nitrate, nitrite and orthophosphate showed marked variations that could be correlated with manure overdoses.     

Aerobic oxidation of hydrogen sulfide by Thiobacillus denitrificans

It has been demonstrated that Thiobacillus denitrificans may be readily cultured aerobically in batch and continuous flow reactors on H(2)S(g) under sulfide limiting conditions. Under these conditions sulfide concentrations in the culture medium were less than 1muM resulting in very low concentrations of H(2)S in the reactor outlet gas. Biomass yield under aerobic conditions was much lower than previously reported for anaerobic conditions, presumably because of oxygen inhibition of growth. However, biomass yield was not affected by steady state oxygen concentration in the range of 45muM-150muM. Biomass yield was also observed to be essentially independent of specific growth rate in the range of 0.030-0.053 h(-1). Indicators of reactor upset were determined and recovery from upset conditions demonstrated. Maximum loading of the biomass for H(2)S oxidation under aerobic conditions was observed to be 15.1-20.9 mmol/h/g biomass which is much higher than previously reported for aerobic conditions. Other aspects of the stoichiometry of aerobic H(2)S oxidation are also reported.     

Photodynamic activity of a new sensitizer derived from porphyrin-C60 dyad and its biological consequences in a human carcinoma cell line

The photokilling activity of a porphyrin-C₆₀ (P-C₆₀) dyad was evaluated on a Hep-2 human larynx-carcinoma cell line. This study represents the first evaluation of a dyad, with high capacity to form a photoinduced charge-separated state, to act as agent to inactivate cells by photodynamic therapy (PDT). Cell treatment was carried out with 1 μM P-C₆₀ incorporated into liposomal vesicles. No dark cytotoxicity was observed using 1 μM P-C₆₀ concentration and during long incubation time (24 h). The uptake of sensitizer into Hep-2 was studied at different times of incubation. Under these conditions, a value of 1.5 nmol/10⁶ cells was found after 4 h of incubation showing practically no change even after 24 h. The cell survival after irradiation of the cells with visible light was dependent upon light exposure level. A high photocytotoxic effect was observed for P-C₆₀, which inactivated 80% of the cells after 54 J/cm² of irradiation. Moreover, the dyad kept a high photoactivity even under argon atmosphere. Thus, depending on the microenviroment where the sensitizer is localized, this compound could produce a biological photodamage through either a ¹O₂-mediated photoreaction process or a free radical mechanism under low oxygen concentration.

The mechanism of cell death was analyzed by Hoechst-33258, toluidine blue staining, TUNEL and DNA fragmentation. Cell cultures treated for 24 h with P-C₆₀ and irradiated with a dose of 54 J/cm² showed a great amount of apoptotic cells (58%). Moreover, changes in cell morphology were analyzed using fluorescence microscopy with Hoechst-33258 under low oxygen concentration. Under this anaerobic condition, necrotic cellular death predominated on apoptotic pathway. There were more apoptotic cells under air irradiation condition than under argon irradiation condition. To determine the apoptotic pathway, caspase-3 activation was studied by caspase-3 activity detection kits. The last results showed that P-C₆₀ induced apoptosis by caspase-3-dependent pathway. These results indicated that molecular dyad, which can form a photoinduced charge-separated state, is a promising model for phototherapeutic agents and they have potential application in cell inactivation by PDT.     

Impact of liquid-to-gas hydrogen mass transfer on substrate conversion efficiency of an upflow anaerobic sludge bed and filter reactor

Efficient anaerobic degradation may be completed only under low levels of dissolved hydrogen in the liquid surrounding the microorganisms. This restraint can be intensified by the limitations of liquid-to-gas H₂ mass transfer, which results in H₂ accumulation in the bulk liquid of the reactor. Dissolved hydrogen proved to be an interesting parameter for reactor monitoring by showing a good correlation with short-chain volatile fatty acid concentration, namely propionate, which was not the case for the H₂ partial pressure. Biogas recycle was performed in a upflow anaerobic sludge bed and filter reactor. The effects of varying the ratio of recycled-to-produced gas from 2:1 (9 l/l reactor per day) to 8:1 (85 l/l reactor per day) were studied. By increasing the liquid—gas interface with biogas recycling, the dissolved hydrogen concentration could be lowered from 1.1 to 0.4 μ . Accordingly, the H₂ sursaturation factor was also reduced, leading to an important improvement of the H₂ mass transfer rate, which reached 20.86 h⁻¹ (±9.79) at a 8:1 gas recycling ratio, compared to 0.72 h⁻¹ (±0.24) for the control experiment. Gas recycling also lowered the propionate concentration from 655 to 288 mg l⁻¹ and improved the soluble chemical oxygen demand removal by 10–15%. The main problem encountered was the shorter solid retention time, which could lead to undesirable biomass washout at high gas recycling ratio. This could be circumvented by improving the reactor design to reduce the turbulence within the biomass bed.     

Biodegradation Kinetic Studies on Phenol in Internal Draft Tube (Inverse Fluidized Bed) Biofilm Reactor Using Pseudomonas fluorescens: Performance Evaluation of Biofilm and Biomass Characteristics

The bioremediation potential of Pseudomonas fluorescens was studied in an internal draft tube (inverse fluidized bed) biofilm reactor (IDTBR) under batch recirculation conditions using synthetic phenol of various concentrations (400, 600, 800, 1000, and 1200 mg/L). The performance of IDTBR was investigated and the characteristics of biomass and biofilm were determined by evaluating biofilm dry density and thickness, bioparticle density, suspended and attached biomass concentration, chemical oxygen demand, and phenol removal efficiency. Biodegradation kinetics had been studied for the suspended biomass culture and biofilm systems. Suspended biomass followed substrate inhibition kinetics, and the experimental data fitted well with the Haldane model. The correlation coefficient, R ², and root-mean-square error (RMSE) obtained for the Haldane model with respect to specific growth rate were .9389 and .00729, respectively, and with respect to specific phenol consumption rate were .9259 and .00972, respectively. It was also observed experimentally that biofilm overcame substrate inhibition effect and fitted the same to the Monod model (R ² = .9831, RMSE = .00884 for specific growth rate and R ² = .9686, RMSE = .00912 for specific phenol consumption rate).     

Oxygen responses of the free-living anaerobic amoeboflagellate Psalteriomonas lanterna

Abstract The effects of O₂ on growth of the anaerobic amoeboflagellate Psalteriomonas lanterna were studied. The organism tolerates low oxygen tensions (about 1% O₂ atm. sat.) and under these conditions growth was stimulated in mixed populations. Catalase could not be found in the cells, whereas superoxide dismutase was present. Addition of O₂ resulted in loss of the methanogenic endosymbionts and favoured the transformation to amoeba cells. Symbiont-free cells did not grow under anaerobic conditions probably due to the accumulation of H₂.     

Growth responses of <Emphasis Type="Italic">Salix gracilistyla</Emphasis> cuttings to a range of substrate moisture and oxygen availability

The objective of this study is to determine the effects of substrate moisture and oxygen availability on growth traits of Salix gracilistyla Miquel, which colonizes gravel bars along rivers, the shoot growth schedule, biomass production, and resource allocation were examined under greenhouse conditions. We used four treatments representing a range of substrate moisture and oxygen availability: drought (D), flooding with standing water (FS), flooding with running water (FR), and control without drought or flooding (C). Cuttings in D stopped flushing and had low biomass production, reduced total leaf mass, and small leaves. Under anaerobic conditions, cuttings in FS stopped flushing and had low biomass production, small root biomass, low biomass allocation to roots, shallow roots, high biomass allocation to hypertrophied lenticels, and a few small, thick leaves. Under aerobic conditions, cuttings in FR showed continuous branch elongation and flushing, large biomass production, and large leaf biomass, similar to cuttings in C, in addition to low allocation to hypertrophied lenticels and many large leaves. The growth of cuttings was not inhibited by flooding of the roots throughout the experiment unless the conditions were anaerobic. Thus, cuttings respond to water stress under low moisture conditions by reducing the transpiration area and respond to flooding under low oxygen conditions by high allocation to hypertrophied lenticels and reduced transpiration area. Plasticity in the shoot growth schedule, biomass production, and resource allocation according to moisture conditions and the ability to develop hypertrophied lenticels upon flooding allow S. gracilistyla to colonize sites in which both desiccation and flooding occur.     

High efficiency of a coupled aerobic-anaerobic recycling biofilm reactor system in the degradation of recalcitrant chloroaromatic xenobiotic compounds

Chloroaromatic compounds are xenobiotics that cause great concern. The degradation of a model molecule, 3,4-dichlorobenzoate (3,4-DCB), was studied using three aerobic (AE)-anaerobic (AN) biofilm reactor systems: a coupled aerobic-anaerobic recycle biofilm reactor (CAR) system, an in-series anaerobic-aerobic biofilm reactor (SAR) system; and an independent aerobic and anaerobic biofilm reactor (IAR) system. In all three systems the inlet substrate concentration was 2.0 g/l and the dilution rates ranged from 0.045 to 0.142 per hour. The results show that the degradation efficiency of the CAR system (expressed as dechlorination and xenobiotic disappearance efficiencies, and biomass yield), was higher at all dilution rates tested than in both SAR and IAR systems. Moreover, dechlorination and xenobiotic disappearance efficiencies for resting suspended aerobic and anaerobic cells or mixed aerobic-anaerobic growing cells under anaerobic conditions were higher than under aerobic conditions. These results suggest that a “cooperative metabolism” between aerobic and anaerobic bacteria (caused by an exchange of cells and metabolites between AE and AN reactors) in the CAR system overcame the metabolic and kinetic limitations of aerobic and anaerobic bacteria in the AE and AN reactors of IAR and SAR systems. Therefore, the degradation efficiency of persistent and recalcitrant chloroaromatic xenobiotic compounds could be enhanced by using a CAR system. Received: 1 March 1999 / Received revision: 11 May 1999 / Accepted: 16 May 1999