Growth and enzymatic activity of Leucoagaricus gongylophorus, a mutualistic fungus isolated from the leaf-cutting ant Atta mexicana, on cellulose and lignocellulosic biomass.

A mutualistic fungus of the leaf-cutting ant Atta mexicana was isolated and identified as Leucoagaricus gongylophorus. This isolate had a close phylogenetic relationship with L. gongylophorus fungi cultivated by other leaf-cutting ants as determined by ITS sequencing. A subcolony started with ~500 A. mexicana workers could process 2 g day-1 of plant material and generate a 135 cm3 fungus garden in 160 days. The presence of gongylidia structures of ~35 µm was observed on the tip of the hyphae. The fungus could grow without ants on semi-solid cultures with α-cellulose and microcrystalline cellulose and in solid-state cultures with grass and sugarcane bagasse, as sole sources of carbon. The maximum CO2 production rate on grass (Vmax  = 17·5 mg CO2  Lg-1  day-1 ) was three times higher than on sugarcane bagasse (Vmax  = 6·6 mg CO2  Lg-1 day-1 ). Recoveries of 32·9 mgglucose  gbiomass-1 and 12·3 mgglucose  gbiomass-1 were obtained from the fungal biomass and the fungus garden, respectively. Endoglucanase activity was detected on carboxymethylcellulose agar plates. This is the first study reporting the growth of L. gongylophorus from A. mexicana on cellulose and plant material. SIGNIFICANCE AND IMPACT OF THE STUDY: According to the best of our knowledge, this is the first report about the growth of Leucoagaricus gongylophorus, isolated from the colony of the ant Atta mexicana, on semisolid medium with cellulose and solid-state cultures with lignocellulosic materials. The maximum CO2 production rate on grass was three times higher than on sugarcane bagasse. Endoglucanase activity was detected and it was possible to recover glucose from the fungal gongylidia. The cellulolytic activity could be used to process lignocellulosic residues and obtain sugar or valuable products, but more work is needed in this direction.

Removal of odorant dimethyl disulfide under alkaline and neutral conditions in biotrickling filters.

The aim of this paper was to evaluate the performance of biotrickling filters (BTFs) for treating low concentrations of dimethyl disulfide (DMDS), using different bacterial consortia adapted to consume reduced sulfur compounds under alkaline (pH ≈ 10) or neutral (pH ≈ 7) conditions. Solubility experiments indicated that the partition of DMDS in neutral and alkaline mineral media was similar to the value with distilled water. Respirometric assays showed that oxygen consumption was around ten times faster in the neutrophilic as compared with the alkaliphilic consortium. Batch experiments demonstrated that sulfate was the main product of the DMDS degradation. Two laboratory-scale BTFs were implemented for the continuous treatment of DMDS in both neutral and alkaline conditions. Elimination capacities of up to 17 and 24 g(DMDS) m(-3) h(-1) were achieved for the alkaliphilic and neutrophilic reactors with 100% removal efficiency after an initial adaptation and biomass build-up.

Effect of VOCs and methane in the biological oxidation of the ferrous ion by an acidophilic consortium.

During the elimination of H2S from biogas in an aqueous ferric sulphate solution, volatile organic compounds (VOCs) and methane are absorbed and may have an effect on the subsequent biological regeneration of ferric ion. This study was conducted to investigate the effect of maximum concentrations of methane and some VOCs found in biogas on the ferrous oxidation of an acidophilic microbial consortium (FO consortium). The presence and impact of heterotrophic microorganisms on the activity of the acidophilic consortium was also evaluated. No effect on the ferrous oxidation rate was found with gas concentrations of 1500 mg toluene m(-3), 1400 mg 2-butanol m(-3) or 1250 mg 1,2-dichloroethane m(-3), nor with methane at gas concentrations ranging from 15-25% (v/v). A tenfold increase in VOCs concentrations totally inhibited the microbial activity of the FO consortium and the heterotrophs. The presence of a heterotrophic fungus may promote the autotrophic growth of the FO consortium.

Production of poly-ß-hydroxybutyrate (PHB) by Methylobacterium organophilum isolated from a methanotrophic consortium in a two-phase partition bioreactor.

The biodegradation of methane, a greenhouse gas, and the accumulation of poly-ß-hydroxybutyrate (PHB) were studied using a methanotrophic consortium and an isolated strain thereof. The specific rates for methane consumption were 100 and [Formula: see text] for the isolate and the consortium, respectively. Also the effect of including 10% (vv(-1)) of silicone oil in a two-phase partitioning bioreactor (TPPB) was assayed for the elimination of 1% methane in air stream. TPPB allowed a 33-45% increase of methane elimination under growing conditions. Nitrogen limitation was assayed in bioreactors to promote PHB production. Under this condition, the specific methane degradation rate remained unchanged for the consortium and decreased to [Formula: see text] for the isolated strain. The accumulated PHB in the reactor was 34% and 38% (ww(-1)) for the consortium and the isolate, respectively. The highest productivity was obtained in the TPPB and was 1.61 mg(PHB)g(x)(-1) h(-1). The CZ-2 isolate was identified as Methylobacterium organophilum, this is the first study that reports this species as being able to grow on methane and accumulate up to 57% (ww(-1)) of PHB under nitrogen limitation in microcosm experiments.

Effect of surfactant and oil additions in the biodegradation of hexane and toluene vapours in batch tests.

The biological treatment of gaseous emissions of hydrophobic volatile organic compounds (VOCs) results in low rates of elimination partially because of the low solubility of VOCs in water. Recently, the use of two-phase partition bioreactors (TPPBs) was proposed to increase the bioavailability and consequently the elimination capacities of this kind of VOC. In the present study, TPPBs operating in a batch feed mode were tested for biodegradation of hexane and toluene vapours with a microbial consortium. The results obtained were compared with single-phase systems (control experiments). The liquid phase used was silicone oil (organic phase) with the surfactant Pluronic F-68. Experiments were named F1 and F2 for one and two phases, respectively, and F(1S) and F(2S) when the surfactant was included. The maximum specific rates (S(rates)) of hydrocarbon consumption for hexane and toluene were 539 and 773 mg(hydrocarbon)/(g(protein) x h), respectively. For both substrates, the systems that showed the highest S(rates) of hydrocarbon consumption were F2 and F(2S). In experiment F(1S) the surfactant Pluronic F-68 increased the solubility of hydrocarbons in the liquid phase, but did not increase the S(rates). The maximum percentages of mineralization were 51% and 72% for hexane and toluene, respectively. The results showed that simultaneous addition of silicone oil and surfactant favours the mineralization, but not the rate ofbiodegradation, of toluene and hexane vapours.

Dimethyl sulphide degradation using immobilized Thiobacillus thioparus in a biotrickling filter.

Gaseous dimethyl sulphide (DMS) was eliminated in a biotrickling filter with Thiobacillus thioparus grown in polyurethane foam cubes as carrier material. The temperature, pH and empty bed residence time of the gas were maintained at 30 degrees C, 7.0 and 40 s, respectively. In the first 45 days, DMS loads of around 2.0 gDMS m(-3) h(-1) were fed to the BTF to adapt T. thioparus to DMS consumption, attaining close to 100% removal efficiency (RE) on day 46, and the maximum elimination capacity (EC) was 4.0 gDMS m(-3) h(-1) with a RE of 77%. The overall performance was enhanced by adding a nitrogen-enriched (9x) medium but was negatively affected by high superficial liquid velocity (8.18 m h(-1)) and high pH (>7.5). Sulphate concentrations (up to 10 g L(-1)) showed no effect. The system supported shock loads up to 58 gDMS m(-3) h(-1) with increased elimination. With nitrogen-enriched medium and a pH of 7.0 it was possible to increase the EC of DMS up to a maximum of around 23 gDMS m(-3) h(-1) with 65% RE.

Biological sulfide removal under alkaline and aerobic conditions in a packed recycling reactor.

The biological sulfide removal from wastewater caustic streams can be achieved without significant dilution by alkaliphilic microorganisms which usually show lower growth and oxidation rates as compared with acidic and neutral bacteria. To improve volumetric removal rates under alkaline condition (pH 10), an Alkaliphilic Sulfide-oxidizing Bacteria Consortium (ASBC) was studied in a Packed Recycling Reactor (PRR). A commercial Nylon fiber resulted to be a convenient packing support for biofilm development as it has high specific area and similar hydrophobic propertie. The PRR reached a maximum sulfide oxidation rate of 100 mmol L(-1) d(-1) with efficiency close to 100%, representing an enhancement of 56% from the maximum sulfide oxidation rate reached for a free cell continuous culture. Higher sulfide loading rates induced oxygen limiting conditions reducing the biological activity despite the considerable biofilm attached on the nylon fiber.

Sulfur formation and recovery in a thiosulfate-oxidizing bioreactor.

This work describes the design and performance of a thiosulfate-oxidizing bioreactor that allowed high elemental sulfur production and recovery efficiency. The reactor system, referred to as a Supernatant-Recycling Settler Bioreactor (SRSB), consisted of a cylindrical upflow reactor and a separate aeration vessel. The reactor was equipped with an internal settler and packing material (structured corrugated PVC sheets) to facilitate both cell retention and the settling of the formed elemental sulfur. The supernatant from the reactor was continuously recirculated through the aerator. An inlet thiosulfate concentration of 100 mmol l(-1) was used. The reactor system was fed with 89 mmol l(-1) d(-1) thiosulfate reaching 98 to 100% thiosulfate conversion with an elemental sulfur yield of 77%. Ninety-three percent of the produced sulfur was harvested from the bottom of the reactor as sulfur sludge. The dry sulfur sludge contained 87% elemental sulfur. The inclusion of an internal settler and packing material in the reactor system resulted in an effective retention of sulfur and biomass inside the bioreactor, preventing the oxidation of thiosulfate and elemental sulfur to sulfate in the aerator and, therefore, improving the efficiency of elemental sulfur formation and recovery.

Oxygen transfer and consumption in a thiosulfate oxidizing bioreactor with sulfur production.

AIMS: To evaluate the contribution of oxygen transfer and consumption in a sulfoxidizing system to increase the elemental sulfur yield from thiosulfate oxidation. METHODS AND RESULTS: A 10 l thiosulfate oxidizing bioreactor with suspended cells operating under microaerophilic conditions and a separated aerator with a variable volume of 0.8--1.7 l were operated with a consortium containing mainly Thiobacillus sp. that oxidizes several sulfide species to elemental sulfur and sulfate. From the gas-liquid oxygen balance, the k(L)a was estimated under different operation conditions. A k(L)a of around 200 h(-1) favoured elemental sulfur production and can serve as scale-up criterion. It was further shown that more than 50% of the oxygen fed to the system was consumed in the aerator. CONCLUSIONS: The performance of the sulfoxidizing system can be improved by controlling oxygen transfer. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed method for the k(L)a determination was based on the oxygen balance, which incorporates the oxygen concentrations measured in the liquid in steady state, reducing the interference of the response time in the traditional non-steady state methods. This approach can be used to optimize reactors where microaerophilic conditions are desirable.

Sulphide and oxygen inhibition over the anaerobic digestion of organic matter: influence of microbial immobilization type.

Two different types of microbial aggregates (granular sludge and biofilm onto a plastic matrix) were evaluated for their susceptibility to sulphide and dissolved oxygen. Their specific methanogenic and sulphate reducing activities were evaluated separately and simultaneously. Total sulphide concentrations that caused 50% loss of methanogenic activity were 800 and 1250 mg l(-1) and for sulphate reduction 750 and 860 mg l(-1) for the granular sludge and the attached biomass, respectively. Simultaneous methanogenesis and sulphate reduction resulted in an increased tolerance of the sulphate reducing process towards sulphide. Results suggest that methanogenesis in granular sludge is less resistant to sulphide than in the attached biomass structure, whereas in sulphate reduction the attached biomass exhibited a better tolerance to high concentrations of total sulphide than the granular sludge. The better sulphate reducing capacity in the attached biomass may suggest that biomass was selectively attached. The dissolved oxygen concentration that inhibited 50% the methanogenic activity was 4.9 and 6.4 mg l(-1) for the granular sludge and attached biomass, respectively. When methanogenesis and sulphate reduction were carried out simultaneously, the whole process was not affected by the supplied oxygen, as produced sulphide was used by sulphide oxidizing microorganisms thus scavenging oxygen. Results showed that the integration of anaerobic/aerobic conditions in a single bioreactor is quite possible and can be used as a good strategy for the complete transformation of sulphate to elemental sulphur.

Sulfur formation by steady-state continuous cultures of a sulfoxidizing consortium and Thiobacillus thioparus ATCC 23645.

The elemental sulfur formation by the partial oxidation of thiosulfate by both a sulfoxidizing consortium and by Thiobacillus thioparus ATCC 23645 was studied under aerobic conditions in chemostat. Steady state was attained with essentially total conversion to sulfate when the dissolved oxygen concentration was 5 mgO2 l(-1) and below a dilution rate (D) of 3.0 d(-1)for the consortium and 0.9 d(-1) for T thioparus. The consortium formed elemental sulfur in steady state under oxygen limitation. Fifty percent of the theoretical elemental sulfur yield was obtained with a dissolved oxygen concentration of 0.2 mgO2 l(-1). Growth of T thioparus was negatively affected with a concentration below 1.9 mgO2 l(-1). Consortium yield from batch cultures was 2.1 g(-1) (protein) mol(-1) (thiosulfate), which was comparable with the values obtained in the chemostat at dilution rates of 0.4 d(-1) and 1.2 d(-1). The consortium showed a maximum degradation rate of 0.105 g(thiosulfate) g(-1) (protein) min(-1) and a saturation rate for S2O3(2-) of 1.9 mM.

Pressure drop and gas distribution in compost based biofilters: medium mixing and composition effects.

The pressure drop and gas distribution in four different filter media for compost biofilters were studied as a function of three superficial loading rates of moist air and by carrying out the filter medium homogenization by mixing. The filter media used were compost, compost with cane bagasse, lava rock and aerobic sludge previously dried to 60% of water content. The pressure drop increased when lava rock and cane bagasse were used as bulking agents. The same trend was observed when water was added to the filter medium. Pressure drop tended to decrease with time as flow channels were formed inthe filter media. Tracer studies were carried out to quantify the gas distribution and the effect of channel formation. For the biofilters submitted to an airflow of 10, 40 and 70 l min(-1), an average normalized time of 0.96, 0.89 and 0.82, respectively were obtained. The results showed that channel formation was increased as the superficial loading rate was also increased. An operational practice that this work proposes and evaluates to improve gas distribution and medium moisture control is to carry out intermittent medium mixing. The medium moisture and void volume achieved under mixing condition were around 50% and 0.40, respectively with an average constant pressure drop of 11, 45 and 78 cm of water m(-1) for air velocities of 75, 300 and 525 m h(-1).

Effects of packing material on the biofiltration of benzene, toluene and xylene vapours.

Biofiltration was used to eliminate volatile organic compounds from air streams in bench-scale reactors inoculated with an adapted consortium. Organic and inert supports were tested on 100 days of operation. The supports were: peat, vermiculite, a mixture of vermiculite and activated carbon, tree bark and, porous glass Rashig rings. A mixture of benzene, toluene and xylene vapors with a load of 200 gC m(-3) h(-1) was fed to the biofilters with an empty bed residence time of 60 s. Removal efficiencies higher than 95% were obtained with the mixture of vermiculite and activated carbon, 85% for peat and bark, 80% for vermiculite and 65% for the Rashig rings. In all cases, drying problems in beds were observed after several days of operation. Water addition with or without nutrients was required to maintain and increase the performance of biofilters. In steady state operation, experiments at loads ranging from 50 to 400 gC m(-3) h(-1) were carried out and a maximum elimination capacity of 260 gC m(-3) h(-1) was obtained for vermiculite-activated carbon support. The three xylene isomers were degraded. Observations of the supports surface by scanning electronic microscopy at the end of the biofiltration experiment showed abundant growth of fungi, which were not in the inoculum, had colonized the biofilter.

Biofiltration of volatile ethanol using sugar cane bagasse inoculated with Candida utilis.

Candida utilis (C. utilis) growing on sugar cane bagasse complemented with a mineral salt solution was studied for gaseous ethanol removal in a biofilter. Ethanol loads from 93.7 to 511.9 g/h m(3) were used, by varying both inlet ethanol concentration (9.72 to 52.4 g/m(3)) and air flow rate (1.59 x 10(-3) to 2.86 x 10(-3) m(3)/h). At a loading rate of 93.7 g/h m(3), a steady-state was maintained for 300 h. Ethanol removal was complete, and 76.3% of the carbon consumed was found in carbon dioxide. At an higher aeration rate (ethanol load=153.8 g/h m(3)), the biofilter displayed an average removal efficiency (RE) of 70%, and an elimination capacity (EC) of 107.7 g/h m(3). Only 64.4% of the carbon consumed was used for CO(2) production. Acetaldehyde and ethyl acetate in the outlet gas attained 7.86 and 20.4% in terms of carbon balance, respectively. In both cases, the transient phase was less than one day. At a high inlet ethanol concentration (52.4 g/m(3)), no steady-state was observed and the process stopped during the third day. In the three cases, final biomass was poor, ranging from 10.5 to 14.8 mg/g dm. Final pH 4.0-4.6, indicated that acidifying non-volatile metabolites, such as acetate, accumulated in the reactor.

Toluene biofiltration by the fungus Scedosporium apiospermum TB1.

The performance of biofilters inoculated with the fungus Scedosporium apiospermum was evaluated. This fungus was isolated from a biofilter which operated with toluene for more than 6 months. The experiments were performed in a 2.9 L reactor packed with vermiculite or with vermiculite-granular activated carbon as packing material. The initial moisture content of the support and the inlet concentration of toluene were 70% and 6 g/m3, respectively. As the pressure drop increased from 5-40 mm H2O a strong initial growth was observed. Stable operation was maintained for 20 days with a moisture content of 55% and a biomass of 33 mg biomass/g dry support. These conditions were achieved with intermittent addition of culture medium, which permitted a stable elimination capacity (EC) of 100 g/m3(reactor)h without clogging. Pressure drop across the bed and CO2 production were related to toluene elimination. Measurement of toluene, at different levels of the biofilter, showed that the system attained higher local EC (200 g/m3(r)h) at the reactor outlet. These conditions were related to local humidity conditions. When the mineral medium was added periodically before the EC decreases, EC of approximately 258 g/m3(r)h were maintained with removal efficiencies of 98%. Under these conditions the average moisture content was 60% and 41 mg biomass/g dry support was produced. No sporulation was observed. Evaluation of bacterial content and activities showed that the toluene elimination was only due to S. apiospermum catabolism.

Influence of mixing and water addition on the removal rate of toluene vapors in a biofilter.

The effects of successive mixing (homogenization) of packing material (peat), with or without water addition, on the removal of toluene vapors in a biofilter were studied. Over a period of 50 days, an increase in the Elimination Capacity (EC) of approximately 240% was obtained by successive mixing and water additions. After each mixing, a high EC of toluene was maintained only for a short period of 3-4 days. After this time, decreased biofilter performance was observed, probably associated with the development of dried and/or clogged zones. In the long-term experiments, an attenuation of the EC recovery was observed after successive mixing. In this case, an increase of 110% over 4 months of experiment was obtained. The global reduction of EC over time could be explained by the colonization of the biofilter by filamentous fungi which was facilitated by the mixing of the packing material. The most frequently observed fungi were identified as Scedosporium sp. and Cladosporium sp.

Cometabolic biodegradation of methyl t-butyl ether by Pseudomonas aeruginosa grown on pentane.

A bacterial strain identified as Pseudomonas aeruginosa was isolated from a soil consortium able to mineralize pentane. P. aeruginosa could metabolize methyl t-butyl ether (MTBE) in the presence of pentane as the sole carbon and energy source. The carbon balance for this strain, grown on pentane, was established in order to determine the fate of pentane and the growth yield (0.9 g biomass/g pentane). An inhibition model for P. aeruginosa grown on pentane was proposed. Pentane had an inhibitory effect on growth of P. aeruginosa, even at a concentration as low as 85 micrograms/l. This resulted in the calculation of the following kinetic parameters (mumax = 0.19 h-1, Ks = 2.9 micrograms/l, Ki = 3.5 mg/l). Finally a simple model of MTBE degradation was derived in order to predict the quantity of MTBE able to be degraded in batch culture in the presence of pentane. This model depends only on two parameters: the concentrations of pentane and MTBE.

An analysis of a trickle-bed bioreactor: carbon disulfide removal.

An analysis of the local processes occurring in a trickle-bed bioreactor (TBB) with a first-order bioreaction shows that the identification of the TBB operating regime requires knowledge of the substrate concentration in the liquid phase. If the substrate liquid concentration is close to 0, the rate-controlling step is mass transfer at the gas-liquid interface; when it is close to the value in equilibrium with the gas phase, the controlling step is the phenomena occurring in the biofilm. CS2 removal rate data obtained in a TBB with a Thiobacilii consortia biofilm are analyzed to obtain the mass transfer and kinetic parameters, and to show that the bioreactor operates in a regime mainly controlled by mass transfer. A TBB model with two experimentally determined parameters is developed and used to show how the bioreactor size depends on the rate-limiting step, the absorption factor, the substrate fractional conversion, and on the gas and liquid contact pattern. Under certain conditions, the TBB size is independent of the flowing phases' contact pattern. The model effectively describes substrate gas and liquid concentration data for mass transfer and biodegradation rate controlled processes.

Microbiological and kinetic aspects of a biofilter for the removal of toluene from waste gases

Microbiological and kinetic aspects of a biofilter inoculated with a consortium of five bacteria and two yeast adapted to remove toluene vapors were investigated. Initially the toluene sorption isotherm on peat and the effect of different environmental conditions on the toluene consumption rates of this consortium were measured. The fast start-up of the biofilter and the decay in the elimination capacity (EC) were reproduced using microcosm assays with toluene successive additions. Nutrient limitation and a large degree of heterogeneity were also detected. EC values, extrapolated from microcosms, were higher than biofilter EC when it was operating close to 100% efficiency but tended to relate better as the biofilter EC diminished. In studies on the microbial evolution in the biofilter, an increase in the cell count and variation in the ecology of the consortium were noted. Bacterial counts up to 10 x 10(11) cfu/gdry peat were found in 88 days, which corresponds to about a 10(4) increase from inoculum. Observations with SEM showed a nonuniform biofilm development on the support and the presence of an extracellular material. The results obtained in this work demonstrated that activity measurement in microcosms concomitant to the biofilter operation could be an important tool for understanding, predicting and improving the biofiltration performance. Copyright 1999 John Wiley & Sons, Inc.

Start-up and the effect of gaseous ammonia additions on a biofilter for the elimination of toluene vapors.

Biotechnological techniques, including biofilters and biotrickling filters are increasingly used to treat air polluted with VOCs (Volatile Organic Compounds). In this work, the start-up, the effect of the gaseous ammonia addition on the toluene removal rate, and the problems of the heat accumulation on the performance of a laboratory scale biofilter were studied. The packing material was sterilized peat enriched with a mineral medium and inoculated with an adapted consortium (two yeast and five bacteria). Start-up showed a short adaptation period and an increased toluene elimination capacity (EC) up to a maximum of 190 g/m3/h. This was related to increased CO2 outlet concentration and temperature gradients between the packed bed and the inlet (Tm-Tin). These events were associated with the growth of the microbial population. The biofilter EC decreased thereafter, to attain a steady state of 8 g/m3/h. At this point, gaseous ammonia was added. EC increased up to 80 g/m3/h, with simultaneous increases on the CO2 concentration and (Tm-Tin). Two weeks after the ammonia addition, the new steady state was 30 g/m3/h. In a second ammonia addition, the maximum EC attained was 40 g/m3/h, and the biofilter was in steady state at 25 g/m3/h. Carbon, heat, and water balances were made through 88 d of biofilter operation. Emitted CO2 was about 44.5% of the theoretical value relative to the total toluene oxidation, but accumulated carbon was found as biomass, easily biodegradable material, and carbonates. Heat and water balances showed strong variations depending on EC. For 88 d the total metabolic heat was -181.2 x 10(3) Kcal/m3, and water evaporation was found to be 56.5 kg/m3. Evidence of nitrogen limitation, drying, and heterogeneities were found in this study.