Treatment of high-concentration gaseous benzene streams using a novel bioreactor system

A continuous treatment system combining a packed-bed column and a two-phase partitioning bioreactor has been designed to treat high-concentration benzene-containing gas streams. 1-Octadecene was used in a closed loop as an absorbant to scrub benzene in the counter-current column, after which it was transferred to the two-phase partitioning bioreactor to partition benzene into the 1 l aqueous phase for degradation by Klebsiella sp. The solvent was then recirculated back to the absorber. A gas stream containing 20 mg l^–1 benzene at a flow rate of 60 l h^–1 was introduced to the system, and the benzene was degraded at a biological removal efficiency of 87% at steady state.     

Influence of nitrogen on the behaviour of nickel in wheat

A glasshouse experiment was conducted to study the effect of Ni on the growth and nutrients concentration in wheat (Triticum aestivum Cv. WH 291) in the presence and absence of applied N as urea. Responses to N application were observed up to 120 g N g^–1 soil. No response to Ni was observed in the dry matter yield of wheat tops (leaves + stem) in the absence of applied N while in the presence of applied N, significant yield increases were obtained at 12.5g Ni g^–1 soil. Nickel was not toxic to wheat up to 50g Ni g^–1 soil in the presence of 120g N g^–1 soil. Nitrogen and Ni concentration in wheat tops and roots increased with increasing levels of applied N and Ni, respectively. Applied Ni had an antagonistic effect on N concentration. Similarly, N reduced the Ni concentration in the wheat tissues. Positive growth responses to Ni were associated with 22 and 15g Ni g^–1 in wheat tops, in the presence of applied N at 60 and 120g N g^–1 soil, while Ni toxicity was associated with 63, 92.5 and 112.5g Ni g^–1 in wheat tops, in the absence and presence of applied N at 60 and 120g N g^–1 soil, respectively.     

The kinetics of the degradation of chloroform and benzene in anaerobic sediment from the River Rhine

In anaerobic methanogenic sediment microcosms¹⁴C labelled chloroform was degraded mainly to carbon dioxide. At a concentration of 4 g.l^–1 the mineralization followed first order kinetics with a half life of 12 days at 10°C and 2.6 days at 20°C. At a concentration of 400 g.l^–1 the mineralization rate increased with time and followed logarithmic kinetics with a _max of 0.02.d^–1 at 10°C. The logarithmic kinetics can be explained by growth of the bacteria on the higher concentration of chloroform with a generation time of 35 days. Shaking and oxygenation did not inhibit the mineralization of chloroform, probably because of bacterial consumption of the dissolved oxygen. ¹⁴C labelled benzene was mineralized only for a small percentage to¹⁴C labelled carbon dioxide while other, not acid extractable, degradation products were formed. Under anaerobic conditions after one day when 5% of the benzene was degraded to carbon dioxide, the mineralization ceased, while the disappearance of benzene proceeded. With air in the headspace of the incubation bottles 25% of the benzene was mineralized to carbon dioxide. The anaerobic degradation of benzene at a concentration of 100 .l^–1 showed similar kinetics as the degradation at 1 g.l^–1. Hence no adaptation of the microflora in the sediment occurred during the 63 days of the experiment at 100 g.l^–1.     

H2 production with anaerobic sludge using activated-carbon supported packed-bed bioreactors

Packed-bed bioreactors containing activated carbon as support carrier were used to produce H₂ anaerobically from a sucrose-limiting medium while acclimated sewage sludge was used as the H₂ producer. The effects of bed porosity (_b) and substrate loading rate on H₂ fermentation were examined using packed beds with _b of 70–90% being operated at hydraulic retention times (HRT) of 0.5–4 h. Higher _b and lower HRT favored H₂ production. With 20 g COD l^–1 of sucrose in the feed, the optimal H₂ production rate (7.4 l h^–1 l^–1) was obtained when the bed with _b=90% was operated at HRT = 0.5 h. Flocculation of cells enhanced the retention of sludge for stable operations of the bioreactor at low HRTs. The gas products resulting from fermentative H₂ production consisted of 30–40% H₂ and 60–70% CO₂. Butyric acid was the primary soluble product, followed by propionic acid and valeric acid.     

Plant uptake and leaching of copper during EDTA-enhanced phytoremediation of repacked and undisturbed soil

EDTA-enhanced phytoremediation of copper contaminated soil was evaluated. Up to 740 g g^–1 of Na₂H₂ EDTA in solution was added to repacked soil columns, and intact cores of a sandy loam of volcanic origin, that was growing Agrostis tenuis. The soil contained up to 400 g g^–1 of copper due to a history of fungicide spraying. EDTA application increased the herbage copper concentration of the grass growing in repacked soil from 30 to 300 g g^–1, but the same application to an intact core only brought about an increase from 10 to 60 g g^–1. More copper accumulated in the herbage when the EDTA was applied in numerous small doses than in just one or two larger amounts. Calculation of the concentration of copper in the water taken up by the grass revealed this to be two orders of magnitude lower than that in the soil solution. As a result of the EDTA applications, about 100 times more copper was leached than was taken up by the herbage. This means that a strategy for managing leaching losses needs to be part of any plan for EDTA-enhanced phytoremediation.     

Volatilization of selenium from a dewatered seleniferous sediment: A field study

Summary One of the major concerns in central California (San Joaquin Valley) is the level of selenium (Se) in evaporation ponds containing agricultural drainage water. The objective of this work was to determine if volatilization of Se could be used as a bioremediation program to detoxify a saline seleniferous sediment of a dewatered evaporation pond. The dewatered sediment was rototilled, divided into subplots, and amended with various organic materials including citrus (orange) peel, cattle manure, barley straw and grape pomance. Some of the subplots were fertilized with nitrogen [(NH₄)₂SO₄] and zinc (ZnSO₄). Selenium volatilization was monitored in the field with a flux chamber system utilizing alkaline peroxide to trap the gas. Overall, the greatest emission of gaseous Se was recorded in the summer months and the lowest emission during the winter months. The background emission of volatile Se averaged 3.0 g Se h^–1 m^–2. The most effective organic amendment was cattle manure with an avg. Se emission of 54 g Se h^–1 m^–2. Composite soil samples from each subplot (upper 15 cm) were analyzed for total Se on a monthly interval during the course of this field study. After 22 months, the application of water plus tillage alone removed 32.2% of the Se content while the cattle manure treatment removed 57.8%. Among the parameters which enhanced volatilization of Se were an available C source, aeration, moisture, and high temperatures. This field study indicates promising results in detoxifying seleniferous sediments via microbial volatilization once environmental conditions have been optimized.     

Hydrocarbon production from secondarily treated piggery wastewater by the green alga Botryococcus braunii

A laboratory study was conducted on the removal of nitrogen and phosphorus from piggery wastewater during growth of Botryococcus braunii UTEX 572, together with measurements of hydrocarbon formation by the alga. The influence was tested of the initial nitrogen and phosphorus concentration on the optimum concentration range for a culture in secondarily treated piggery wastewater. A high cell density (> 7 g L^–1 d. wt) was obtained with 510 mg L^–1 NO₃-N. Growth increased with nitrogen concentration at the basal phosphorus concentration (14 mg P L^–1). The growth rate was nearly independent ( = 0.027 0.030 h^–1) of the initial phosphate concentration, except under conditions of phosphate deficiency ( = 0.019 h^–1). B. braunii grew well in piggery wastewater pretreated by a membrane bioreactor (MBR) with acidogenic fermentation. A dry cell weight of 8.5 mgL^–1 and hydrocarbon level of 0.95 gL^–1 were obtained, and nitrate was removed at a rate of 620 mg NL^–1. These results indicate that pretreated piggery wastewater provides a good culture medium for the growth and hydrocarbon production by B. braunii.     

Fate of 15N-labelled nitrogen fertilizer applied to kiwifruit (Actinidia deliciosa) vines

The fate of ¹⁵N-labelled ammonium fertilizer applied once to six-year-old field-grown kiwifruit (Actinidia deliciosa Hayward) vines was measured over three years. The three main treatments were nitrogen (N) applied singularly at 100 or 200 kg N ha^–1 in early spring (two weeks before bud burst) or split with 100 kg N ha^–1 (unlabelled) in early spring and 100 kg N ha^–1 (¹⁵N-labelled) ten weeks later. All N treatments were applied to vines with a history of either 50 or 200 kg N ha^–1 yr^–1. For three years after ¹⁵n application, components of the vines and soil (0–600 mm depth) were sampled at harvest in late autumn and the N and ¹⁵N contents determined.By the first harvest, all plant uptake of ¹⁵N had occurred and this represented 48–53% of the ¹⁵N applied. There was no significant effect of current N fertilizer treatment or of N history on ¹⁵N recovery by vines. Removal of ¹⁵N in harvested fruit was small at 5–6% in the first year and 8% over 3 years. After 2–3 years, most plant ¹⁵N occurred in the roots and this component declined only slowly over time. In contrast, there was a large temporal decline in ¹⁵N in above-ground plant components due to the annual removal in leaf fall and pruning. An associated experiment showed that when ¹⁵N-labelled prunings and leaves were mulched and returned to the soil, only about 9% was recovered by plants within 2 years. Almost all remaining mulched material had been immobilised into the soil organic N.In all treatments, about 20% of the added ¹⁵N remained in soil at the first harvest. This was almost entirely in organic fractions (<0.4% in inorganic N) and mostly in the surface 150-mm layer. The ¹⁵N content in soil changed little over time (from 20 to 17% between the first and third harvests respectively) and indicated that most of the N had been immobilised into stable humus forms.     

Adventitious shoot regeneration from leaf explants of tissue cultured and greenhouse-grown raspberry

Adventitious shoot regeneration was observed using leaf-petiole explants from shoot-proliferating cultures of Comet red raspberry (Rubus idaeus L.). A maximum regeneration rate of 70% (3.7 shoots/explant) was obtained using 4.5–9.1 M (1–2 mg l^–1) N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ) with 2.5–4.9 M (0.5–1 mg l^–1) 1H-indole-3-butanoic acid (IBA) or 2.3 M (0.5 mg l^–1) TDZ with 4.9 M (1 mg l^–1) IBA in modified Murashige-Skoog medium. TDZ was more effective than N-(phenylmethyl)-1H-purin-6-amine (BA) at promoting regeneration in combinations tested with IBA (maximum 50% regeneration rate; 1.8 shoots/explant). Variation in the agar concentration or incubation temperature, orientation or scoring of the leaf-petiole explants and use of separate leaf or petiole explants had no effect on shoot regeneration. Incubation in the dark for 1, 2 or 3 weeks prior to growth in the light did not influence the percent regeneration rate but depressed the number of adventitious shoots. Explant source, from micropropagated shoots or greenhouse-grown plants, had an effect on shoot regeneration that was genotype dependent. Only 8 of 22 (36%) raspberry cultivars were capable of regeneration from leaf explants derived from greenhouse-grown plants.     

Metabolic responses of microbiota to diesel fuel addition in vegetated soil

The effects of trees and contamination on microbial metabolic activity, especially that of hydrocarbon degrading bacteria, were compared during phytoremediation to find which conditions increase diesel fuel removal. Diesel fuel utilisation, microbial extracellular enzyme activities and utilisation of Biolog ECO plate carbon sources by soil bacteria were determined during phytoremediation experiments consisting of two separate diesel applications. Diesel fuel removal after 28 days of second diesel application was 20–30% more than after the first application 1 year earlier. Soil microbiota utilised 26–31 of the 31 Biolog ECO plate carbon sources. Carbon source utilisation profiles indicated minor differences in microbiota in soil vegetated with pine compared to microbiota in soil vegetated with poplar. The potential maximum rates of aminopeptidase activity were 10–10² M AMC/h/g dry soil prior to and after second diesel application, except 14days after the second diesel addition, where the rates were at the scale of 10³M AMC/h/g dry soil. The potential maximum rates of esterase activity were 10³–10⁴M MUF/h/g dry soil. The presence of plants did not influence the activity of esterases. The utilisation of diesel by soil bacteria in Biolog MT2 plate assay was higher in contaminated soil, especially when vegetated, than in uncontaminated soil, measured both as lag times and maximum specific utilisation rates. MT2 plate assay detected the biological response after diesel fuel addition better than general activity methods.     

Impact of picolinic acid on the chromium accumulation in fodder radish and komatsuna

Effects of picolinic acid (2-pyridinecarboxylic acid) and chromium(III) picolinate was studied on the chromium (Cr) accumulation of fodder radish (Raphanus sativus L. convar. oleiformis Pers., cv. Leveles olajretek) and komatsuna (Brassica campestris L. subsp. napus f. et Thoms. var. komatsuna Makino, cv. Kuromaru ) grown in a pot experiment. Control cultures, grown in an uncontaminated soil (UCS; humous sand with pH_KCl 7.48, sand texture with 12.4% clay+silt content, organic carbon 0.56%, CaCO₃ 2.2%, CEC 6.2 cmol_c kg^–1, Cr 10.6 mg kg^–1), accumulated low amounts of chromium (less than 5.4 g g^–1) in their roots or shoots. When this UCS was artificially contaminated with 100 mg kg^–1 Cr (CrCl₃) later picolinic acid treatment promoted the translocation of chromium into the shoots of both species. In fodder radish shoots Cr concentration reached 30.4 g g^–1 and in komatsuna shoots 44.5 g g^–1. Application of ethylene diamine tetra-acetic acid (EDTA) to this Cr contaminated soil had similar effect to picolinic acid. When the UCS was amended with leather factory sewage sediment (which resulted in 853 mg kg^–1 Cr in soil), Cr mobilization was observed only after repeated soil picolinic acid applications. From a galvanic mud contaminated soil (brown forest soil with pH_KCl 6.77, loamy sand texture with 26.6% clay+silt content, organic carbon 1.23%, CaCO₃ 0.7%, CEC 24.5 cmol_c kg^–1, Cd 5.0 mg kg^–1, Cr 135 mg kg^–1, and Zn 360 mg kg^–1) the rate of Cr mobilization was negligible, only a slight increase was observed in Cr concentration of fodder radish shoots after repeated picolinic acid treatments of soil. Presumably picolinic acid forms a water soluble complex (chromium(III) picolinate) with Cr in the soil, which promotes translocation of this element (and also Cu) into the shoots of plants. The rate of complex formation may be related to the binding forms and/or concentration of Cr in soil and also to soil characteristics (i.e. pH, CEC), since the rate of Cr translocation was the following: artificially contaminated soil > leather factory sewage sediment amended soil > galvanic mud contaminated soil. Four times repeated 10 mg kg^–1 chromium(III) picolinate application to UCS multiplied the transport of chromium to shoots, as compared to single 10 mg kg^–1 CrCl₃ treatment. This also suggests that chromium(III) picolinate is forming in the picolinic acid treated Cr-contaminated soils, and plants more readily accumulates and translocates organically bound Cr than ionic Cr. Picolinic acid promotes Cr translocation in soil-plant system. This could be useful in phytoextraction (phytoremediation) of Cr contaminated soils or in the production of Cr enriched foodstuffs.     

Effects of copper on algal communities at different current velocities

This study examines the influence of current velocity in the toxiceffect of copper in diatom-dominated biofilms grown in artificial channels.Effects on community structure, algal biomass and photosynthesis (carbonincorporation) caused by 15 g L^–1 of copperwere tested at contrasting (1 and 15 cm s^–1)velocities. Moreover, a possible threshold on the effect of copper on algalbiomass and photosynthesis related to current velocity was examined by usingprogressively increasing current velocity (1 to 50 cms^–1) at 15 g L^–1 Cu.Chlorophyll-a decreased ca. 50% as a result of addition of15 g L^–1 Cu. Chlorophyll decrease occurredearlier at 15 cm s^–1 than at 1 cms^–1 when adding 15 g L^–1Cu. Copper also caused a remarkable decrease in carbon incorporation(from 30 to ca. 50%), which was produced earlier at 15 cms^–1 (three days) than at 1 cms^–1 (seven days). Some taxa were affected by thecombination of copper and current velocity. Both Achnanthesminutissima and Stigeoclonium tenue becomedominant at 15 cm s^–1 in the presence of copper.Significant inhibition of algal growth in 15 g L^–1Cu occurred at low (1 cm s^–1) and highvelocities (50 cm s^–1), but not at intermediatevelocity (20 cm s^–1). The experiments indicatethat current velocity triggers the effect that copper has on diatom-dominatedbiofilms, and that the effect is more remarkable at low and high than atintermediate current velocities.     

Mold colonization during use of preservative-treated and untreated air filters, including HEPA filters from hospitals and commercial locations over an 8-year period (1996–2003)

High efficiency particulate arrestance (HEPA; 99.97% efficient at 0.3 m) filters, filters with ASHRAE particulate arrestance rating of 90–95% at 1 m (90–95% filters), and lower efficiency cellulosic-polyester filters from air conditioning systems in hospitals and commercial buildings were removed from the systems and examined microscopically for mold colonization. Cellulosic-type filters from systems with water entrainment problems typically were colonized, or became colonized upon incubation in moisture chambers. Species of Acremonium, Aspergillus, and Cladosporium were most common. With air filters of all types, treatment of filter media with an antimicrobial preservative tended to reduce or delay colonization. Mold colonization of HEPA and 90–95% filters was observed most often on the load surfaces, but two untreated HEPA filters were permeated with fungi, one with Aspergillus flavus, the other with Cladosporium sp. Air filters in heating, ventilating, and air conditioning (HVAC) systems, particularly those with chronic or periodic exposure to moisture, may serve as point sources for indoor molds.     

Biodegradation of nonylphenol in a continuous packed-bed bioreactor

A packed bed bioreactor, with 170 ml glass bead carriers and 130 ml medium, was tested for the removal of the endocrine disrupter, nonylphenol, with a Sphingomonas sp. The bioreactor was first continuously fed with medium saturated with nonylphenol in an attempt to simulate groundwater pollution. At best, nonylphenol was degraded by 99.5% at a feeding rate of 69 ml h^–1 and a removal rate of 4.3 mg nonylphenol day^–1, resulting in a 7.5-fold decrease in effluent toxicity according to the Microtox. The bioreactor was then fed with soil leachates at 69 ml h^–1 from artificially contaminated soil (1 g nonylphenol kg^–1 soil) and a real contaminated soil (0.19 g nonylphenol kg^–1 soil). Nonylphenol was always completely removed from the leachates of the two soils. It was removed by 99% from the artificial soil but only 62% from real contaminated soil after 18 and 20 d of treatment, respectively, showing limitation due to nonylphenol adsorption.     

Phosphate in the sediments of the Gulf of Lions (NW Mediterranean Sea), relationship with input by the river Rhone

We investigated P-input by the Rhone river into the Mediterranean Sea taking into account P trapped in the surface sediment of the Gulf of Lions. Total phosphate concentration was determined every cm in the upper 10 cm-layer of sediments sampled at 11 stations in the Gulf of Lions during two cruises (March 1998 and January 1999). Two low downward gradients, one East–West and another North–South, with distance to the Rhone river mouth were found. Except at one station, total phosphate concentration in surface sediments was found to be constant with depth down to 10 cm. Values for individual stations ranged between 400 and 700 g g^–1 with an average value of 547 g g^–1 (st. dev. = 63 g g^–1) for the whole gulf. The low variability in total-P concentration in sediments is in contrast to the large variability in suspended matter load of the river Rhone and suggests the dominance of authigenic P removal mechanisms in P burial. The total P-pool in the upper 10 cm-layer of the sediments in the gulf was estimated at 562 kt, with about 80% trapped into the shelf and 20% into the slope. Annual P-deposition was estimated as 7.2–12.4 kt y^–1, from the P-pool in the sediment and the sedimentation rates. This is equivalent to a previous estimation of the river Rhone input, estimated to be about 6.5–12.2 kt y^–1. As the Rhone is the major river flowing into the Mediterranean Sea, total P in surface sediments of the Gulf of Lions should be taken into account in P-budgets at the scale of the Mediterranean Sea.     

The Soil Flagellate Heteromita globosa Accelerates Bacterial Degradation of Alkylbenzenes through Grazing and Acetate Excretion in Batch Culture

The impact of grazing by soil flagellates Heteromita globosa on aerobic biodegradation of benzene by Pseudomonas strain PS+ was examined in batch culture. Growth of H. globosa on these bacteria obeyed Monod kinetics (_max, 0.17 ± 0.03 h^–1; K_s, 1.1 ± 0.2 × 10⁷ bacteria mL^–1) and was optimal at a bacteria/ flagellate ratio of 2000. Carbon mass balance showed that 5.2% of total [ring-U-¹⁴C]benzene fed to bacteria was subsequently incorporated into flagellate biomass. Growth-inhibiting concentrations (IC₅₀) of alkylbenzenes (benzene, toluene, ethylbenzene) were inversely related with their octanol/ water partitioning coefficients, and benzene was least toxic for bacteria and flagellates with IC₅₀ values of 4392 (± 167) M and 2770 (± 653) M, respectively. The first-order rate constant for benzene degradation (k₁, 0.48 ± 0.12 day^–1) was unaffected by the presence or absence of flagellates in cultures. However, the rate of benzene degradation by individual bacteria averaged three times higher in the presence of flagellates (0.73 ± 0.13 fmol cell^–1 h^–1) than in their absence (0.26 ± 0.03 fmol cell^–1 h^–1). Benzene degradation also coincided with higher levels of dissolved oxygen and a higher rate of nitrate reduction in the presence of flagellates (p < 0.02). Grazing by flagellates may have increased the availability of dissolved oxygen to a smaller surviving population of bacteria engaged in the aerobic reactions initiating benzene degradation. In addition, flagellates may also have increased the rate of nitrate reduction through the excretion of acetate as an additional electron donor for these bacteria. Indeed, acetate was shown to progressively accumulate in cultures where flagellates grazed on heat-killed bacteria. This study provided evidence that grazing flagellates stimulate bacterial degradation of alkylbenzenes and provide a link for carbon cycling to consumers at higher trophic levels. This may have important implications for bioremediation processes.     

Exopolysaccharide biosynthesis and related enzyme activities of the medicinal fungus, Ganoderma lucidum, grown on lactose in a bioreactor

Exopolysaccharide (EPS) production and biosynthesis were studied in Ganoderma lucidum, a fungus used in traditional Chinese medicine, grown with lactose in a bioreactor. -Galactosidase activity, which implies the existence of a lactose permease system, was induced by lactose. Lactose feeding also increased -phosphoglucomutase activity and EPS accumulation but decreased phosphoglucose isomerase activity and lactate concentration in the culture broth. A maximum cell density of 22 g l^–1 and EPS at 1.25 g l^–1 were obtained in fed-batch bioreactor culture.     

Soybean, cowpea, groundnut, and pigeonpea response to soils, rainfall, and cropping season in the forest margins of Cameroon

Yields of groundnut, the traditional grain legume grown in central and southern Cameroon and in much of the humid zone of Central Africa, are generally low. Other food legumes may provide alternatives to groundnut. On-farm experiments examined the relative yields of up to 15 pigeonpea, 10 groundnut, 7 soybean, and 4 cowpea varieties over three growing seasons in four to six rural communities. Soil analytical values and rainfall data from all seasons were used as covariates in the analysis of variance. In the first two trials, variety-within-species interactions were significant (P<0.0001 and 0.04). Groundnut var. JL-24 yielded 60% more than local groundnuts in the first season of 2000, while soybean var. TGx1838-5E, local cowpea var. `Mefak' and pigeonpea var. ICEAP 00436 outyielded several other varieties of their respective species. Comparing these selected varieties over three seasons, significant species×community and species×season effects (P<0.0001) were observed. Covariate analysis showed that soybean yields increased with increasing soil Mg saturation and P levels. Groundnut yielded more in the first season of 2000 compared to the second seasons of 1999 and 2000 (average yields of 927 kg ha^–1 vs. 422 and 522 kg ha^–1, respectively). Improved yields were related to soil exchangeable Ca levels greater than 5 cmol(+) kg^–1 in both second seasons, but not during the first season. Cowpea yields were superior in both second seasons. Pigeonpea yields were unrelated to soil factors, showing its wide adaptability to soil conditions. Pigeonpea, which matured in February during the dry season, was severely affected by the early cessation of rains in 2000. In 1999 yields averaged 820 kg ha^–1 across communities. The results show that good food legume alternatives to groundnut exist, particularly for second season production. Species can be targeted to communities based on soil properties and season of production.     

Degradation of organic pollutants by methane grown microbial consortia

Microbial consortia were enriched from various environmental samples with methane as the sole carbon and energy source. Selected consortia that showed a capacity for co-oxidation of naphthalene were screened for their ability to degrade methyl-tert-butyl-ether (MTBE), phthalic acid esters (PAE), benzene, xylene and toluene (BTX). MTBE was not removed within 24 h by any of the consortia examined. One consortium enriched from activated sludge (AAE-A2), degraded PAE, including (butyl-benzyl)phthalate (BBP), and di-(butyl)phthalate (DBP). PAE have not previously been described as substrates for methanotrophic consortia. The apparent K_m and V_max for DBP degradation by AAE-A2 at 20 °C was 3.1 ± 1.2 mg l^–1 and 8.7 ± 1.1 mg DBP (g protein × h)^–1, respectively. AAE-A2 also showed fast degradation of BTX (230 ± 30 nmol benzene (mg protein × h)^–1 at 20 °C). Additionally, AAE-A2 degraded benzene continuously for 2 weeks. In contrast, a pure culture of the methanotroph Methylosinus trichosporium OB3b ceased benzene degradation after only 2 days. Experiments with methane mono-oxygenase inhibitors or competitive substrates suggested that BTX degradation was carried out by methane-oxidizing bacteria in the consortium, whereas the degradation of PAE was carried out by non-methanotrophic bacteria co-existing with methanotrophs. The composition of the consortium (AAE-A2) based on polar lipid fatty acid (PLFA) profiles showed dominance of type II methanotrophs (83–92% of biomass). Phylogeny based on a 16S-rRNA gene clone library revealed that the dominating methanotrophs belonged to Methylosinus/Methylocystis spp. and that members of at least 4 different non-methanotrophic genera were present (Pseudomonas, Flavobacterium, Janthinobacterium and Rubivivax).     

Continuous culture with deep seawater of a benthic food diatom Nitzschia sp.

A continuous culture system for a benthic food diatom Nitzschia sp. wasestablished by using properties of high nutrient and clean of deep seawater(DSW). DSW collected from 320 m depth in Muroto City, Japan, was introducedinto a glass-pipe bioreactor (14 cm length, 3 cm diam.) containing glassbeads of 0.5 cm diam. as substrata for the alga, and it was incubated at18°C · 80Em^–2sec^–1 · L:D=14:10. The chlorophyll a yield of benthicdiatoms in a reactor as a unit of surface area of the substratum was only0.001–0.003 g cm^–2 when the flow rate of DSW was 0(batch culture conditions). However, when DSW was supplied continuously to areactor, the yield increased to 1.4 g-chl.a cm^–2 alongwith the increase in flow rate of DSW. Moreover, amounts of chl.a washed outof the system were negligible, 0.0014 to 0.0045%, even though theflow rate of DSW was as much as 25 times h^–1, suggesting thatsloughing of benthic diatoms from the substratum was minimized. Although theyield of diatoms fluctuated significantly at the time that the DSW wascollected, the variation could be minimized by increasing the flow rate ofDSW. These results indicate that the continuous culturing system with DSWsupports the stable and effective mass culture of benthic food diatom.