Extracellular enzymes in a polyhumic lake: important regulators in detritus processing

Eight extracellular enzymes and their corresponding natural substrates were studied in an acid polyhumic lake. Highest activities were found for phosphatases (100–150 nmol 1⁻¹ h⁻¹), glucosidase (70–120 nmol 1⁻¹ h⁻¹), and aminopeptidases (20–30 nmol l⁻¹ h⁻¹), Significant relationships were found for natural polymeric substrate composition, variation and enzyme activities. Identified carbohydrates and amino acids contributed 1–5% to the DOC pool and are assumed to undergo significant processing by microbial glycosidases and aminopeptidases. Measured enzymes are partially modified in their activity by lake water acidity, temperature and humic matter. Extracellular enzymes are regarded as important regulators in microbial detritus processing and substrate utilization.     

Metabolic energy utilization during development of Antarctic naked dragonfish (Gymnodraco acuticeps)

We have capitalised on the availability of eggs and adults of the naked dragonfish Gymnodraco acuticeps (Sub-order Notothenioidei, F. Bathydraconidae) near McMurdo Station, Antarctica to examine metabolic energy utilization at different stages of its life cycle. Average egg respiration rates were found to increase from 2.17±1.02 nmol O₂ h⁻¹ ind⁻¹ at about 17 h post-fertilization (hpf) to 5.72±0.56 nmol h⁻¹ ind⁻¹ at about 24 hpf, during which time the eggs underwent first cleavage. The respiration rates of embryos from 2–20 days post-fertilization (dpf) averaged 4.11±1.47 nmol O₂ h⁻¹ ind⁻¹. About 10 months post-fertilization, oxygen consumption rates of 27.14±3.92 nmol O₂ h⁻¹ ind⁻¹ were recorded immediately prior to hatching, with a peak of 112.41±31.38 nmol O₂ h⁻¹ ind⁻¹ at the time of hatch. Larvae aged 46–63 days post-hatch had an average respiration rate of 64.4±15.11 nmol O₂ h⁻¹ ind⁻¹. Mass-specific respiration rates of hatched larvae (approximately 1–2 months old) were calculated using dry weights (DW) and averaged 16.1±3.4 nmol O₂ h⁻¹ mg⁻¹ DW. Adult dragonfish respiration rates (corrected for a 100 g fish and using a 0.8 scaling exponent) averaged 0.91±0.36 mmol O₂ kg⁻¹ h⁻¹ after a 48 h acclimatization period, which is not indicative of significant metabolic cold adaptation. The energy contents of dragonfish eggs and larvae were also measured by microbomb calorimetry and used, along with the respiration data, in an initial approach to estimate an energy budget. In order to balance the budget, the bulk of the available post-gastrulation respiratory energy (during 213 days of embryonic incubation) must be consumed at a relatively low average rate (7.1 nmol O₂ h⁻¹ ind⁻¹), which supports the possibility that advanced dragonfish embryos overwinter in a relatively quiescent metabolic state while awaiting a suitable stimulus (such as the return of the sun) to initiate hatching.     

Nitrite-driven anaerobic ATP synthesis in barley and rice root mitochondria

Mitochondria isolated from the roots of barley (Hordeum vulgare L.) and rice (Oryza sativa L.) seedlings were capable of oxidizing external NADH and NADPH anaerobically in the presence of nitrite. The reaction was linked to ATP synthesis and nitric oxide (NO) was a measurable product. The rates of NADH and NADPH oxidation were in the range of 12–16 nmol min⁻¹ mg⁻¹ protein for both species. The anaerobic ATP synthesis rate was 7–9 nmol min⁻¹ mg⁻¹ protein for barley and 15–17 nmol min⁻¹ mg⁻¹ protein for rice. The rates are of the same order of magnitude as glycolytic ATP production during anoxia and about 3–5% of the aerobic mitochondrial ATP synthesis rate. NADH/NADPH oxidation and ATP synthesis were sensitive to the mitochondrial inhibitors myxothiazol, oligomycin, diphenyleneiodonium and insensitive to rotenone and antimycin A. The uncoupler FCCP completely eliminated ATP production. Succinate was also capable of driving ATP synthesis. We conclude that plant mitochondria, under anaerobic conditions, have a capacity to use nitrite as an electron acceptor to oxidize cytosolic NADH/NADPH and generate ATP.     

Localization of chitinolytic activities in Fagus sylvatica mycorrhizas

 Localization of chitinolytic activities in Fagus sylvatica (beech) mycorrhizas was examined using a range of fluorogenic 4-methylumbelliferyl [4-MU-(GlcNAc)_1–4] substrates in order to distinguish between exochitinase, endochitinase and β-N–acetylglucosaminidase activities. The validity of the technique was confirmed using onion epidermis cells. In the beech mycorrhiza, endochitinase activity was not detectable above background fluorescence. Exochitinase activity was detected in the fungal sheath and the Hartig net. β-N–Acetylglucosaminidase activity was also mainly associated with the fungal sheath and Hartig net. Individual fungal hyphae extending from these structures also showed substantial β-N–acetylglucosaminidase activity. The cortical cell walls of the host in the Hartig net region also fluoresced brightly. The localization of β-N–acetylglucosaminidase activity was confirmed using a chromogenic histochemical reagent, 5-bromo-4-chloro-3-indolyl-N–acetyl-β-d-glucosaminide (X-GlcNAc). Accepted: 5 December 1995     

Nitrogen fixation by the benthic freshwater cyanobacterium Lyngbya wollei

The ability of the benthic cyanobacterium Lyngbya wollei to fix nitrogen was studied using field samples and axenic cultures. L. wollei was collected and isolated from Lake Okeechobee, Florida, where it forms extensive mats. Rates of acetylene reduction up to 39.1 nmol mg dry wt⁻¹ h⁻¹ were observed for field samples. The maximum observed rate of acetylene reduction in axenic laboratory cultures was 200 nmol mg dry wt⁻¹ h⁻¹. Aerobic conditions limited nitrogen fixation activity, but dark/light cycles promoted the development of activity. Reduced oxygen levels appeared to be required for the development of significant levels of nitrogenase activity. The level of irradiance also had a significant impact on the level of activity. The potential significance of nitrogen fixation to Lyngbya production is discussed.     

Factors Affecting the Antibacterial Activity of the Ruminal Bacterium,Streptococcus bovis HC5

Streptococcus bovis HC5 inhibits a variety of S. bovis strains and other Gram-positive bacteria, but factors affecting this activity had not been defined. Batch culture studies indicated that S. bovis HC5 did not inhibit S. bovis JB1 (a non-bacteriocin-producing strain) until glucose was depleted and cells were entering stationary phase, but slow-dilution-rate, continuous cultures (0.2 h⁻¹) had as much antibacterial activity as stationary-phase batch cultures. Because the activity of continuous cultures (0.2–1.2 h⁻¹) was inversely related to the glucose consumption rate, it appeared that the antibacterial activity was being catabolite repressed by glucose. When the pH of continuous cultures (0.2 h⁻¹) was decreased from 6.7 to 5.4, antibacterial activity doubled, but this activity declined at pH values less than 5.0. Continuous cultures (0.2 h⁻¹) that had only ammonia as a nitrogen source had antibacterial activity, and large amounts of Trypticase (10 mg ml⁻¹) caused only a 2.0-fold decline in the amount of HC5 cell-associated protein that was needed to prevent S. bovis JB1 growth. Because S. bovis HC5 was able to produce antibacterial activity over a wide range of culture conditions, there is an increased likelihood that this activity could have commercial application. Received: 6 February 2002 / Accepted: 27 March 2002     

Seasonal changes in digestive enzyme (trypsin) activity of the copepods <Emphasis Type="Italic">Pseudocalanus minutus</Emphasis> (Calanoida) and <Emphasis Type="Italic">Oithona similis</Emphasis> (Cyclopoida) in the Arctic Kongsfjorden (Svalbard)

Seasonal activities of the digestive enzyme trypsin were measured between August 1998 and May 1999 to study different nutritional strategies of the two copepods Pseudocalanus minutus and Oithona similis in the Arctic Kongsfjorden (Svalbard) using a highly sensitive fluorescence technique. Stage-, depth- and season-specific characteristics of digestive activity were reflected in the trypsin activity. P. minutus females and stage V copepodids (C) had highest trypsin activities in spring during reproduction (197.5 and 145.7 nmol min⁻¹ ng C⁻¹, respectively). In summer stages CIII–V and in autumn stages CIV and V had high activities (80–116 nmol min⁻¹ ng C⁻¹) in the shallow layer (< 100 m) presumably as a consequence of prolonged feeding before descending to overwintering depth. Trypsin activities at depth (> 100 m) in summer and autumn were low in stages CIII and CIV (29–60 nmol min⁻¹ ng C⁻¹) and in winter in all stages in both layers (20–43 nmol min⁻¹ ng C⁻¹). Based on low trypsin activity, males most likely did not feed. In O. similis, the spring phytoplankton bloom did not significantly affect trypsin activity as compared to the other seasons. O. similis CV and females had high trypsin activities in summer in the deep stratum (304.5 nmol min⁻¹ ng C⁻¹), which was concomitant with reproductive processes and energy storage for overwintering. In autumn, stage CV and female O. similis had significantly higher activities than stage CIV (130–152 versus 78 nmol min⁻¹ ng C⁻¹), which is in accordance with still ongoing developmental and reproductive processes in CVs and females. Comparisons of both species revealed different depth-related responses emphasizing different nutritional preferences: the mainly herbivorous P. minutus is more actively feeding in the shallow layer, where primary production occurs, whereas the omnivorous O. similis is not as much restricted to a certain depth layer, when searching for food. P. minutus had lower levels of trypsin activity during all seasons. In contrast to P. minutus, higher enzyme activities in males of O. similis suggest that they continue to feed and survive after fertilization of females.     

Influence of UV-light on carthamin accumulation and floret elongation in a cultivar of saffron thistle (Carthamus tinctorius L.)

Pre-matured florets of Benibana, a cultivar of saffron thistle (Carthamus tinctorius L.) was irradiated with UV-B (280–320 nm) or UV-C (254 nm) light for 48 h at 23±1 °C and the influence of UV-light on carthamin accumulation and floret elongation was investigated. UV-C light enhances carthamin accumulation most prominently, showing a specific value of 52.3 nmol carthamin·dm⁻³·h⁻¹·25μm⁻² (13.9 times of control), while it restricts floret elongation by a light-suppression manner (net elongation: 0.058 mm·h⁻¹, one ninth of control). UV-B light is also promotive for the red colour appearance (25.0 nmol carthamin·dm⁻³·h⁻¹·25 μm⁻², 6.7 times of control) with suppressing floret elongation (net elongation: 0.17 mm·h⁻¹, one third of control). Heterogeneous productivity of carthamin was seen in floret tissues after continuous treating under UV-C light. Carthamin accumulation, heterogeneous carthamin productivity and decrease of floret elongation restraint under UV-lights are discussed.     

Efficient induction of formate hydrogen lyase of aerobically grown <Emphasis Type="Italic">Escherichia coli</Emphasis> in a three-step biohydrogen production process

A three-step biohydrogen production process characterized by efficient anaerobic induction of the formate hydrogen lyase (FHL) of aerobically grown Escherichia coli was established. Using E. coli strain SR13 (fhlA ⁺⁺, ΔhycA) at a cell density of 8.2 g/l medium in this process, a specific hydrogen productivity (28.0 ± 5.0 mmol h⁻¹ g⁻¹ dry cell) of one order of magnitude lower than we previously reported was realized after 8 h of anaerobic incubation. The reduced productivity was attributed partly to the inhibitory effects of accumulated metabolites on FHL induction. To avoid this inhibition, strain SR14 (SR13 ΔldhA ΔfrdBC) was constructed and used to the effect that specific hydrogen productivity increased 1.3-fold to 37.4 ± 6.9 mmol h⁻¹ g⁻¹. Furthermore, a maximum hydrogen production rate of 144.2 mmol h⁻¹ g⁻¹ was realized when a metabolite excretion system that achieved a dilution rate of 2.0 h⁻¹ was implemented. These results demonstrate that by avoiding anaerobic cultivation altogether, more economical harvesting of hydrogen-producing cells for use in our biohydrogen process was made possible.     

Characterization of an ecto-ATPase activity in Fonsecaea pedrosoi

In this work, we characterized an ecto-ATPase activity in intact mycelial forms of Fonsecaea pedrosoi, the primary causative agent of chromoblastomycosis. In the presence of 1 mM EDTA, fungal cells hydrolyzed adenosine-5′-triphosphate (ATP) at a rate of 84.6 ± 11.3 nmol Pi h⁻¹ mg⁻¹ mycelial dry weight. The ecto-ATPase activity was increased at about five times (498.3 ± 27.6 nmol Pi h⁻¹ mg⁻¹) in the presence of 5 mM MgCl₂, with values of V _max and apparent K _m for Mg-ATP²⁻corresponding to 541.9 ± 48.6 nmol Pi h⁻¹ mg⁻¹ cellular dry weight and 1.9 ± 0.2 mM, respectively. The Mg²⁺-stimulated ecto-ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A₁ (V-ATPases)_, and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate, and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The surface of the Mg²⁺-stimulated ATPase in F. pedrosoi was confirmed by assays in which 4,4′-diisothiocyanostylbene-2,2′-disulfonic acid (DIDS), a membrane impermeant inhibitor, and suramin, an inhibitor of ecto-ATPase and antagonist of P₂ purinoreceptors. Based on the differential expression of ecto-ATPases in the different morphological stages of F. pedrosoi, the putative role of this enzyme in fungal biology is discussed.     

Continuous nisin production with bioengineered <Emphasis Type="Italic">Lactococcus lactis</Emphasis> strains

Nisin production in continuous cultures of bioengineered Lactococcus lactis strains that incorporate additional immunity and regulation genes was studied. Highest nisin activities were observed at 0.2 h^–1 dilution rate and 12.5 g l^–1 fructose concentration for all strains. Recombinant strains were able to produce greater amounts of nisin at dilution rates below 0.3 h⁻¹ compared to the control strain. However, this significant difference disappeared at dilution rates of 0.4 and 0.5 h^–1. For the strains LL27, LAC338, LAC339, and LAC340, optimum conditions for nisin production were determined to be at 0.29, 0.26, 0.27, and 0.27 h^–1 dilution rates and 11.95, 12.01, 11.63, and 12.50 g l^–1 fructose concentrations, respectively. The highest nisin productivity, 496 IU ml^–1 h^–1, was achieved with LAC339. The results of this study suggest that low dilution rates stabilize the high specific nisin productivity of the bioengineered strains in continuous fermentation. Moreover, response surface methodology analysis showed that regulation genes yielded high nisin productivity at wide ranges of dilution rates and fructose concentrations.     

Bioremoval of hexavalent chromium from water by a salt tolerant bacterium, Exiguobacterium sp. GS1

Pollution of terrestrial surfaces and aquatic systems by hexavalent chromium, Cr(VI), is a worldwide public health problem. A chromium resistant bacterial isolate identified as Exiguobacterium sp. GS1 by 16S rRNA gene sequencing displayed high rate of removal of Cr(VI) from water. Exiguobacterium sp. GS1 is 99% identical to Exiguobacterium acetylicum. The isolate significantly removed Cr(VI) at both high and low concentrations (1–200 μg mL⁻¹) within 12 h. The Michaelis–Menten K _m and V _max for Cr(VI) bioremoval were calculated to be 141.92 μg mL⁻¹ and 13.22 μg mL⁻¹ h⁻¹, respectively. Growth of Exiguobacterium sp. GS1 was indifferent at 1–75 μg mL⁻¹ Cr(VI) in 12 h. At initial concentration of 8,000 μg L⁻¹, Exiguobacterium sp. GS1 displayed rapid bioremoval of Cr(VI) with over 50% bioremoval in 3 h and 91% bioremoval in 8 h. Kinetic analysis of Cr(VI) bioremoval rate revealed zero-order in 8 h. Exiguobacterium sp. GS1 grew and significantly reduced Cr(VI) in cultures containing 1–9% salt indicating high salt tolerance. Similarly the isolate substantially reduced Cr(VI) over a wide range of temperature (18–45  °C) and initial pH (6.0–9.0). The T _opt and initial pH_opt were 35–40  °C and 7–8, respectively. Exiguobacterium sp. GS1 displayed a great potential for bioremediation of Cr(VI) in diverse complex environments.     

21科41种(变种)植物叶片几丁质酶系的研究

对广州地区常见的21科41种（变种）植物叶片几丁质酶系研究的结果表明,所有受试植物都具有几丁质酶活性。几丁质酶不仅存在于被子植物的双子叶植物和单子叶植物中,而且也存在于裸子植物及蕨类植物中。几丁质酶活性及比活性均较高的有蕹菜、葱、蕨类植物、蒜、茄科植物、玉米、菜豆、番木瓜等。植物一般都具有两种几丁质酶:外切酶和内切酶。几丁质外切酶活性及比活性均较高的有蕨类植物、葱、茄科植物等。几丁质内切酶活性及比活性均较高的有蕹菜、裸子植物等。不同植物几丁质外切酶与内切酶的比例相差较大。有些植物的几丁质酶系以外切酶为主,如茄科植物、大部分豆科植物、番木瓜等;有些植物以内切酶为主,如裸子植物、伞形科植物、蕹菜等;有些植物的外切酶与内切酶含量相差不大。     

Molecular cloning,sequence analysis,expression and characterization of the endochitinase gene from <Emphasis Type="Italic">Trichoderma</Emphasis> sp. in <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21

The endochitinase DNA and cDNA from Trichoderma sp. were cloned, sequenced and expressed. The cloned DNA and cDNA sequences were 1,476 and 1,275 bp in length, respectively. There were three introns in DNA sequence in comparison with the cDNA sequence. The endochitinase protein contained three regions: the signal peptide, the prepro-region and the mature protein region. The gene fragment encoding the mature endochitinase was ligated into the expression vector pET-28a+, yielding pET-1. The plasmid pET-1 was transformed into the Escherichia coli BL21 (DE3). The clone bearing pET-1 was picked and cultured at 30°C for the expression of endochitinase. SDS-PAGE analysis showed that the endochitinase was expressed in the periplasmic space and the purified protein showed a single band. The activity of 70.2 U/mg was obtained from the cellular extract of the recombinant strain. The activity of endochitinase was 2.5-fold higher at 24 h than at 16 h in the periplasmic space. The optimal pH and temperature of the recombinant endochitinase were determined to be 7.0 and 35°C, respectively. It was relatively stable within the pH range of 5–8. Significant activity stimulation by 1 mM Mg²⁺ and 5 mM Fe²⁺ and inhibition by 5 mM Co²⁺ and 5 mM Hg²⁺ were observed. The kinetic constants K_m, V_max and K_cat for the hydrolysis of the colloidal chitin were 1.5 mM, 1.37 μmol min⁻¹ and 6.23 min⁻¹, respectively.     

Occurrence and Activity of Microorganisms in Shrimp Waste

This study aimed to determine the occurrence and respiration activity of heterotrophic bacteria and fungi in shrimp shell waste and to evaluate the role of chitinolytic bacteria and fungi in its decomposition. The highest levels of bacteria were found in shrimp heads sections and the lowest in exoskeletons. The level of fungi was much lower, with the highest proportion present in heads sections and the lowest in exoskeletons. Chitinolytic bacteria constituted a small percentage of the total heterotrophic bacteria in fresh shrimp waste, averaging 4% in exoskeletons, 2.4% in all parts, and 2% in heads. No chitinolytic bacteria were detected in stored waste. In contrast, the percentage of chitinolytic fungi in shrimp waste was much higher than that of bacteria. Chitinolytic fungi constituted 25–60% of the total fungi in fresh waste and 15–40% in stored waste. Chitinolytic bacteria isolated from heads sections were characterized by the highest chitinolytic activity, averaging 11.2 nmol of methylumbelliferyl · mg⁻¹ protein · h⁻¹, whereas the lowest activity was in strains from exoskeletons, averaging 3.2 nmol of methylumbelliferyl · mg⁻¹ protein · h⁻¹. The chitinolytic activity of fungi isolated from all parts waste, head sections, and exoskeletons was similar. The respiration activity of microorganisms in fresh and stored waste was similar. Oxygen consumption activity increased during incubation and approached a saturation value between days 4 and 5. No correlation between the end value of respiratory activity in the analyzed section of shrimp discard after 5 days and the level of bacteria and fungi was observed. The only significant correlation observed was between the respiratory activity of the shrimp and the level of fungi. The respiration activity significantly depended on the analyzed section of shrimp discard (p < 0.000).     

Influence of Interfacial Composition on in Vitro Digestibility of Emulsified Lipids: Potential Mechanism for Chitosan's Ability to Inhibit Fat Digestion

The objective of this study was to investigate the influence of interfacial composition and electrical charge on the in vitro digestion of emulsified fats by pancreatic lipase. An electrostatic layer-by-layer deposition technique was used to prepare corn oil-in-water emulsions (3 wt% oil) that contained droplets coated by (1) lecithin, (2) lecithin–chitosan, or (3) lecithin–chitosan–pectin. Pancreatic lipase (1.6 mg mL⁻¹) and/or bile extract (5.0 mg mL⁻¹) were added to each emulsion, and the particle charge, droplet aggregation, and free fatty acids released were measured. In the presence of bile extract, the amount of fatty acids released per unit amount of emulsion was much lower in the emulsions containing droplets coated by lecithin–chitosan (38 ± 16 μmol mL⁻¹) than those containing droplets coated by lecithin (250 ± 70 μmol mL⁻¹) or lecithin–chitosan–pectin (274 ± 80 μmol mL⁻¹). In addition, there was much more extensive droplet aggregation in the lecithin–chitosan emulsion than in the other two emulsions. We postulated that lipase activity was reduced in the lecithin–chitosan emulsion as a result of the formation of a relatively thick cationic layer around each droplet, as well as the formation of large flocs, which restricted the access of the pancreatic lipase to the lipids within the droplets. Our results also suggest that droplets initially coated by a lecithin–chitosan–pectin layer did not inhibit lipase activity, which may have been because the chitosan–pectin desorbed from the droplet surfaces thereby allowing the enzyme to reach the lipids; however, further work is needed to establish this. This information could be used to create food emulsions with low caloric level, or to optimize diets for individuals with lipid digestion problems.     

Isolation of algicidal compounds from the red alga <Emphasis Type="Italic">Corallina pilulifera</Emphasis> against red tide microalgae

We determined the structure of two compounds, namely, 5,8,11,14,17-eicosapentaenoic acid (EPA) and di-n-octylphthalate (DnOP), which have algicidal activity against the toxic dinoflagellate, Cochlodinium polykrikoides. The polyunsaturated fatty acid EPA and the anthropogenic DnOP were isolated from the MeOH extract of the red alga Corallina pilulifera. We also found that a commercial EPA has algicidal activity identical to that of the EPA purified from C. pilulifera. At low inoculum (5.0 × 10² cells mL⁻¹), the highest algicidal activity of a commercial EPA exhibited approximately 92.6% algicidal activity after 1 h and 96.8% after 6 h treatment at 6 μg mL⁻¹, respectively. At high inoculum (1.0 × 10⁴ cells mL⁻¹), the strongest algicidal activity of EPA showed 69.5% after 1 h and 75.5% algicidal activity after 6 h treatment at 6 μg mL⁻¹, respectively. However, EPA did not show algicidal activity against several microalgae used in aquaculture such as Pavlova lutheri, Tetraselmis suecica, Isochrysis galbana, and Nannochloris oculata for 6 h treatment at 6 μg mL⁻¹. The algicidal activity of the five red tide strains to EPA (3 μg mL⁻¹) showed about 86.6%, 86.6%, and 67.3% algicidal activity against Skeletonema costatum, Chaetoceros curvisetus, and C. polykrikoides after 1 h treatment at low inoculum (5.0 × 10² cells mL⁻¹), respectively, but not against Prorocentrum minimum and Scrippsiella trochoidea. We concluded that EPA might be useful as a controlling agent of harmful algal blooms.     

Perchlorate reduction by a mixed culture in an up-flow anaerobic fixed bed reactor

Wolinella succinogenes HAP-1 is a Gram-negative microaerophile which reduces perchlorate to chloride by the proposed pathway ClO₄ to ClO₃ to ClO₂ to Cl + O₂. A cost-effective perchlorate treatment process has been established using a consortium of facultative anaerobic organisms and W. succinogenes HAP-1. The mixed anaerobic bacterial culture containing W. succinogenes HAP-1 was examined for the ability to form a biofilm capable of perchlorate reduction. An up-flow anaerobic fixed bed reactor (UAFBR) was packed with diatomaceous earth pellets and operated at residence times of 1.17 and 0.46 h to insure a viable biofilm had attached to the diatomaceous earth pellets. Reduction rates of perchlorate to chloride in the UAFBR could be maintained at 1 g of perchlorate reduced h⁻¹ L⁻¹. Studies with the same bacterial consortium in continuously stirred tank reactors (CSTR) generally reduced 0.5–0.7 g of perchlorate h⁻¹. Viable cell counts were performed periodically on the diatomaceous earth pellets and demonstrated that the W. succinogenes HAP-1 population was maintained at 28–47% of the total microbial population. Scanning electron micrographs demonstrated that the external and internal surfaces of the diatomaceous pellets were densely colonized with microbial cells of multiple cell types. This is the first report of an anaerobic mixed culture forming a biofilm capable of perchlorate reduction. Received 22 May 1997/ Accepted in revised form 07 January 1998     

Molecular characterization of an endochitinase from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. konkukian

A chitinase gene from Bacillus thuringiensis serovar konkukian S4 was cloned, sequenced, and heterologously expressed in Escherichia coli M15. Recombinant enzyme (Chi74) was purified by Ni-NTA affinity column chromatography. The chi74 gene contains an open reading frame (ORF), with a capacity to encode an endochitinase with a deduced molecular weight 74 kDa and predicted isoelectric point of 5.67. Comparison of Chi74 with other chitinases has shown that it contains a modular structure with an N-terminal family 18 catalytic-domain, a Fibronectin-III like domain and a C-terminal carbohydrate binding module (CBM-II). Turn over rate (K _cat ) of the enzyme was determined using colloidal chitin (28.3 ± 0.70 S⁻¹) as substrate. The Purified enzyme was active at a broad range of pH (pH 3.5–7.5) and temperature (20–70°C) with a peak activity at pH 5.5 and 55°C. However, the enzyme was found to be stable up to 30°C for longer incubation periods. Moreover, the purified enzyme was shown to inhibit fungal spore germination and hyphal growth in the pathogenic fungi Fusarium oxysporum and Aspergillus niger. These studies will lead us to develop broad spectrum resistance in the crop plants via co-expression of the chitinases and the insecticidal proteins.     

Physiology and kinetics of trimethylamine conversion by two methylotrophic strains in continuous cultivation systems

The change of dilution rate (D) on both Methylophilus methylotrophus NCIMB11348 and Methylobacterium sp. RXM CCMI908 growing in trimethylamine (TMA) chemostat cultures was studied in order to assess their ability to remove odours in fish processing plants. M. methylotrophus NCIMB11348 was grown at dilution rates of 0.012–0.084 h⁻¹ and the biomass level slightly increased up to values of D around 0.07 h⁻¹. The maximum cell production rate was obtained at 0.07 h⁻¹ corresponding to a maximum conversion of carbon into cell mass (35%). The highest rate of TMA consumption was 3.04 mM h⁻¹ occurring at D=0.076 h⁻¹. Methylobacterium sp. RXM CCMI908 was grown under similar conditions. The biomass increased in a more steep manner up to values of D around 0.06 h⁻¹. The maximum cell production rate (0.058 g l⁻¹h⁻¹) was obtained in the region close to 0.06 h⁻¹ where a maximum conversion of the carbon into cell mass (40%) was observed. The maximum TMA consumption was 2.33 mM h⁻¹ at D=0.075 h⁻¹. The flux of carbon from TMA towards cell synthesis and carbon dioxide in both strains indicates that the cell is not excreting products but directing most of the carbon source to growth. Carbon recovery levels of approximately 100% show that the cultures are carbon-limited. Values for theoretical maximum yields and maintenance coefficients are presented along with a kinetic assessment based on the determination of the substrate saturation constant and maximum growth rate for each organism. Received: 25 February 1999 / Received revision: 14 May 1999 / Accepted: 17 May 1999