Immobilization of Serratia marcescens lipase onto amino-functionalized magnetic nanoparticles for repeated use in enzymatic synthesis of Diltiazem intermediate

Magnetic Fe₃O₄ nanoparticles were prepared by chemical coprecipitation method and subsequently coated with 3-aminopropyltriethoxysilane (APTES) via silanization reaction. The synthesized materials were characterized by transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR). With glutaraldehyde as the coupling agent, the lipase from Serratia marcescens ECU1010 (SmL) was successfully immobilized onto the amino-functionalized magnetic nanoparticles. The results showed that the immobilized protein load could reach as high as 35.2 mg protein g⁻¹ support and the activity recovery was up to 62.0%. The immobilized lipase demonstrated a high enantioselectivity toward (+)-MPGM (with an E-value of 122) and it also displayed the improved thermal stability as compared to the free lipase. When the immobilized lipase was employed to enantioselectively hydrolyze (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] in water/toluene biphasic reaction system for 11 consecutive cycles (totally 105 h), still 59.6% of its initial activity was retained, indicating a high stability in practical operation.     

Study of the experimental conditions for the lipase production by a newly isolated strain of Pseudomonas aeruginosa for the enantioselective hydrolysis of (±)-methyl trans-3(4-methoxyphenyl) glycidate

A lipase producing bacterium has been isolated from the soil to enantiospecifically hydrolyze the (±)-methyl trans-3(4-methoxyphenyl) glycidate (MPGM), an intermediate in the synthesis of cardiovascular drug, diltiazem. This hydrolysis provided the desired (−)-MPGM in 44% yield with 99% enantiomeric excess. The organism was identified and confirmed as Pseudomonas aeruginosa by 16S rRNA sequencing. The various physiochemical parameters have been optimized for the maximum production of lipase in shake flask. Beef extract was found to be the best nitrogen source for lipase production. The optimized cultivation conditions were 30°C with an initial medium pH 8 in shake flask. Both inoculum age and inoculum concentration have positive effect on the lipase production and (±)-MPGM (3 mM) was found to be the optimal inducer.An erratum to this article can be found at     

Refolding,purification and characterization of an organic solvent-tolerant lipase from Serratia marcescens ECU1010

Expression of recombinant proteins as inclusion bodies in bacteria is one of the most efficient ways to produce cloned proteins, as long as the inclusion bodies can be successfully refolded. In this study, the different parameters were investigated and optimized on the refolding of denatured lipase. The maximum lipase activity of 5000 U/L was obtained after incubation of denatured enzyme in a refolding buffer containing 20 mM Tris–HCl (pH 7.0), 1 mM Ca²⁺ at 20 °C. Then, the refolded lipase was purified to homogeneity by anion exchange chromatography. The purified refolded lipase was stable in broad ranges of temperatures and pH values, as well as in a series of water-miscible organic solvents. In addition, some water-immiscible organic solvents, such as petroleum ether and isopropyl ether, could reduce the polarity and increase the nonpolarity of the refolding system. The results of Fourier transform infrared (FT-IR) microspectroscopy were the first to confirm that lipase refolding could be further improved in the presence of organic solvents. The purified refolded lipase could enantioselectively hydrolyze trans-3-(4-methoxyphenyl) glycidic acid methyl ester [(±)-MPGM]. These features render the lipase attraction for biotechnological applications in the field of organic synthesis and pharmaceutical industry.     

Stability of lipase immobilized on <Emphasis Type="Italic">O</Emphasis>-pentynyl dextran

O-Pentynyl dextran (PyD), an amphiphilic polysaccharide derivative with a degree of substitution (DS) of 0.43 was compared with ion exchange resins Lewatit VP OC 1600, Amberlite XAD 761 and Duolite A568 for immobilization of Lipase from Rhizopus arrhizus by adsorption method. The immobilized enzymes were employed for esterification of octanoic acid with geraniol in n-hexane as model reaction. PyD showed higher lipase adsorption and with 249 μmol min⁻¹ g⁻¹ significant higher esterification activity than the other supports (67–83 μmol min⁻¹ g⁻¹). Biocatalysts from all types of supports except PyD became completely inactive within 8 weeks storing at −10 °C while lipase immobilized on PyD retained its full esterification activity for at least 14 weeks. In repeated use, yield decreased rapidly after two cycles for all supports except for PyD. For this biopolymeric support, constantly 90% yield was achieved even after eight cycles, when the biocatalyst was washed with n-hexane and water and then freeze-dried. To achieve this yield, prolonged reaction times were required, partly on the account of an increasing delay period, probably to adapt active conformation, until the reaction starts.     

Serratia marcescens H30脂肪酶的重组可溶表达、固定化及其酶学性质

为了提高Serratia marcescens H30脂肪酶的可溶表达水平,分别将目的基因与p GEX-4T-1、p ET28a和p ET32a构建重组表达载体,转入大肠杆菌BL21(DE3),通过优化诱导过程,发现可溶性酶的最高活性可达25 000 U/L。再经Ni2+亲和柱纯化、LH-EP固定化后,固定化酶的比酶活为214 U/g(以1 g湿质量计),酶活回收率为51%。固定化后重组脂肪酶的最适温度由30℃提高到35℃,最适p H从7.0偏移至8.0左右,并且稳定性也有所增加。该固定化重组脂肪酶同样能够拆分消旋体反式-4-甲氧苯基缩水甘油酸甲酯,光学选择性没有改变。反应14 h,转化率为48.5%,底物的e.e.值为99.2%,表明该固定化脂肪酶能有效拆分消旋体反式-4-甲氧苯基缩水甘油酸甲酯,为工业生物催化制备地尔硫卓提供了可能。     

Bioaugmentation of UASB reactors with immobilized <Emphasis Type="Italic">Sulfurospirillum barnesii</Emphasis> for simultaneous selenate and nitrate removal

Whole-cell immobilization of selenate-respiring Sulfurospirillum barnesii in polyacrylamide gels was investigated to allow the treatment of selenate contaminated (790 μg Se × L⁻¹) synthetic wastewater with a high molar excess of nitrate (1,500 times) and sulfate (200 times). Gel-immobilized S. barnesii cells were used to inoculate a mesophilic (30°C) bioreactor fed with lactate as electron donor at an organic loading rate of 5 g chemical oxygen demand (COD) × L⁻¹ day⁻¹. Selenate was reduced efficiently (>97%) in the nitrate and sulfate fed bioreactor, and a minimal effluent concentration of 39 μg Se × L⁻¹ was obtained. Scanning electron microscopy with energy dispersive X-ray (SEM–EDX) analysis revealed spherical bioprecipitates of ≤2 μm diameter mostly on the gel surface, consisting of selenium with a minor contribution of sulfur. To validate the bioaugmentation success under microbial competition, gel cubes with immobilized S. barnesii cells were added to an Upflow Anaerobic Sludge Bed (UASB) reactor, resulting in earlier selenate (24 hydraulic retention times (HRTs)) and sulfate (44 HRTs) removal and higher nitrate/nitrite removal efficiencies compared to a non-bioaugmented control reactor. S. barnesii was efficiently immobilized inside the UASB bioreactors as the selenate-reducing activity was maintained during long-term operation (58 days), and molecular analysis showed that S. barnesii was present in both the sludge bed and the effluent. This demonstrates that gel immobilization of specialized bacterial strains can supersede wash-out and out-competition of newly introduced strains in continuous bioaugmented systems. Eventually, proliferation of a selenium-respiring specialist occurred in the non-bioaugmented control reactor, resulting in simultaneous nitrate and selenate removal during a later phase of operation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A novel cold-adapted lipase from <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. XMZ-26: gene cloning and characterisation

Acinetobacter sp. XMZ-26 (ACCC 05422) was isolated from soil samples obtained from glaciers in Xinjiang Province, China. The partial nucleotide sequence of a lipase gene was obtained by touchdown PCR using degenerate primers designed based on the conserved domains of cold-adapted lipases. Subsequently, a complete gene sequence encoding a 317 amino acid polypeptide was identified. Our novel lipase gene, lipA, was overexpressed in Escherichia coli. The recombinant protein (LipA) was purified by Ni-affinity chromatography, and then deeply characterised. The LipA resulted to hydrolyse pNP esters of fatty acids with acyl chain length from C2 to C16, and the preferred substrate was pNP octanoate showing a k _cat = 560.52 ± 28.32 s⁻¹, K _m = 0.075 ± 0.008 mM, and a k _cat/K _m = 7,377.29 ± 118.88 s⁻¹ mM⁻¹. Maximal LipA activity was observed at a temperature of 15°C and pH 10.0 using pNP decanoate as substrate. That LipA peaked at such a low temperature and remained most activity between 5°C and 35°C indicated that it was a cold-adapted enzyme. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents, including Ninol, Triton X-100, methanol, PEG-600, and DMSO. This cold-adapted lipase may find applications in the detergent industry and organic synthesis.     

Chemoinformatics-assisted development of new anti-biofilm compounds

The fuel oxygenate, methyl tert-butyl ether (MTBE), although now widely banned or substituted, remains a persistent groundwater contaminant. Multidimensional compound-specific isotope analysis (CSIA) of carbon and hydrogen is being developed for determining the extent of MTBE loss due to biodegradation and can also potentially distinguish between different biodegradation pathways. Carbon and hydrogen isotopic fractionation factors were determined for MTBE degradation in aerobic and anaerobic laboratory cultures. The carbon isotopic enrichment factor (εC) for aerobic MTBE degradation by a bacterial consortium containing the aerobic MTBE-degrading bacterium, Variovorax paradoxus, was −1.1 ± 0.2‰ and the hydrogen isotope enrichment factor (εH) was −15 ± 2‰. This corresponds to an approximated lambda value (Λ = εH/εC) of 14. Carbon isotope enrichment factors for anaerobic MTBE-degrading enrichment cultures were −7.0 ± 0.2‰ and did not vary based on the original inoculum source, redox condition of the enrichment, or supplementation with syringic acid as a co-substrate. The hydrogen enrichment factors of cultures without syringic acid were insignificant, however a strong hydrogen enrichment factor of −41 ± 3‰ was observed for cultures which were fed syringic acid during MTBE degradation. The Λ = 6 obtained for NYsyr cultures might be diagnostic for the stimulation of anaerobic MTBE degradation by methoxylated compounds by an as yet unknown pathway and mechanism. The stable-isotope enrichment factors determined in this study will enhance the use of CSIA for monitoring anaerobic and aerobic MTBE biodegradation in situ.     

The Antarctic notothenioid fish <Emphasis Type="Italic">Pagothenia borchgrevinki</Emphasis> is thermally flexible: acclimation changes oxygen consumption

Antarctic marine organisms are considered to have extremely limited ability to respond to environmental temperature change. However, here we show that the Antarctic notothenioid fish Pagothenia borchgrevinki is an exception to this theory. P. borchgrevinki was able to acclimate its resting metabolic rate and resting ventilation frequency after a 5°C rise in temperature. Acute exposure to 4°C resulted in an elevation in metabolic rate (57.8 ± 4.79 mg O₂ kg⁻¹ h⁻¹) and resting ventilation rate (40.38 ± 1.61 breaths min⁻¹) compared with fish at −1°C (metabolic rate 34.45 ± 3.12 mg O₂ kg⁻¹ h⁻¹; ventilation rate 29.88 ± 3.72 breaths min⁻¹). However, after a 1-month acclimation period, there was no significant difference in the metabolic rate (cold fish 29.52 ± 3.01; warm fish 31.13 ± 2.30 mg O₂ kg⁻¹ h⁻¹), or the resting ventilation rate (cold fish 28.75 ± 0.98; warm fish 34.25 ± 2.28 breaths min⁻¹) of cold and warm acclimated fish. Acclimation changes to the rate of oxygen consumption following exhaustive exercise were complex. The pattern of oxygen consumption during recovery from exhaustive exercise was not significantly different in either cold or warm acclimated fish.     

Lipase-catalyzed kinetic resolution of (R,S)-1-phenylethyl propionate in an enzyme membrane reactor

Enzymatic stereoselective hydrolysis of (R,S)-1-phenylethyl propionate was performed in a stirred tank and in a biphasic enzyme membrane reactor. Lipase from Pseudomonas sp. was proved to be a good enantioselective catalyst for this reaction. The enzyme was covalently immobilized in a porous polyamide membrane (flat sheet as well as hollow-fibres) via glutaraldehyde. An influence of membrane hydrophobicity on reactor performance was observed. Initial lipase activity and productivity in the processes were equal to 1.05 × 10^?4, 1.3 × 10^?5 and 1.0 × 10^?5 mole/(h × mg of enzyme) in the case of native lipase, in the aromatic polyamide hydrophobic membrane reactor and in the hydrophilic polyamide-6 membrane reactor, respectively. The influence of some factors such as temperature, pH, buffer concentration, initial substrate concentration and addition of β-cyclodextrin derivatives on reaction rate and enantioselectivity was investigated and discussed. In the enzyme membrane reactor both organic and aqueous phases circulated countercurrently on both sides of the membrane. At a conversion degree of under 55–60%, pure enantiomer of the remaining ester (i.e. > 98%) was obtained.     

Leaf traits variation during leaf expansion in <Emphasis Type="Italic">Quercus ilex</Emphasis> L.

The morphological, anatomical and physiological variations of leaf traits were analysed during Quercus ilex L. leaf expansion. The leaf water content (LWC), leaf area relative growth rate (RGR_l) and leaf dry mass relative growth rate (RGR_m) were the highest (76±2 %, 0.413 cm² cm⁻² d⁻¹, 0.709 mg mg⁻¹ d⁻¹, respectively) at the beginning of the leaf expansion process (7 days after bud break). Leaf expansion lasted 84±2 days when air temperature ranged from 13.3±0.8 to 27.6±0.9 °C. The net photosynthetic rate (P _N), stomatal conductance (g _s), and chlorophyll content per fresh mass (Chl) increased during leaf expansion, having the highest values [12.62±1.64 μmol (CO₂) m⁻² s⁻¹, 0.090 mol (H₂O) m⁻² s⁻¹, and 1.03±0.08 mg g⁻¹, respectively] 56 days after bud break. Chl was directly correlated with leaf dry mass (DM) and P _N. The thickness of palisade parenchyma contributed to the total leaf thickness (263.1±1.5 μm) by 47 %, spongy layer thickness 38 %, adaxial epidermis and cuticle thickness 9 %, and abaxial epidermis and cuticle thickness 6 %. Variation in leaf size during leaf expansion might be attributed to a combination of cells density and length, and it is confirmed by the significant (p<0.001) correlations among these traits. Q. ilex leaves reached 90 % of their definitive structure before the most severe drought period (beginning of June — end of August). The high leaf mass area (LMA, 15.1±0.6 mg cm⁻²) at full leaf expansion was indicative of compact leaves (2028±100 cells mm⁻²). Air temperature increasing might shorten the favourable period for leaf expansion, thus changing the final amount of biomass per unit leaf area of Q. ilex.     

Kinetics of BTEX degradation in a packed-bed anaerobic reactor

The ever-increasing diversity of industrial activity is responsible for the discharge of compounds that are toxic or difficult to degrade into the environment. Some of the compounds found in surface and ground waters, usually deriving from the contamination of oil-based products, are benzene, toluene, ethylbenzene and xylenes (BTEX). To remove these compounds from contaminated water, a bench-scale horizontal-flow anaerobic immobilized biomass reactor, containing anaerobic biomass from various sources immobilized in polyurethane foam matrices, was employed to treat a synthetic substrate composed of protein, carbohydrates and BTEX solution in ethanol, as well as a BTEX solution in ethanol as the sole carbon source. The reactor removed up to 15.0 mg/l of each BTEX compound over a hydraulic detention time of 11.4 h. A first-order kinetic model fitted the experimental data well, showing correlation coefficients higher than 0.994. The apparent first-order coefficient values, , ranged from 8.4±1.5 day⁻¹ for benzene to 10.7±1.4 day⁻¹ for o-xylene in the presence of ethanol, protein and carbohydrates, and from 10.0±2.0 day⁻¹ for benzene to 13.0±1.7 day⁻¹ for o-xylene in the presence of ethanol. The BTEX degradation rates estimated here were 10- to 94-fold higher than those found in reports on microcosm studies.     

Combined effects of zinc and algal food on the competition between planktonic rotifers, <Emphasis Type="Italic">Anuraeopsis fissa</Emphasis> and <Emphasis Type="Italic">Brachionus rubens</Emphasis> (Rotifera)

We evaluated the combined effects of algal (Chlorella vulgaris) food levels (low, 0.5 × 10⁶ (or 2.9 μg C ml⁻¹); and high, 1 × 10⁶ cells ml⁻¹ (or 5.8 μg C ml⁻¹)) and zinc concentrations (0, 0.125, and 0.250 mg l⁻¹ of ZnCl₂) on the competition between two common planktonic rotifers Anuraeopsis fissa and Brachionus rubens using their population growth. Median lethal concentration data (LC₅₀) (mean ± 95% confidence intervals) showed that B. rubens was more resistant to zinc (0.554 ± 0.08 mg l⁻¹) than A. fissa (0.315 ± 0.07 mg l⁻¹). A. fissa when grown alone or with Zn was always numerically more abundant than B. rubens. When grown in the absence of zinc, under low- and high-food levels, the peak abundances of A. fissa varied from 251 ± 24 to 661 ± 77 ind. ml⁻¹, respectively, and the corresponding maxima for B. rubens were 52 ± 3 and 102 ± 18 ind. ml⁻¹. At a given food level, competition for food reduced the peak abundances of both rotifers considerably. Increase in Zn concentration also lowered the rotifer abundances. The impact of zinc on competition between the two-rotifer species was evident at low-food level, mainly for A. fissa. At zinc concentrations of 0 and 0.125 mg l⁻¹, the populations of both rotifers continued to grow for about 10 days, but thereafter B. rubens began to decline. Role of zinc on the competitive outcome of the two species is discussed in relation to the changing algal densities in natural water bodies.     

Development and mathematical modeling of a two-stage reactor system for trichloroethylene degradation using <Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b

A two-stage reactor system was developed for the continuous degradation of gas-phase trichloroethylene (TCE). Methylosinus trichosporium OB3b was immobilized on activated carbon in a TCE degradation reactor, trickling biofilter (TBF). The TBF was coupled with a continuous stirred tank reactor (CSTR) to allow recirculation of microbial cells from/to the TBF for the reactivation of inactivated cells during TCE degradation. The mass transfer aspect of the TBF was analyzed, and mass transfer coefficient of 3.9 h⁻¹ was estimated. The loss of soluble methane monooxygenase (sMMO) activity was modeled based on a material balance on the CSTR and TBF, and transformation capacity (T _c) was determined to be 20.2 mol mg⁻¹. Maximum TCE degradation rate of 525 mg 1⁻¹ d⁻¹ was obtained and reactor has been stably operated for more than 270 days.     

Phenol biodegradation by the thermoacidophilic archaeon <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis> 98/2 in a fed-batch bioreactor

Toxic at low concentrations, phenol is one of the most common organic pollutants in air and water. In this work, phenol biodegradation was studied in extreme conditions (80°C, pH = 3.2) in a 2.7 l bioreactor with the thermoacidophilic archaeon Sulfolobus solfataricus 98/2. The strain was first acclimatized to phenol on a mixture of glucose (2000 mg l⁻¹) and phenol (94 mg l⁻¹) at a constant dissolved oxygen concentration of 1.5 mg l⁻¹. After a short lag-phase, only glucose was consumed. Phenol degradation then began while glucose was still present in the reactor. When glucose was exhausted, phenol was used for respiration and then for biomass build-up. After several batch runs (phenol < 365 mg l⁻¹), specific growth rate (μ_X) was 0.034 ± 0.001 h⁻¹, specific phenol degradation rate (q_P) was 57.5 ± 2 mg g⁻¹ h⁻¹, biomass yield (Y_X/P) was 52.2 ± 1.1 g mol⁻¹, and oxygen yield factor ( \textY_{\textX/\textO 2} ) \left( {{\text{Y}}_{{{\text{X}}/{\text{O}}_{ 2} }} } \right) was 9.2 ± 0.2 g mol⁻¹. A carbon recovery close to 100% suggested that phenol was exclusively transformed into biomass (35%) and CO₂ (65%). Molar phenol oxidation constant ( \textY_{\textO 2 /\textP} ) \left( {{\text{Y}}_{{{\text{O}}_{ 2} /{\text{P}}}} } \right) was calculated from stoichiometry of phenol oxidation and introducing experimental biomass and CO₂ conversion yields on phenol, leading to values varying between 4.78 and 5.22 mol mol⁻¹. Respiratory quotient was about 0.84 mol mol⁻¹, very close to theoretical value (0.87 mol mol⁻¹). Carbon dioxide production, oxygen demand and redox potential, monitored on-line, were good indicators of growth, substrate consumption and exhaustion, and can therefore be usefully employed for industrial phenol bioremediation in extreme environments.     

Ion currents induced by ATP and angiotensin II in cultured follicular cells of <Emphasis Type="Italic">Xenopus laevis</Emphasis>

Xenopus laevis oocytes are commonly used to study the biophysical and pharmacological properties of foreign ion channels and receptors, but little is known about those endogenously expressed in their enveloping layer of follicular cells (FCs). Whole-cell recordings and the perforated patch-clamp technique in cultured FCs held at −60 mV revealed that ATP (20–250 μM) generates inward currents of 465 ± 93 pA (mean ± standard error) in ∼60% of the FCs studied, whereas outward currents of 317 ± 100 pA were found in ∼5% of the cells. The net effect of ATP on the FCs was to activate both mono- and biphasic inward currents, with an associated increase in membrane chloride conductance. Two-microelectrode voltage-clamp recordings of nude oocytes held at −60 mV disclosed that ATP elicited biphasic inward currents, corresponding to the well-known F_in and S_in-like currents. ATP receptor antagonists like suramin, TNP-ATP, and RB2 did not inhibit any of these responses. On the other hand, when using wholecell recordings, 1 μM Ang II yielded smooth inward currents of 157 ± 45 pA in ∼16% of the FC held at −60 mV. The net Ang II response, mediated by the activation of the AT₁ receptor, was a chloride current inhibited by 10 nM ZD7155. This study will help to better understand the roles of ATP and Ang II receptors in the physiology of X. laevis oocytes.     

An alginate-layer technique for culture of Brassica oleracea L. protoplasts

Ten accessions belonging to the Brassica oleracea subspecies alba and rubra, and to B. oleracea var. sabauda were used in this study. Protoplasts were isolated from leaves and hypocotyls of in vitro grown plants. The influence of selected factors on the yield, viability, and mitotic activity of protoplasts immobilized in calcium alginate layers was investigated. The efficiency of protoplast isolation from hypocotyls was lower (0.7 ± 0.1 × 10⁶ ml⁻¹) than for protoplasts isolated from leaf mesophyll tissue (2 ± 0.1 × 10⁶ ml⁻¹). High (70–90%) viabilities of immobilized protoplasts were recorded, independent of the explant sources. The highest proportion of protoplasts undergoing divisions was noted for cv. Reball F1, both from mesophyll (29.8 ± 2.2%) and hypocotyl (17.5 ± 0.3%) tissues. Developed colonies of callus tissue were subjected to regeneration and as a result plants from six accessions were obtained.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

Estimation of the fraction of biologically active methyl <Emphasis Type="Italic">tert</Emphasis>-butyl ether degraders in a heterogeneous biomass sample

The fraction of biologically active methyl tert-butyl ether degraders in reactors is just as important for prediction of removal rates as knowledge of the kinetic parameters. The fraction of biologically active methyl tert-butyl ether degraders in a heterogeneous biomass sample, taken from a packed bed reactor, was determined using a batch kinetic based approach. The procedure involved modeling of methyl tert-butyl ether removal rates from batch experiments followed by parameter estimations. It was estimated to be 5–14% (w/w) of the measured volatile suspended solids concentration in the reactor.     

Carbon nutrition of mature green orchid <Emphasis Type="Italic">Serapias strictiflora</Emphasis> and its mycorrhizal fungus <Emphasis Type="Italic">Epulorhiza</Emphasis> sp.

We studied the nutritional modes of the orchid Serapias strictiflora and its mycorrhizal fungus Epulorhiza sp. using the differences in carbon isotopic composition (δ¹³C) of C₃ orchid and C₄ maize tissues. We found that if cultivated in substrate lacking any organic compounds, the mycorrhizal extraradical mycelia (δ¹³C = −26.3 ± 0.2 ‰) developed well, despite being fully dependent on nutrition from orchid roots (δ¹³C = −28.6 ± 0.1 ‰). If the mycorrhizal fungus had additional access to and colonized decaying maize roots (δ¹³C = −14.6 ± 0.1 ‰), its isotopic composition (δ¹³C = −21.6 ± 0.4 ‰) reflected a mixture of biotrophy and saprotrophy. No statistically significant differences in δ¹³C of new storage tubers were found between Epulorhiza-associated orchids with (δ¹³C = -28.2 ± 0.1 ‰) and without access to maize roots (δ¹³C = −28.6 ± 0.2 ‰). We conclude that autotrophy is the predominant nutritional mode of mature S. strictiflora plants and that they supply their mycorrhizal fungus with substantial amount of carbon (69 ± 3 % of the fungus demand), even if the fungus feeds saprotrophically.