Supplementation of 2nd break mushroom compost with isoleucine, leucine, valine, phenylalanine, Fermenten® and SoyPlus®

Three mushroom (Agaricus bisporus) crops (Crops 1, 2, 3) were grown to evaluate the effects of re-supplementing “spent” mushroom compost (MC) with the crystalline amino acids isoleucine (ile), leucine (leu), valine (val) and phenylalanine (phe) singly or in combination with Fermenten^® or SoyPlus^® on mushroom yield. Fermenten^® is a rumen fermentation enhancer while SoyPlus^® is a commercial delayed release mushroom nutrient. The most important single amino acid found for stimulating mushroom yield from 2nd break MC was ile. Crystalline ile added to 2nd break MC at 3.6% (dry wt) increased mushroom yields by 28.3% and 68.7% (average 48.5%) in Crops 1 and 2, respectively, compared to the non-supplemented control. In Crop 3, the addition of 5% or 10% ile to Fermenten^® and SoyPlus^® (3.6% total combined dry wt) did not significantly improve mushroom yield over treatments containing Fermenten^® or SoyPlus^® (3.6% total dry wt) alone. However, mixtures of equal quantities of Fermenten^®, ile and val significantly increased yield over Fermenten^® alone. Use of ile and val as supplements to stimulate mushroom yield from 2nd break MC is not economically viable because these amino acids are not commercially available at feed grade prices.     

Re-supplementing and re-casing mushroom (<Emphasis Type="Italic">Agaricus bisporus</Emphasis>) compost for a second crop

Experiments were performed to determine the effect of adding nutrient supplements to colonized mushroom compost (MC) for the production of a second crop of mushrooms. Mushrooms were harvested for 1, 2 or 3 flushes, the casing removed and the MC then was fragmented and re-supplemented with delayed release supplements treated or non-treated with fungicide (thiophanate-methyl; Topsin M 70WP) and re-cased. Overall double-crop yields were higher when MC was re-supplemented after 1st flush (1st flush MC) as compared to re-supplementation after the 2nd or 3rd flushes. Mean double-crop BEs were 128, 119 and 109% when 1st-, 2nd- and 3rd-flush MCs were used, respectively. Treatment of delayed release supplement with thiophanate-methyl fungicide did not affect mushroom yields. Soluble salts and potassium concentrations increased 350 and 900%, respectively, in the casing overlay through three flushes suggesting that removal of the casing would help to alleviate the build up of these potential growth-limiting materials. Re-supplementing and re-casing of MC represents a potential opportunity for growers to increase revenues and reduce costs associated with preparation and disposal of compost. The ability to double-crop mushroom compost would provide growers a chance to increase yields by 40% or more, depending on whether they re-supplement and re-case after 1st, 2nd or 3rd flush.     

Activation of the human red cell calcium ATPase by calcium pretreatment

Some kinetic parameters of the human red cell Ca²⁺-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V _max of about 0.4 μmol P_i/mg × hr and a K _s of 0.3 μm Ca²⁺ were found. When Mg²⁺ (10 mm) or Ca²⁺(10 μm) were also added during preincubation, V _maxbut not Kwas altered. Ca²⁺ was more effective than Mg²⁺, thus increasing V _max to about 1.3 μmol P_i/mg × hr. The presence of both Ca²⁺ and Mg²⁺ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V _max. Conversely, addition of ATP (2 mm) with either Ca²⁺ or Ca²⁺ plus Mg²⁺increased V_max without affecting K. Preincubation with Ca²⁺ for periods longer than 30 min further increased V_maxand reduced Kto levels as low as found with calmodulin treatment. The Ca²⁺ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg²⁺, Ca²⁺ or Ca²⁺ plus Mg²⁺ did not differ significantly from each other. Moreover, immunodetection of Ca²⁺-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca²⁺ plus ATP in the presence of Mg²⁺ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca²⁺ effect. Ca²⁺ preincubation may stimulate the Ca²⁺-ATPase activity by stabilizing or promoting the E₁ conformation.     

Yield,size, and mushroom solids content of <Emphasis Type="Italic">Agaricus bisporus</Emphasis> produced on non-composted substrate and spent mushroom compost

Three crops of Agaricus bisporus were grown on non-composted substrate (NCS), spent mushroom compost (SMC), a 50/50 mixture of NSC/SMC, or pasteurized Phase II compost. NCS consisted of oak sawdust (28% oven dry wt), millet (29%), rye (8%), peat (8%), ground alfalfa (4%), ground soybean (4%), wheat bran (9%) and CaCO₃ (10%). Substrates were non-supplemented or supplemented with Target^® (a commercial delayed release nutrient for mushroom culture) or soybean meal at spawning or casing, or with Micromax^® (a mixture of nine micronutrients) at spawning. Mushroom yield (27.2 kg/m²) was greatest on a 50/50 mixture of NCS/SMC supplemented with 10% (dry wt) Target^® at casing. The same substrate supplemented with Target^® at spawning yielded 20.1 kg/m². By comparison, mushroom yield on Phase II compost supplemented at casing or at spawning with Target^® was 21.6 kg/m² and 20.6 kg/m², respectively. On NCS amended with 0.74% or 0.9% Micromax^® at spawning, yields increased by 51.8% (12.9 kg/m²) and 71.8% (14.6 kg/m²), respectively, over non-amended NCS (8.5 kg/m²). Conversely, mushroom yields were not affected when Micromax^® was added to a 50/50 mixture of NCS/SMC. Mushroom solids content was higher in mushrooms harvested from NCS amended with 0.74% Micromax^® (9.6%) compared to non-amended NCS (8.3%).     

d-Lactic acid biosynthesis from biomass-derived sugars via Lactobacillus delbrueckii fermentation

Poly-lactic acid (PLA) derived from renewable resources is considered to be a good substitute for petroleum-based plastics. The number of poly l-lactic acid applications is increased by the introduction of a stereocomplex PLA, which consists of both poly-l and d-lactic acid and has a higher melting temperature. To date, several studies have explored the production of l-lactic acid, but information on biosynthesis of d-lactic acid is limited. Pulp and corn stover are abundant, renewable lignocellulosic materials that can be hydrolyzed to sugars and used in biosynthesis of d-lactic acid. In our study, saccharification of pulp and corn stover was done by cellulase CTec2 and sugars generated from hydrolysis were converted to d-lactic acid by a homofermentative strain, L. delbrueckii, through a sequential hydrolysis and fermentation process (SHF) and a simultaneous saccharification and fermentation process (SSF). 36.3 g L^?1 of d-lactic acid with 99.8 % optical purity was obtained in the batch fermentation of pulp and attained highest yield and productivity of 0.83 g g^?1 and 1.01 g L^?1 h^?1, respectively. Luedeking–Piret model described the mixed growth-associated production of d-lactic acid with a maximum specific growth rate 0.2 h^?1 and product formation rate 0.026 h^?1, obtained for this strain. The efficient synthesis of d-lactic acid having high optical purity and melting point will lead to unique stereocomplex PLA with innovative applications in polymer industry.     

Recombinant production and characterization of an N-acyl-d-amino acid amidohydrolase from Streptomyces sp. 64E6

N-Acyl-d-amino acid amidohydrolases (d-aminoacylases) are often used as tools for the optical resolution of d-amino acids, which are important products with applications in industries related to medicine and cosmetics. For this study, genes encoding d-aminoacylase were cloned from the genomes of Streptomyces spp. using sequence-based screening. They were expressed by Escherichia coli and Streptomyces lividans. Almost all of the cell-free extracts exhibit hydrolytic activity toward N-acetyl-(Ac-)d-Phe (0.05–6.32 μmol min^?1 mg^?1) under conditions without CoCl₂. Addition of 1 mM CoCl₂ enhanced their activity. Among them, the highest activity was observed from cell-free extracts prepared from S. lividans that possess the d-aminoacylase gene of Streptomyces sp. 64E6 (specific activities were, respectively, 7.34 and 9.31 μmol min^?1 mg^?1 for N-Ac-d-Phe and N-Ac-d-Met hydrolysis). Furthermore, when using glycerol as a carbon source for cultivation, the recombinant enzyme from Streptomyces sp. 64E6 was produced in 4.2-fold greater quantities by S. lividans than when using glucose. d-Aminoacylase from Streptomyces sp. 64E6 showed optimum at pH 8.0–9.0. It was stable at pH 5.5–9.0 up to 30 °C. The enzyme hydrolyzed various N-acetyl-d-amino acids that have hydrophobic side chains. In addition, the activity toward N-chloroacetyl-d-Phe was 2.1-fold higher than that toward N-Ac-d-Phe, indicating that the structure of N-acylated portion of substrate altered the activity.     

The arthropodBundenbachiellus giganteus from the Lower Devonian Hunsrück Slate, Germany

The large Hunsrück Slate arthropodBundenbachiellus giganteus (Broili, 1929) has been interpreted as a myriapod, a possible arthropleurid, and most recently as a distorted specimen of the arachnomorph arthropodCheloniellon. The Single specimen lacks the head region and is characterised by a narrow trunk bearing a series of biramous appendages with a segmented endopod and filamentous exopod. A reinvestigation demonstrates that it is not referable toCheloniellon but represents a distinct taxon of whichEschenbachiellus wuttkensis Briggs &;Bartels, 2001, also based on a Single Hunsrück Slate specimen, is a junior synonym. Minor features ofStürmer &;Bergström’s (1978) reconstruction ofCheloniellon, which were based on the holotype ofBundenbachiellus giganteus, are revised.     

Enhanced saccharification of rice straw using hypochlorite-hydrogen peroxide

Rice straw is a lignocellulosic biomass, and has been recognized as a renewable organic substance and alternative energy source. In this study, rice straw was pretreated with hypochlorite-hydrogen peroxide (Ox-B) solution. The optimal pretreatment conditions were determined via response surface methodology, and the pretreated rice straw was hydrolyzed with exo-glucanase, endoglucanase, hemicellulase, and β-glucosidase Accellerase 1000? (endo-glucanase equivalent activity of 1,250 carboxy methyl cellulose (CMC) U/g of rice straw pretreated for 24 h). The optimal conditions were as follows: 60 min pretreatment using Ox-B solution containing 0.6% hypochlorite and 25% hydrogen peroxide for 1 g of rice straw in a total reaction volume of 240 mL. Under these conditions, 406.8 mg of d-glucose and 224.0 mg of d-xylose were obtained from 1 g of rice straw. The fermentation of enzymatic hydrolysates containing 8.14 g/L d-glucose and 4.49 g/L d-xylose with Pichia stipitis generated 3.65 g/L of ethanol with a corresponding yield of 0.37 g/g. The maximum possible ethanol conversion rate is 72.54%.     

Preparation of eutectic substrate mixtures for enzymatic conversion of ATC to l-cysteine at high concentration levels

High concentration eutectic substrate solutions for the enzymatic production of l-cysteine were prepared. Eutectic melting of binary mixtures consisting of d,l-2-amino-Δ²-thiazoline-4-carboxylic acid (ATC) as a substrate and malonic acid occurred at 39 °C with an ATC mole fraction of 0.5. Formation of eutectic mixtures was confirmed using SEM, SEM–EDS, and XPS surface analyses. Sorbitol, MnSO₄, and NaOH were used as supplements for the enzymatic reactions. Strategies for sequential addition of five compounds, including a binary ATC mixture and supplements, during preparation of eutectic substrate solutions were established. Eutectic substrate solutions were stable for 24 h. After 6 h of enzymatic reactions, a 550 mM l-cysteine yield was obtained from a 670 mM eutectic ATC solution.     

Metabolic engineering of Escherichia coli for biosynthesis of d-galactonate

d-galactose is an attractive substrate for bioconversion. Herein, Escherichia coli was metabolically engineered to convert d-galactose into d-galactonate, a valuable compound in the polymer and cosmetic industries. d-galactonate productions by engineered E. coli strains were observed in shake flask cultivations containing 2 g L^?1 d-galactose. Engineered E. coli expressing gld coding for galactose dehydrogenase from Pseudomonas syringae was able to produce 0.17 g L^?1 d-galactonate. Inherent metabolic pathways for assimilating both d-galactose and d-galactonate were blocked to enhance the production of d-galactonate. This approach finally led to a 7.3-fold increase with d-galactonate concentration of 1.24 g L^?1 and yield of 62.0 %. Batch fermentation in 20 g L^?1 d-galactose of E. coli ?galK?dgoK mutant expressing the gld resulted in 17.6 g L^?1 of d-galactonate accumulation and highest yield of 88.1 %. Metabolic engineering strategy developed in this study could be useful for industrial production of d-galactonate.