Spent mushroom substrates as component of growing media for germination and growth of horticultural plants

This research work was conducted in order to investigate the possibility of using spent mushroom substrate (SMS) in the production of horticultural seedlings replacing part of the peat in the growing media. Three vegetable species with different salt sensitivities, the less sensitive being tomato (Lycopersicon esculentum var. Muchamiel), the moderately salt-sensitive being courgette (Cucurbita pepo L. var. Afrodite F1) and the most salt-sensitive being pepper (Capsicum annum L. var. Lamuyo F1) were grown in 12 media containing SMS of two types of mushroom (Agaricus bisporus (SMS-AB) and Pleurotus ostreatus (SMS-PO)) or a mixture of both 50% (v/v) (SMS-50), as well as peat in various ratios. The proportions of each residue in the mixtures elaborated with peat were 25%, 50%, 75% and 100% v/v residue. A substrate of 100% peat was used as control. The experiment was arranged in a completely-randomised design with two replicates per treatment under greenhouse conditions. Prior to sowing, some physical, physico-chemical and chemical properties of the growing media were determined and seed germination and fresh weight of seedling were also measured. In most of the cases, the addition of SMS to the growing media produced an increase in the pH values, salt contents, macro and micronutrient concentrations and a decrease in the water holding capacity contents in comparison to peat, whereas great differences were found in the air capacity values between SMS-based substrates and peat. Up to 75% SMS can be used in mixtures with peat for seed germination of the plant species studied. Regarding the most suitable SMS-based substrates for plant growth, any substrate could be used for tomato seedling production. However, all SMS-AB-based substrates and the media containing low dose of SMS-PO and SMS-50 were adequate for growth of courgette and pepper.     

Regeneration of Three <Emphasis Type="Italic">Sphagnum</Emphasis> species

Sphagnum capillifolium var. tenellum, S. magellanicum, and S. recurvum var. brevifolium were regenerated from stem pieces grown in containers to assess their potential for use in peatland restoration projects. The effect of two water levels; peat, peat/sand or peat/clay substrates; and peat decomposition level on the species’ regeneration was evaluated. S. magellanicum attained the greatest cover on the peat or peat/sand mixture using decomposed peat when the growing surface was occasionally inundated. S. recurvum attained the greatest cover grown on the peat or peat/sand mixture using undecomposed peat when the water level was kept below the surface. S. capillifolium showed an affinity for the peat/clay mixture, and overall attained a greater total cover than the other species when grown under the lower water level on all substrate types, with total cover approximately three to five times that of the others. When developing management plans for restoration of mined peatlands, species-specific responses to water level, type and extent of mineral soil mixed with the peat surface, and peat decomposition level should be considered.     

Effects of Some Environmental Factors on Growth Characteristics of Candida utilis on Peat Hydrolysates

Samples of peat from Pine Island and Brookston bogs in Minnesota were hydrolyzed with various concentrations of HCl or H₂SO₄ solutions, before or after debituminization (an extraction process used to remove waxy materials, bitumens, from peat), to produce peak hydrolysates as growth substrates for Candida utilis. Hydrolysates were neutralized with concentrated NaOH solution to pH 3.5, 4.5, 5.0, 5.5, 6.0, and 7.0. The precipitated humates were removed by filtration. The resulting peat hydrolysates were amended with reagent-grade K₂HPO₄, K₂SO₄, and MgSO₄, 200, 100, and 50 mg per liter of peat hydrolysate, respectively. The debituminized peat produced more total nitrogen (TN) and reducing substances (RS) than the nondebituminized peat. Peat hydrolysates produced by HCl solutions contained slightly higher RS and TN than those produced by H₂SO₄ solutions; however, the latter were better growth substrates than the former. The yield coefficients in both H₂SO₄ and HCl hydrolysates initially decreased at 12 to 24 h and then increased gradually over the remaining incubation period (24 to 96 h). As TN and RS were increased, an increase in cell density, biomass, and productivity was observed. In contrast, a decrease in specific growth rate occurred as the RS and TN were increased. The generation time of C. utilis was affected by the concentrations of RS and TN. A peak substrate yield coefficient was found at pH 5.0 in HCl hydrolysates and at pH 6.0 to 6.5 in H₂SO₄ hydrolysates. Good linear correlation coefficients were found between RS and biomass of C. utilis. The coefficients of correlation increased as the TN level in hydrolysates was increased.     

Mechanisms involved in the re-establishment of Sphagnum-dominated vegetation in rewetted bog remnants

Restoration of peat bog vegetation inhighly degraded peatlands is generallyattempted by improving the hydrology ofthese areas. The present paper discussesand explains various restoration strategiesrelating to peat quality, water chemistryand hydrology. In some cases, (shallow)inundation of bog remnants leads to a rapidredevelopment of (floating) Sphagnumvegetation, usually when poorly humifiedSphagnum peat is still present. Afterinundation, the peat either swells up tothe newly created water table or becomesbuoyant, in both cases creating a favorablesubstrate for Sphagnum mosses. Bulkdensity and methane production rate play animportant role in the buoyancy of floatingpeat, methane providing buoyancy to thesubstrates. The presence of (slightly)calcareous groundwater in the peat base mayenhance the development of floating raftsby stimulating decomposition processes.Alternatively, the growth of submerged Sphagnum species can also lead to thedevelopment of floating rafts. This dependson the penetration of light into the waterlayer and the availability of carbondioxide in the water layer.Many bog remnants, however, only havestrongly humified peat, which does notfavor the redevelopment of Sphagnumcarpets after deep inundation. On the otherhand, most peat moss species appear to dovery well on surface soaked black peat,which is why shallow inundation (< 0.3 m)is to be preferred in such cases.Compartmentalization of the terrain willprobably be necessary to ensure a more orless constant water table.An important prerequisite for thesuccessful restoration of bog remnants isthe development of a hydrologicallyself-regulating acrotelm. Key speciesinvolved in this development are Sphagnum magellanicum, Sphagnumpapillosum and Sphagnum rubellum.These typical hummock and lawn species areusually very slow colonizers compared tohollow species such as Sphagnumcuspidatum and Sphagnum fallax.Introduction of key species in carpetsdominated by hollow species or on baresubstrates appears to be very successful,indicating that the main constraint iscolonization.     

A palaeoecological study of holocene peat bog sections in Germany and The Netherlands,based on the analysis of pollen,spores and macro- and microscopic remains of fungi,algae, cormophytes and animals

A Holocene peat section of the Engbertsdijksveen (The Netherlands) was analysed at centrimetre intervals for all kinds of micro- and macrofossils in order to obtain maximum information regarding local vegetation and animal succession and regional changes in the prevailing vegetation types. The analysed micro- and macrofossils are illustrated, described and interpreted. Among them are about 70 fungal taxa, about 25 animal taxa, at least 4 algae, and about 15 fossils of unknown identity or origin.The taxonomic designation, matrix, host- or substrate specificity and indicator value of representatives of five different groups of fossils have been tabulated.The fruit-bodies of several taxa distinguished as spore types were found during the macrofossil analysis, e.g., ascospores, mycelium and fruit-bodies of the parasite Meliola cf. niessleana (Type 14) are present in the levels in which Calluna vulgaris played a role in the local vegetation.The ascomycete Geoglossum sphagnophilum was, according to its spore distribution, a common element in the bog vegetation during the Atlantic and Subboreal periods.Zygospores of Mougeotia cf. gracillima (Type 61) and other Zygnemataceae indicate the levels where these algae could grow during the local succession in the peat bog. The zygospores indicate phases of shallow, stagnant, mesotrophic open water during spring.Conventional analysis of pollen and macro-remains with a high resolution in time (centimetre diagrams), the above-mentioned new information, and the correlation of events inside and outside the site of the peat bog lead to the following main conclusions:The Atlantic-Subboreal transition, well known for its elm decline, could be correlated with a local change in bog vegetation. Following the alteration of wet Scheuchzeria palustris vegetation with hummock vegetation in the Atlantic period, the appearance of a Molinia coerulea peat coincides with the elm decline. According to the present ecology of M. coerulea, this change must have been caused by a seasonally recurring, considerable lowering of the groundwater level during summer. During winter the groundwater rose so far that in spring shallow open water prevailed and Mougeotia cf. gracillima could develop. This local vegetational succession indicates an unstable climatic period with pronounced dry seasons during the early Subboreal.During the climatic deterioration of the Subboreal-Subatlantic transition the incidence of Corylus avellana decreased. The Corylus pollen curve shows several cyclic fluctuations during this period (1400-600 B.C.). Depressions in the Corylus pollen curve correspond with particularly wet local vegetation (more oceanic, possibly cooler phases), Corylus pollen maxima correspond with relatively dry bog vegetation (less oceanic, possibly warmer phases). These cyclic fluctuations correspond to approximately 150–200 historical years each. The increasing oceanicity ultimately resulted in the formation of peat by Sphagnum species of the section Cymbifolia. In this Subatlantic Cymbifolia peat the fluctuations of the minor constituent Sphagnum cf. rubellum are clearly correlated with the fluctuations of the curve of the rhizopod Amphitrema flavum. This phenomenon is interpreted as a reflection of more and less oceanic phases during the Subatlantic period.All available data were expressed in curves, in order to indicate changes in local and perhaps regional moisture conditions.Ther percentages of plants indicating human influence are relatively low during the Neolithic and Bronze Age. During the Iron Age the influence of prehistoric man was relatively important. Secale cereale was already grown during the secondary century B.C. Population density decreased again during the second century A.D.The present study is intended as a first step towards a more detailed knowledge of the changing ecosystems of bogs and forests and in their response to changing environmental factors, such as minor and major climatic cycles.     

Expression,characterization, and improvement of a newly cloned halohydrin dehalogenase from Agrobacterium tumefaciens and its application in production of epichlorohydrin

A gene encoding halohydrin dehalogenase (HHDH) from Agrobacterium tumefaciens CCTCC M 87071 was cloned and expressed in Escherichia coli. To increase activity and stability of HHDH, 14 amino acid residues around the active site and substrate-binding pocket based on the structural analysis and molecular docking were selected as targets for site-directed mutagenesis. The studies showed that the mutant HHDH (Mut-HHDH) enzyme had a more accessible substrate-binding pocket than the wild-type HHDH (Wt-HHDH). Molecular docking revealed that the distance between the substrate and active site was closer in mutant which improved the catalytic activity. The expressed Wt-HHDH and Mut-HHDH were purified and characterized using 1,3-dichloro-2-propanol (1,3-DCP) as substrates. The specific activity of the mutant was enhanced 26-fold and the value of k _cat was 18.4-fold as compared to the Wt-HHDH, respectively. The Mut-HHDH showed threefold extension of half-life at 45 °C than that of Wt-HHDH. Therefore it is possible to add 1,3-DCP concentration up to 100 mM and epichlorohydrin (ECH) was produced at a relatively high conversion and yield (59.6 %) using Mut-HHDH as catalyst. This Mut-HHDH could be a potential candidate for the upscale production of ECH.     

Inoculant Production with Diluted Liquid Cultures of Rhizobium spp. and Autoclaved Peat: Evaluation of Diluents, Rhizobium spp., Peats, Sterility Requirements, Storage, and Plant Effectiveness

Fully grown broth cultures of various fast- and slow-growing rhizobia were deliberately diluted with various diluents before their aseptic incorporation into autoclaved peat in polypropylene bags (aseptic method) or mixed with the peat autoclaved in trays (tray method). In a factorial experiment with the aseptic method, autoclaved and irradiated peat samples from five countries were used to prepare inoculants with water-diluted cultures of three Rhizobium spp. When distilled water was used as the diluent, the multiplication and survival of rhizobia in the peat was similar to that with diluents having a high nutrient status when the aseptic method was used. In the factorial experiment, the mean viable counts per gram of inoculant were log 9.23 (strain TAL 102) > log 8.92 (strain TAL 82) > log 7.89 (strain TAL 182) after 24 weeks of storage at 28°C. The peat from Argentina was the most superior for the three Rhizobium spp., with a mean viable count of log 9.0 per g at the end of the storage period. The quality of inoculants produced with diluted cultures was significantly (P = 0.05) better with irradiated than with autoclaved peat, as shown from the factorial experiment. With the tray method, rhizobia in cultures diluted 1,000-fold or less multiplied and stored satisfactorily in the presence of postinoculation contaminants, as determined by plate counts, membrane filter immunofluorescence, and plant infection procedures. All strains of rhizobia used in both the methods showed various degrees of population decline in the inoculants when stored at 28°C. Fast- and slow-growing rhizobia in matured inoculants produced by the two methods showed significant (P < 0.01) decline in viability when stored at 4°C, whereas the viability of some strains increased significantly (P < 0.01) at the same temperature. The plant effectiveness of inoculants produced with diluted cultures and autoclaved peat did not differ significantly from that of inoculants produced with undiluted cultures and gamma-irradiated peat.     

The Challenge of Peat Substitution in Organic Seedling Production: Optimization of Growing Media Formulation through Mixture Design and Response Surface Analysis

Peat replacement is an increasing demand in containerized and transplant production, due to the environmental constraints associated to peat use. However, despite the wide information concerning the use of alternative materials as substrates, it is very complex to establish the best materials and mixtures. This work evaluates the use of mixture design and surface response methodology in a peat substitution experiment using two alternative materials (green compost and palm fibre trunk waste) for transplant production of tomato (Lycopersicon esculentum Mill.); melon, (Cucumis melo L.); and lettuce (Lactuca sativa L.) in organic farming conditions. In general, the substrates showed suitable properties for their use in seedling production, showing the best plant response the mixture of 20% green compost, 39% palm fibre and 31% peat. The mixture design and applied response surface methodology has shown to be an useful approach to optimize substrate formulations in peat substitution experiments to standardize plant responses.     

Enhancing planting stock quality of Italian cypress (Cupressus sempervirens L.) by pre-cultivation in mini-plugs

Native species are important in ecological restoration of degraded forest ecosystems provided that their ecological engineering potential is exploited. Their failure to get established is usually due to the use of low-quality planting stock and adverse site conditions. Mini-plug transplants are a relatively new and promising production system in the forest nursery area. Objectives of our study were the evaluation of seedling production of Italian cypress (Cupressus sempervirens) in mini-plug containers, the study of the effect of mini-plug density and substrate on planting stock quality, the comparison of the performance between mini-plug seedlings and the standard planting stock produced by Greek nurseries and the evaluation of physiological and morphological variables as predictive indicators of Italian cypress field performance. Our results showed that cypress seedlings could be produced using the mini-plug technique, resulting in higher quality seedlings than the standard stock type. Pre-cultivation of cypress seedlings under favorable conditions for a period of 5 weeks using peat and high mini-plug densities (1800–3500 mini-plugs m^?2) could be recommended. Grading of seedlings by using both easily measured morphological variables (root length, leaf area, root and shoot dry weight) and physiological tests, such as shoot electrolyte leakage, may improve survival and transplanting success. It is concluded that mini-plugs can serve ecological restoration of degraded forest ecosystems more efficiently than standard planting stock.     

Expression of Plant Flavor Genes in Lactococcus lactis

Lactic acid bacteria, such as Lactococcus lactis, are attractive hosts for the production of plant-bioactive compounds because of their food grade status, efficient expression, and metabolic engineering tools. Two genes from strawberry (Fragaria x ananassa), encoding an alcohol acyltransferase (SAAT) and a linalool/nerolidol synthase (FaNES), were cloned in L. lactis and actively expressed using the nisin-induced expression system. The specific activity of SAAT could be improved threefold (up to 564 pmol octyl acetate h⁻¹ mg protein⁻¹) by increasing the concentration of tRNA₁^Arg, which is a rare tRNA molecule in L. lactis. Fermentation tests with GM17 medium and milk with recombinant L. lactis strains expressing SAAT or FaNES resulted in the production of octyl acetate (1.9 μM) and linalool (85 nM) to levels above their odor thresholds in water. The results illustrate the potential of the application of L. lactis as a food grade expression platform for the recombinant production of proteins and bioactive compounds from plants.     

Sulfate Reduction in Peat from a New Jersey Pinelands Cedar Swamp

Microbial sulfate reduction rates in acidic peat from a New Jersey Pine Barrens cedar swamp in 1986 were similar to sulfate reduction rates in freshwater lake sediments. The rates ranged from a low of 1.0 nmol cm⁻³ day⁻¹ in February at 7.5- to 10.0-cm depth to 173.4 nmol cm⁻³ day⁻¹ in July at 5.0- to 7.5-cm depth. The presence of living Sphagnum moss at the surface generally resulted in reduced rates of sulfate reduction. Pore water sulfate concentrations and water table height also apparently affected the sulfate reduction rate. Concentrations of sulfate in pore water were nearly always higher than those in surface water and groundwater, ranging from 26 to 522 μM. The elevated pore water sulfate levels did not result from the evapotranspiratory concentration of infiltrating stream water or groundwater, but probably resulted from oxidation of reduced sulfur compounds, hydrolysis of ester sulfates present in the peat, or both. The total sulfur content of peat that had no living moss at the surface was 164.64 ± 1.5 and 195.8 ± 21.7 μmol g (dry weight)⁻¹ for peat collected from 2.5 to 5.0 and 7.5 to 10.0 cm, respectively. Organosulfur compounds accounted for 84 to 88% of the total sulfur that was present in the peat. C-bonded sulfur accounted for 91 to 94% of the organic sulfur, with ester sulfate being only a minor constituent. Reduced inorganic sulfur species in peat from 2.5 to 7.5 cm were dominated by H₂S-FeS (68%), while pyritic sulfide was the predominant inorganic sulfur species in the peat from depths of 7.5 to 10.0 cm (75%).     

Climate change effects on carbon and nitrogen mineralization in peatlands through changes in soil quality

Climate change will directly affect carbon and nitrogen mineralization through changes in temperature and soil moisture, but it may also indirectly affect mineralization rates through changes in soil quality. We used an experimental mesocosm system to examine the effects of 6‐year manipulations of infrared loading (warming) and water‐table level on the potential anaerobic nitrogen and carbon (as carbon dioxide (CO₂) and methane (CH₄) production) mineralization potentials of bog and fen peat over 11 weeks under uniform anaerobic conditions. To investigate the response of the dominant methanogenic pathways, we also analyzed the stable isotope composition of CH₄ produced in the samples. Bog peat from the highest water‐table treatment produced more CO₂ than bog peat from drier mesocosms. Fen peat from the highest water‐table treatment produced the most CH₄. Cumulative nitrogen mineralization was lowest in bog peat from the warmest treatment and lowest in the fen peat from the highest water‐table treatment. As all samples were incubated under constant conditions, observed differences in mineralization patterns reflect changes in soil quality in response to climate treatments. The largest treatment effects on carbon mineralization as CO₂ occurred early in the incubations and were ameliorated over time, suggesting that the climate treatments changed the size and/or quality of a small labile carbon pool. CH₄ from the fen peat appeared to be predominately from the acetoclastic pathway, while in the bog peat a strong CH₄ oxidation signal was present despite the anaerobic conditions of our incubations. There was no evidence that changes in soil quality have lead to differences in the dominant methanogenic pathways in these systems. Overall, our results suggest that even relatively short‐term changes in climate can alter the quality of peat in bogs and fens, which could alter the response of peatland carbon and nitrogen mineralization to future climate change.     

Microbial Species Diversity,Community Dynamics,and Metabolite Kinetics of Water Kefir Fermentation

Water kefir is a sour, alcoholic, and fruity fermented beverage of which the fermentation is started with water kefir grains. These water kefir grains consist of polysaccharide and contain the microorganisms responsible for the water kefir fermentation. In this work, a water kefir fermentation process was followed as a function of time during 192 h to unravel the community dynamics, the species diversity, and the kinetics of substrate consumption and metabolite production. The majority of the water kefir ecosystem was found to be present on the water kefir grains. The most important microbial species present were Lactobacillus casei/paracasei, Lactobacillus harbinensis, Lactobacillus hilgardii, Bifidobacterium psychraerophilum/crudilactis, Saccharomyces cerevisiae, and Dekkera bruxellensis. The microbial species diversities in the water kefir liquor and on the water kefir grains were similar and remained stable during the whole fermentation process. The major substrate, sucrose, was completely converted after 24 h of fermentation, which coincided with the production of the major part of the water kefir grain polysaccharide. The main metabolites of the fermentation were ethanol and lactic acid. Glycerol, acetic acid, and mannitol were produced in low concentrations. The major part of these metabolites was produced during the first 72 h of fermentation, during which the pH decreased from 4.26 to 3.45. The most prevalent volatile aroma compounds were ethyl acetate, isoamyl acetate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate, which might be of significance with respect to the aroma of the end product.     

Physiological Changes in Rhizobia after Growth in Peat Extract May Be Related to Improved Desiccation Tolerance

Improved survival of peat-cultured rhizobia compared to survival of liquid-cultured cells has been attributed to cellular adaptations during solid-state fermentation in moist peat. We have observed improved desiccation tolerance of Rhizobium leguminosarum bv. trifolii TA1 and Bradyrhizobium japonicum CB1809 after aerobic growth in water extracts of peat. Survival of TA1 grown in crude peat extract was 18-fold greater than that of cells grown in a defined liquid medium but was diminished when cells were grown in different-sized colloidal fractions of peat extract. Survival of CB1809 was generally better when grown in crude peat extract than in the control but was not statistically significant (P > 0.05) and was strongly dependent on peat extract concentration. Accumulation of intracellular trehalose by both TA1 and CB1809 was higher after growth in peat extract than in the defined medium control. Cells grown in water extracts of peat exhibit morphological changes similar to those observed after growth in moist peat. Electron microscopy revealed thickened plasma membranes, with an electron-dense material occupying the periplasmic space in both TA1 and CB1809. Growth in peat extract also resulted in changes to polypeptide expression in both strains, and peptide analysis by liquid chromatography-mass spectrometry indicated increased expression of stress response proteins. Our results suggest that increased capacity for desiccation tolerance in rhizobia is multifactorial, involving the accumulation of trehalose together with increased expression of proteins involved in protection of the cell envelope, repair of DNA damage, oxidative stress responses, and maintenance of stability and integrity of proteins.     

In-gel protein phosphatase assay using fluorogenic substrates

We developed a method for the detection of phosphatase activity using fluorogenic substrates after polyacrylamide gel electrophoresis. When phosphatases such as Ca²⁺/calmodulin-dependent protein kinase phosphatase (CaMKP), protein phosphatase 2C (PP2C), protein phosphatase 5 (PP5), and alkaline phosphatase were resolved by polyacrylamide gel electrophoresis in the absence of SDS and the gel was incubated with a fluorogenic substrate such as 4-methylumbelliferyl phosphate (MUP), all of these phosphatase activities could be detected in situ. Although 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) as well as MUP could be used as a fluorogenic substrate for an in-gel assay, MUP exhibited lower background fluorescence. Using this procedure, several fluorescent bands that correspond to endogenous phosphatases were observed after electrophoresis of various crude samples. The in-gel phosphatase assay could also be used to detect protein phosphatases resolved by SDS-polyacrylamide gel electrophoresis. In this case, however, the denaturation/renaturation process of resolved proteins was necessary for the detection of phosphatase activity. This procedure could be used for detection of renaturable protein phosphatases such as CaMKP and some other phosphatases expressed in cell extracts. The present fluorescent in-gel phosphatase assay is very useful, since no radioactive compounds or no special apparatus are required.     

Effects of Standard Humic Materials on Relative Bioavailability of NDL-PCBs in Juvenile Swine

Young children with their hand-to-mouth activity may be exposed to contaminated soils. However few studies assessing exposure of organic compounds sequestrated in soil were realized. The present study explores the impact of different organic matters on retention of NDL-PCBs during digestive processes using commercial humic substances in a close digestive model of children: the piglet. Six artificial soils were used. One standard soil, devoid of organic matter, and five amended versions of this standard soil with either fulvic acid, humic acid, Sphagnum peat, activated carbon or a mix of Sphagnum peat and activated carbon (95∶5) (SPAC) were prepared. In order to compare the different treatments, we use spiked oil and negative control animals. Forty male piglets were randomly distributed in 7 contaminated and one control groups (n  = 5 for each group). During 10 days, the piglets were fed artificial soil or a corn oil spiked with 19 200 ng of Aroclor 1254 per g of dry matter (6 000 ng.g⁻¹ of NDL-PCBs) to achieve an exposure dose of 1 200 ng NDL-PCBs.Kg⁻¹ of body weight per day. NDL-PCBs in adipose tissue were analyzed by GC-MS. Fulvic acid reduced slightly the bioavailability of NDL-PCBs compared to oil. Humic acid and Sphagnum peat reduced it significantly higher whereas activated carbon reduced the most. Piglets exposed to soil containing both activated carbon and Shagnum peat exhibited a lower reduction than soil with only activated carbon. Therefore, treatment groups are ordered by decreasing value of relative bioavailability as following: oil ≥ fulvic acid>Sphagnum peat ≥ Sphagnum peat and activated carbon ≥ Humic acid>>activated carbon. This suggests competition between Sphagnum peat and activated carbon. The present study highlights that quality of organic matter does have a significant effect on bioavailability of sequestrated organic compounds.     

Hydrogenotrophic Methanogenesis by Moderately Acid-Tolerant Methanogens of a Methane-Emitting Acidic Peat

The emission of methane (1.3 mmol of CH₄ m⁻² day⁻¹), precursors of methanogenesis, and the methanogenic microorganisms of acidic bog peat (pH 4.4) from a moderately reduced forest site were investigated by in situ measurements, microcosm incubations, and cultivation methods, respectively. Bog peat produced CH₄ (0.4 to 1.7 μmol g [dry wt] of soil⁻¹ day⁻¹) under anoxic conditions. At in situ pH, supplemental H₂-CO₂, ethanol, and 1-propanol all increased CH₄ production rates while formate, acetate, propionate, and butyrate inhibited the production of CH₄; methanol had no effect. H₂-dependent acetogenesis occurred in H₂-CO₂-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H₂ that were subsequently consumed. The accumulation of H₂ was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H₂; these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H₂-utilizing methanogens. A total of 10⁹ anaerobes and 10⁷ hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H₂-CO₂-supplemented, fatty acid-enriched defined medium. CH₄ production rates by the enrichment were similar at pH 4.5 and 6.5, and acetate inhibited methanogenesis at pH 4.5 but not at pH 6.5. A total of 27 different archaeal 16S rRNA gene sequences indicative of Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae were retrieved from the highest CH₄-positive serial dilutions of bog peat and methanogenic enrichments. A total of 10 bacterial 16S rRNA gene sequences were also retrieved from the same dilutions and enrichments and were indicative of bacteria that might be responsible for the production of H₂ that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H₂ is an important substrate for acid-tolerant methanogens, (ii) interspecies hydrogen transfer is involved in the degradation of organic carbon, (iii) the accumulation of protonated volatile fatty acids inhibits methanogenesis, and (iv) methanogenesis might be due to the activities of methanogens that are phylogenetic members of the Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae.     

Phenylcoumaran Benzylic Ether Reductase Prevents Accumulation of Compounds Formed under Oxidative Conditions in Poplar Xylem

Phenylcoumaran benzylic ether reductase (PCBER) is one of the most abundant proteins in poplar (Populus spp) xylem, but its biological role has remained obscure. In this work, metabolite profiling of transgenic poplar trees downregulated in PCBER revealed both the in vivo substrate and product of PCBER. Based on mass spectrometry and NMR data, the substrate was identified as a hexosylated 8–5-coupling product between sinapyl alcohol and guaiacylglycerol, and the product was identified as its benzyl-reduced form. This activity was confirmed in vitro using a purified recombinant PCBER expressed in Escherichia coli. Assays performed on 20 synthetic substrate analogs revealed the enzyme specificity. In addition, the xylem of PCBER-downregulated trees accumulated over 2000-fold higher levels of cysteine adducts of monolignol dimers. These compounds could be generated in vitro by simple oxidative coupling assays involving monolignols and cysteine. Altogether, our data suggest that the function of PCBER is to reduce phenylpropanoid dimers in planta to form antioxidants that protect the plant against oxidative damage. In addition to describing the catalytic activity of one of the most abundant enzymes in wood, we provide experimental evidence for the antioxidant role of a phenylpropanoid coupling product in planta.     

Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k_cat, the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.