Optimization of Lipase Production from <Emphasis Type="Italic">Aspergillus terreus</Emphasis> by Response Surface Methodology and Its Potential for Synthesis of Partial Glycerides Under Solvent Free Conditions

Aspergillus terreus produces lipase 7.01 IU/ml in 96 h after optimization by one variable at a time method. Using the significant factors i.e. corn oil (A), sodium nitrate (B), casein (C), agitation rate (D) and incubation period (E) RSM was carried out resulting in 19.65 IU/ml from the combination +1(A), −1(B), −1(C), +1(D) and 0(E). The interactions between sodium nitrate, casein and agitation with corn oil were most significant. Scale up of production from 250 ml shake flask to 30 l bioreactor resulted in increased productivity of 0.52 IU/ml/h as against 0.2 IU/ml/h obtained in shake flasks. This lipase could carryout solvent free synthesis of partial glycerides of oleic acid with 96% efficiency in 12 h.     

Toxicity, repellency, and effects of acetamiprid on western subterranean termite (Isoptera: Rhinotermitidae)

The insecticidial and biological activity of the cyano-substituted neonicotinoid acetamiprid was determined against the western subterranean termite, Reticulitermes hesperus Banks (Isoptera: Rhinotermitidae). Acetamiprid was very active against termites in topical applications, with an LD50 = 0.02 ng per termite. Even though acetamiprid was extremely toxic in topical applications, deposits > or =50 ppm on sand were required to consistently provide >90% kill of termites within 7 d after a 1-h exposure. Termites were quickly affected by brief exposures to sand treated with 1 ppm acetamiprid and within 1 h, their locomotion was dramatically impaired. Acetamiprid was transferred from donors to recipients only when donors were held on deposits > or =50 ppm for 1 h. Deposits even as low as 1 ppm were repellent and termites failed to tunnel into treated sand, and there was no significant mortality. Exposure to acetamiprid impaired locomotion of termites as did other slow-acting neonicotinoids, such as imidacloprid. Acetamiprid was repellent at all concentrations tested, acting like type I pyrethroid treatments in soil. A new subcategory of type III soil termiticides is proposed that incorporates the sublethal and delayed effects observed in neonicotinoid insecticides, and repellency at certain concentrations.     

Identification of three distinct Clostridium thermocellum xylanase genes by molecular cloning

Three genes coding for xylanase synthesis in Clostridium thermocellum were cloned and expressed in Escherichia coli. Genomic DNA from Clostridium thermocellum was digested to completion with HindIII, BamHI, and SalI. The fragments were ligated into the corresponding sites of pUC19 and transformed into Escherichia coli. Two of the genes encoded for xylanases which depolymerized xylans but were unable to extensively convert these substrates to reducing sugar. The third gene encoded for an enzyme that extensively hydrolyzed xylan. The insert containing the latter gene was subjected to extensive mapping and was found to encode for a xylanase with a molecular weight of approximately 25,000. The protein product of the cloned gene was obtained in a relatively pure form by heat treatment, ion exchange and gel permeation steps. The enzyme was quite stable to high temperatures with a half-life of 24 h at 70°C.Issued as National Research Council of Canada No. 30545     

Microclimatic differentiation of gene pools in the Lobaria pulmonaria symbiosis in a primeval forest landscape

Population genetics of the tree‐colonizing lichen Lobaria pulmonaria were studied in the largest primeval beech forest of Europe, covering 10 000 ha. During an intensive survey of the area, we collected 1522 thallus fragments originating from 483 trees, which were genotyped with eight mycobiont‐ and 14 photobiont‐specific microsatellite markers. The mycobiont and photobiont of L. pulmonaria were found to consist of two distinct gene pools, which are co‐existing within small areas of 3–180 ha in a homogeneous beech forest. The small‐scale distribution pattern of the symbiotic gene pools show habitat partitioning of lineages associated with either floodplains or mountain forests. Using approximate Bayesian computation (ABC), we dated the divergence of the two fungal gene pools of L. pulmonaria as the Early Pleistocene. Both fungal gene pools survived the Pleistocene glacial cycles in the Carpathians, although possibly in climatically different refugia. Fungal diversification prior to these cycles and the selection of photobionts with different altitudinal distributions explain the current sympatric, but ecologically differentiated habitat partitioning of L. pulmonaria. In addition, the habitat preferences of the mycobiont are determined by other factors and are rather independent of those of the photobiont at the landscape level. The distinct gene pools should be considered evolutionarily significant units and deserve specific conservation priorities in the future, for example gene pool A, which is a Pliocene relict.     

A statistical approach to study the interactive effects of process parameters on succinic acid production from Bacteroides fragilis

A statistical approach response surface methodology (RSM) was used to study the production of succinic acid from Bacteroides fragilis. The most influential parameters for succinic acid production obtained through one-at-a-time method were glucose, tryptone, sodium carbonate, inoculum size and incubation period. These resulted in the production of 5.4gL(-1) of succinic acid in 48h from B. fragilis under anaerobic conditions. Based on these results, a statistical method, face-centered central composite design (FCCCD) falling under RSM was employed for further enhancing the succinic acid production and to monitor the interactive effect of these parameters, which resulted in a more than 2-fold increase in yield (12.5gL(-1) in 24h). The analysis of variance (ANOVA) showed the adequacy of the model and the verification experiments confirmed its validity. On subsequent scale-up in a 10-L bioreactor using conditions optimized through RSM, 20.0gL(-1) of succinic acid was obtained in 24h. This clearly indicated that the model stood valid even on large scale. Thus, the statistical optimization strategy led to an approximately 4-fold increase in the yield of succinic acid. This is the first report on the use of FCCCD to improve succinic acid production from B. fragilis. The present study provides useful information about the regulation of succinic acid synthesis through manipulation of various physiochemical parameters.     

Toxicity, uptake, and transfer efficiency of fipronil in western subterranean termite (Isoptera: Rhinotermitidae)

The potential horizontal transfer ofnonrepellent termiticides has become an important paradigm to control termites in recent years. In this study, 14C-radiolabeled fipronil was used in a series of laboratory experiments to demonstrate the extent and ability of termites to transfer lethal amounts of fipronil to unexposed nestmates. Fipronil is an active and nonrepellent termiticide against western subterranean termites, Reticulitermes hesperus Banks, on sand at relevant doses. It exhibited delayed toxicity with the lowest LD50 approximately 0.2 ng/termite expressed between day 4 and 7. Both continuous and brief exposures to fipronil-treated sand seriously impaired the termite's ability to move and respond to a dodecatrienol trail, limiting potential horizontal transfer. In tunneling studies, fipronil prevented termite tunneling at concentrations as low as 0.5 ppm and was nonrepellent even at 500 ppm. Greater than 90% mortality was recorded by day 7 with concentrations ranging from 0.5 to 500 ppm. There was a linear relationship between the time of exposure and uptake of [14C]fipronil when termites were continuously exposed to 0.5, 1.0, and 5.0 ppm for 24 h. However, uptake discontinued when the termites were immobilized. Maximum transfer of fipronil from donors to recipients occurred within the first 24 h. Fipronil was transferred by body contact and trophallaxis did not play a major role in horizontal transfer. In successive transfer studies, there was not enough fipronil on recipients for them to serve as secondary donors and kill other termites. In a linear arena study, there was an inverse relationship between the amount offipronil on dead termites and their distance from the treated zone. Maximum mortality was observed within 1.5 m from the treated zone. Results in our laboratory studies suggest that horizontal transfer was not a major factor contributing to the efficacy of fipronil in the field.     

Biochar – synergies and trade‐offs between soil enhancing properties and C sequestration potential

The characterization of biochar has been predominantly focused around determining physicochemical properties including chemical composition, porosity and volatile content. To date, little systematic research has been done into assessing the properties of biochar that directly relate to its function in soil and how production conditions could impact these. The aim of this study was to evaluate how pyrolysis conditions can influence biochar's potential for soil enhancing benefits by addressing key soil constraints, and identify potential synergies and restrictions. To do this, biochar produced from pine wood chips (PC), wheat straw (WS) and wheat straw pellets (WSP) at four highest treatment temperatures (HTT) (350, 450, 550 and 650 °C) and two heating rates (5 and 100 °C min^?1) were analysed for pH, extractable nutrients, cation exchange capacity (CEC), stable‐C content and labile‐C content. Highest treatment temperature and feedstock selection played an important role in the development of biochar functional properties while overall heating rate (in the range investigated) was found to have no significant effect on pH, stable‐C or labile‐C concentrations. Increasing the HTT reduced biochar yield and labile‐C content while increasing the yield of stable‐C present within biochar. Biochar produced at higher HTT also demonstrated a higher degree of alkalinity improving biochar's ability to increase soil pH. The concentration of extractable nutrients was mainly affected by feedstock selection while the biochar CEC was influenced by HTT, generally reaching its highest values between 450–550 °C. Biochar produced at ≥550 °C showed high combined values for C stability, pH and CEC while lower HTTs favoured nutrient availability. Therefore attempts to maximize biochar's C sequestration potential could reduce the availability of biochar nutrients. Developing our understanding of how feedstock selection and processing conditions influence key biochar properties can be used to refine the pyrolysis process and design of ‘bespoke biochar’ engineered to deliver specific environmental functions.