An industrial scale process for the enzymatic removal of steryl glucosides from biodiesel

Background

Biodiesels produced from transesterification of vegetable oils have a major quality problem due to the presence of precipitates, which need to be removed to avoid clogging of filters and engine failures. These precipitates have been reported to be mostly composed of steryl glucosides (SGs), but so far industrial cost-effective methods to remove these compounds are not available. Here we describe a novel method for the efficient removal of SGs from biodiesel, based on the hydrolytic activity of a thermostable β-glycosidase obtained from Thermococcus litoralis.

Results

A steryl glucosidase (SGase) enzyme from T. litoralis was produced and purified from Escherichia coli cultures expressing a synthetic gene, and used to treat soybean-derived biodiesel. Several optimization steps allowed for the selection of optimal reaction conditions to finally provide a simple and efficient process for the removal of SGs from crude biodiesel. The resulting biodiesel displayed filterability properties similar to distilled biodiesel according to the total contamination (TC), the cold soak filtration test (CSFT), filter blocking tendency (FBT), and cold soak filter blocking tendency (CSFBT) tests. The process was successfully scaled up to a 20 ton reactor, confirming its adaptability to industrial settings.

Conclusions

The results presented in this work provide a novel path for the removal of steryl glucosides from biodiesel using a cost-effective, environmentally friendly and scalable enzymatic process, contributing to the adoption of this renewable fuel.

     

Genetic polymorphisms of <Emphasis Type="Italic">T-1131C APOA5</Emphasis> and <Emphasis Type="Italic">ALOX5AP SG13S114</Emphasis> with the susceptibility of ischaemic stroke in Morocco

Ischaemic stroke is a multifactorial disease. Genetic polymorphisms involved in lipid, inflammatory and thrombotic metabolisms play an important role in the development of ischaemic stroke. The present study aimed to assess the relationship between T1131C APOA5 and SG13S114 ALOX5AP polymorphisms and the risk of ischemic stroke in 175 cases and 201 controls. Genotyping was performed by high resolution melting and polymerase chain reaction restriction fragment length polymorphism methods. In the case of T-1131C APOA5, a modest risk of ischaemic stroke was noticed with CC (OR: 2.86; 95% CI = 1.24–6.58; Pc = 0.039) and C allele (OR: 1.54; 95% CI = 1.01–2.33; Pc = 0.014). For SG13S114ALOX5AP, a significant association was observed among subjects with TT (OR: 2.57; 95% CI =1.49–4.83; Pc = 0.009) and T allele (OR: 1.59; 95% CI = 1.16–2.19; Pc = 0.008). According to the risk factors of ischaemic stroke, a positive correlation was observed only between SG13S114 variant of ALOX5AP gene and hypertension (Pc = 0.026). Despite lower sample size, T-1131C APOA5 and SG13S114 variants could be considered an independent genetic risk factor of ischaemic stroke in Moroccan population.     

Strain engineering and process optimization for enhancing the production of a thermostable steryl glucosidase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Biodiesels produced from transesterification of vegetable oils have a major problem in quality due to the presence of precipitates, which are mostly composed of steryl glucosides (SGs). We have recently described an enzymatic method for the efficient removal of SGs from biodiesel, based on the activity of a thermostable β-glycosidase from Thermococcus litoralis. In the present work, we describe the development of an Escherichia coli-based expression system and a high cell density fermentation process. Strain and process engineering include the assessment of different promoters to drive the expression of a codon-optimized gene, the co-expression of molecular chaperones and the development of a high cell density fermentation process. A 200-fold increase in the production titers was achieved, which directly impacts on the costs of the industrial process for treating biodiesel.     

Aquaporin gene expression and physiological responses of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L. to the mycorrhizal fungus <Emphasis Type="Italic">Rhizophagus irregularis</Emphasis> and drought stress

The influence of arbuscular mycorrhiza (AM) and drought stress on aquaporin (AQP) gene expression, water status, and photosynthesis was investigated in black locust (Robinia pseudoacacia L.). Seedlings were grown in potted soil inoculated without or with the AM fungus Rhizophagus irregularis, under well-watered and drought stress conditions. Six full-length AQP complementary DNAs (cDNAs) were isolated from Robinia pseudoacacia, named RpTIP1;1, RpTIP1;3, RpTIP2;1, RpPIP1;1, RpPIP1;3, and RpPIP2;1. A phylogenetic analysis of deduced amino acid sequences demonstrated that putative proteins coded by these RpAQP genes belong to the water channel protein family. Expression analysis revealed higher RpPIP expression in roots while RpTIP expression was higher in leaves, except for RpTIP1;3. AM symbiosis regulated host plant AQPs, and the expression of RpAQP genes in mycorrhizal plants depended on soil water condition and plant tissue. Positive effects were observed for plant physiological parameters in AM plants, which had higher dry mass and lower water saturation deficit and electrolyte leakage than non-AM plants. Rhizophagus irregularis inoculation also slightly increased leaf net photosynthetic rate and stomatal conductance under well-watered and drought stress conditions. These findings suggest that AM symbiosis can enhance the drought tolerance in Robinia pseudoacacia plants by regulating the expression of RpAQP genes, and by improving plant biomass, tissue water status, and leaf photosynthesis in host seedlings.     

Using hydrothermal time concept to describe sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.) seed germination response to temperature and water potential

To quantify both temperature (T) and water potential (ψ) effects on sesame (Sesamum indicum L.) seed germination (SG) and also to determine the cardinal T _s for this plant, a laboratory experiment was carried out using hydrothermal time model (HTT). For this purpose, four sesame cultivars (‘Asbomahalleh’, ‘Darab’, ‘Dashtestan’ and ‘Yellowhite’) were germinated at seven constant T _s (20, 25, 30, 35, 37, 39 and 43 °C) at each of the following ψ _s (0, ? 0.12, ? 0.24 and ? 0.36 MPa; provided by PEG 8000). Germination rate (GR) and germination percentage (GP) significantly influenced by ψ, T and their interactions in all cultivars (P ≤ 0.01). There was no significant difference, based on the confidence intervals of the model coefficients, between cultivars, so an average of cardinal T _s was 14.7, 35.4 and 47.2 °C for the minimum (T _b), optimum (T _o) and maximum (T _c) T _s, respectively, in the control condition (0 MPa). Hydrotime values in all cultivars decreased when T was increased to T _o and then remained constant at T _s > T _o (15 MPa h^?1). An average value of ψ _b(50) was estimated to be ? 1.23 MPa at T _s ≤ T _o and then increased linearly (0.1041 MPa°Ch^?1, the slope of the relationship between ψ _b(50) and supra-optimal T _s) with T when T _s increased above T _o and finally reached to zero at T _c. The T _b and T _o values were not influenced by ψ, but T _c value decreased (from 47.2 for zero to 43.5 °C for ? 0.36 MPa) at supra-optimal T _s as a result of the effect of ψ on GR. Based on our findings, this model (as a predictive tool) and or the estimated parameter values in this study can easily be used in sesame SG simulation models to quantitatively characterize the physiological status of sesame seed populations at different T _s and ψ _s.     

Ecological requirements of Ostracoda (Crustacea) in a heavily polluted shallow lake,Lake Yeniçağa (Bolu,Turkey)

Although the area of Lake Yeniça?a is a potential candidate for RAMSAR convention, several anthropogenic factors compromise its biological diversity. This is mostly due to nutrient-rich water released from both point and nonpoint sources. Thirteen ostracod taxa (Candona neglecta, C. candida, Ilyocypris bradyi, Darwinula stevensoni, Cypridopsis vidua, Physocypria kraepelini, Cypria ophtalmica, Prionocypris zenkeri, Eucypris virens, Herpetocypris reptans, Pseudocandona compressa, Fabaeformiscandona fabaeformis Potamocypris cf. fulva) were found during this study. Potamocypris cf. fulva is a new record for the Turkish freshwater ostracod fauna. The first nine of these species have broad geographic ranges, implying high tolerance levels to different environmental variables. Based on the estimated species optima and tolerance levels, two species exhibited higher effective number of occurrences (C. neglecta, and D. stevensoni, respectively) than the other species. Three species (C. neglecta, D. stevensoni, I. bradyi) did not show significant correlation with any environmental variable we used. Both Canonical correspondence (CCA) and Pearson correlation analyses showed that temperature was the most effective predictor of species occurrence, followed by electrical conductivity and redox potential. In contrast, pH and dissolved oxygen of water were the least effective predictors. Approximately 71% of the correlation between community composition and environmental variables was explained by the first axis of the CCA diagram, which had a relatively low (7.7%) cumulative variance of species. The lower (560 μg/l) and the upper (2030 μg/l) levels of ammonia (NH₃) exceeded the limits during winter season. The concentrations of total coliform and Escherichia coli bacteria were measured up to 10 × 10⁷ cfu/ml and 10 × 10³ cfu/ml, respectively. Results of physicochemical measurements, microbiological counts, and species data indicate that water quality of Lake Yeniça?a has been rapidly deteriorated by anthropogenic factors that are the main threat for not only the lake’s aquatic diversity but also human health around the lake.     

Microorganisms of low-temperature heavy oil reservoirs (Russia) and their possible application for enhanced oil recovery

Physicochemical and microbiological characteristics of formation waters low-temperature heavy oil reservoirs (Russia) were investigated. The Chernoozerskoe, Yuzhno-Suncheleevskoe, and Severo-Bogemskoe oilfields, which were exploited without water-flooding, were shown to harbor scant microbial communities, while microbial numbers in the water-flooded strata of the Vostochno-Anzirskoe and Cheremukhovskoe oilfields was as high as 10⁶ cells/mL. The rates of sulfate reduction and methanogenesis were low, not exceeding 1982 ng S^2–/(L day) and 9045 nL СН₄/(L day), respectively, in the samples from water-flooded strata. High-throughput sequencing of microbial 16S rRNA gene fragments in the community of injection water revealed the sequences of the Proteobacteria (74.7%), including Betaproteobacteria (40.2%), Alphaproteobacteria (20.7%), Gammaproteobacteria (10.1%), Deltaproteobacteria (2.0%), and Epsilonproteobacteria (1.6%), as well as Firmicutes (7.9%), Bacteroidetes (4.1%), and Archaea (0.2%). DGGE analysis of microbial mcrA genes in the community of injection water revealed methanogens of the genera Methanothrix, Methanospirillum, Methanobacterium, Methanoregula, Methanosarcina, and Methanoculleus, as well as unidentified Thermoplasmata. Pure cultures of bacteria of the genera Rhodococcus, Pseudomonas, Gordonia, Cellulomonas, etc., capable of biosurfactant production when grown on heavy oil, were isolated. Enrichment cultures of fermentative bacteria producing significant amounts of volatile organic acids (acetic, propionic, and butyric) from sacchariferous substrates were obtained. These acids dissolve the carbonates of oil-bearing rock efficiently. Selection of the efficient microbial technology for enhanced recovery of heavy oil from terrigenous and carbonate strata requires model experiments with microbial isolates and the cores of oil-bearing rocks.     

Reliability of the search for 19 common mutations in the <Emphasis Type="Italic">CFTR</Emphasis> gene in Russian cystic fibrosis patients and the calculated frequency of the disease in Russian Federation

A study of Russian cystic fibrosis (CF) patient DNA was conducted to assess the incidence frequency of 19 mutations, namely CFTRdele2,3(21kb), F508del, I507del, 1677delTA, 2143delT, 2184insA, 394delTT, 3821delT, L138ins, 604insA, 3944delGT, G542X, W1282X, N1303K, R334W, and 3849 + 10kbC > T, S1196X, 621 + 1g > t, and E92K of the CFTR gene. We also sought to determine the estimated CF frequency in Russian Federation. In addition, we determined the total information content of the approach for 19 common mutations registration in the CFTR gene, 84.6%, and the allelic frequencies of the examined mutations: three mutations were observed with a frequency exceeding 5% (F508del, 53.98%, E92K, 6.47%, CFTRdele2,3(21kb), 5.35%); other mutations were observed with frequencies ranging from 0.13 to 3.0%. The CF population carrier frequency was 1 in 38 subjects, while the predicted CF frequency was 1 in 5776 newborns.     

Family Juncaceae: promising source of biologically active natural phenanthrenes

Phenanthrenes represent a relatively small group of aromatic secondary metabolites, which can be divided into three main subgroups (mono-, di-, and triphenanthrenes). Phenanthrenes are reported as an intensively researched field in phytochemistry according to their structural diversity and promising biological activities. Because of their limited occurrence phenanthrenes are considered to be as important taxonomic markers. Juncaceae is a relatively large plant family divided into seven genera of which Juncus and Luzula are the most important ones from phytochemical and pharmacological points of view. To date, almost one hundred natural phenanthrenes have been isolated but only from eight (Juncus acutus, J. effusus, J. inflexus, J. maritimus, J. roemerianus, J. setchuensis, J. subulatus, and Luzula luzuloides) Juncaceae species, including mono-, and diphenanthrenes, and phenanthrene glucosides. Great deal of the isolated compounds are substituted with a vinyl group. This substitution is characteristic exclusively to Juncaceae species. Juncusol (2) was isolated from every investigated species. The richest source of phenanthrenes, as well as the most extensively investigated species is J. effusus. Several isolated compounds possessed different biological activities, e.g. antiproliferative, antimicrobial, anti-inflammatory, antioxidant, spasmolytic, anxiolytic, and antialgal effects. Among them, dehydroeffusol (60) is the most promising one, as it showed antimicrobial, anxiolytic, sedative, spasmolytic, cellular protective and antiproliferative activities. The aim of this review is to summarize the occurrence of phenanthrenes in the family Juncaceae, and give a comprehensive overview of their isolation, structural characteristics and biological activities.     

Isolation of endophytic fungi from tropical forest in Indonesia

Endophytic fungi (EPF) are an important contributor to fungal diversity. It is surmised that EPF colonizing plant roots have high diversity. This study aimed to alleviate the scarcity of information regarding EPF in tropical forests, by isolationg and identifying EPF from a tropical forests in Indonesia. Soils were collected from five forests: (1) Tectona grandis monoculture; (2) Swietenia macrophylla monoculture; (3) Gmelina sp., Artocarpus champeden, Dipterocarp mixed; (4) Dipterocarp primary; (5) Macaranga sp. secondary. Four trees (Calliandra calothyrsus, Paraserianthes falcataria, Sesbania grandiflora, and Cassia siamea) and three crops (Sorghum bicolor, Allium fistulosum, and Trifolium repens) were grown in the forest soils to trap EPF. EPF were isolated from roots and isolation rates were calculated. Based on the isolation rates, P. falcataria and S. bicolor were chosen and grown again in forest soils. EPF were isolated and identified by their rDNA ITS1 region. Twelve and 21 EPF were isolated from 250 roots of P. falcataria and 300 roots of S. bicolor, respectively. Identified EPF were from genera Acrocalymma, Fusarium, Tolypocladium, Penicillium, Talaromyces, Exophiala, Dictyosporium, Pseudochaetosphaeronema, Mariannaea, Trichoderma, and Mycoleptodiscus. Acrocalymma, Tolypocladium, Penicillium, Exophiala, Pseudochaetosphaeronema, Mariannaea, and Mycoleptodiscus spp. were isolated from only one forest. Fusarium, Talaromyces, and Trichoderma spp. were isolated from more than one forest. The numbers of EPF isolated from Gmelina sp., Artocarpus champeden, Dipterocarp mixed forest, and Macaranga sp. secondary forest were higher than those from other forests, suggesting that different plant species in forests affect the root EPF community.     

Water-related environments: a multistep procedure to assess the diversity and enzymatic properties of cultivable bacteria

Studying the culturable portion of environmental bacterial populations is valuable for understanding the ecology, for discovering taxonomically interesting isolates and for exploiting their enzymatic abilities. In this study, diverse water-related samples, iced water (3 °C) from river, the sediment (29 °C) and water (55 °C) of a hot-spring, were investigated by two cultivation strategies, Dry and novel Wet approach. The isolates were clustered by fluorescent internal transcribed spacer PCR and identified by 16S rRNA sequencing. Several bacterial groups were also sub-typed through the application of Random Amplified Microsatellite Polymorphisms method. A broad enzymatic screening of all bacterial isolates was performed in order to assess the proteolytic, cellulolytic, lipolytic, esterolytic, amylolytic properties, as well as catalase and peroxidase activities. The Wet cultivation demonstrated to be suitable for the isolation of potential new species belonging to genera Massilia, Algoriphagus, Rheinheimera and Pandoraea. Valuable microbial resources with extensive enzymatic activities were recognized among the psychrophilic (Pantoea brenneri and Serratia sp.), mesophilic (Pandoraea, Massilia, Pseudomonas, Stenotrophomonas, Bacillus and Aeromonas) and thermophilic bacteria (Aeribacullus pallidus and Geobacillus kaustophilus). The experimental strategy developed in this study includes simple investigation tools able to reveal the genetic and enzymatic peculiarities of isolated microorganisms. It can be applied to different kinds of aquatic samples and extreme environments similar to those described in this study.     

Risk reduction strategies in <Emphasis Type="Italic">Branchipus schaefferi</Emphasis> (Crustacea: Anostraca: Branchiopoda) as adaptation to a variable environment

The effect of long-term changes in total precipitation on physical and chemical parameters of the water and the structure of phytoplankton community during a year were studied in a deep hard water lake. With respect to total precipitation, two different periods were distinguished: dry and wet. In the wet period, the water level rose and caused an increase in the water colour and a decrease in the electrolytic conductivity and concentration of nutrients. These changes were reflected in the composition and amount of phytoplankton. Certain phytoplankton groups, e.g. Cryptophyceae, Dinophyceae, Chrysophyceae, Bacillariophyceae and Chlorophyta/Charophyta, were positively affected by the environmental changes; instead, these effects were not observed in Cyanobacteria and Euglenophyta. The development of flagellates, such as Cryptomonas curvata, Plagioselmis nannoplanctica and Ceratium hirundinella, was enhanced during the wet period, whereas the dry period favoured non-flagellates, such as Planktothrix agardhii, Planktothrix rubescens and Limnothrix planctonica. Hence, the long-term variability in total precipitation can be a switch from non-flagellate- to flagellate-dominated phytoplankton in lakes. Moreover, a short time of ice cover duration in winter linked with the wet period promoted phytoplankton groups typical for spring and autumn, e.g. Cryptophyceae and Bacillariophyceae.     

Isolation and molecular characterization of the fungal endophytic microbiome from conventionally and organically grown avocado trees in South Florida

Fungal endophytes are the most ubiquitous and highly diverse microorganisms that inhabit the interior of healthy plants. They are important in plant ecology and offer untapped potential to improve plant health and productivity in agroecosystems. The endophytic assemblage of avocado is poorly understood; therefore, surveys of fungal endophytes of Persea americana Mill. (Avocado) in South Florida organic and conventional orchards were conducted. A total of 17 endophytic fungal species were recovered from healthy avocado terminal branches. Endophytic fungal species were identified by rDNA sequencing of the internal transcribed spacer (ITS) region, using UNITE Species Hypotheses to reliably assign a taxon name, and determined as belonging to the genera Alternaria, Cladosporium, Colletotrichum, Corynespora, Diaporthe, Lasiodiplodia, Neofusicoccum, Neopestalotiopsis, Phyllosticta, and Strelitziana. Endophyte community assemblage differed between organic and conventional agroecosystems. This is the first report of Alternaria eichhorniae, Cladosporium tenuissimum, Corynespora cassiicola, Colletotrichum alatae, Diaporthe fraxini-angustifoliae, Lasiodiplodia gonubiensis, Neofusicoccum algeriense, Neofusicoccum andinum, Neopestalotiopsis foedans, Phyllosticta capitalensis, and Strelitziana africana as endophytes of avocado. Evaluation using pathogenicity tests on avocado leaves and terminal branches showed that endophytic fungal isolates did not cause disease symptoms.     

<Emphasis Type="Italic">In Vitro</Emphasis>-<Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of Novel Co-spray Dried Rifampicin Phospholipid Lipospheres for Oral Delivery

The objective of this study comprises of developing novel co-spray dried rifampicin phospholipid lipospheres (SDRPL) to investigate its influence on rifampicin solubility and oral bioavailability. Solid-state techniques were employed to characterize the liposphere formulation. SDRPL solubility was determined in distilled water. BACTEC 460TB System was employed to evaluate SDRPL antimycobacterial activity. The oral bioavailability of the lipospheres was evaluated in Sprague Dawley rats. Lipospheres exhibited amorphous, smooth spherical morphology with a significant increase (p?<?0.001) in solubility of SDRPL (2:1), 350.9?±?23 versus 105.1?±?12 μg/ml and SDRPL (1:1) 306.4?±?20 versus 105.1?±?12 μg/ml in comparison to rifampicin (RMP). SDRPL exhibited enhanced activity against Mycobacterium tuberculosis, H37Rv strain, with over twofolds less minimum inhibitory concentration (MIC) than the free drug. Lipospheres exhibited higher peak plasma concentration (109.92?±?25 versus 54.31?±?18 μg/ml), faster T _max (two versus four hours), and enhanced area under the curve (AUC_0–∞) (406.92?±?18 versus 147.72?±?15 μg h/L) in comparison to pure RMP. Thus, SDRPL represents a promising carrier system exhibiting enhanced antimycobacterial activity and oral bioavailability of rifampicin.     

Deep sequencing analysis of bacterial community structure of Soldhar hot spring,India

We investigated the bacterial community structure of Soldhar hot spring with extreme high temperature 95°C located in Uttarakhand, India using high throughput sequencing. Bacterial phyla Proteobacteria (88.8%), Deinococcus-Thermus (7.5%), Actinobacteria (2.3%), and Firmicutes (1.07%) were predominated in the sequencing survey, whereas Bacteroidetes, Verrucomicrobia, Aquificae and Acidobacteria were detected in relatively lower abundance in Soldhar hot spring. At the family level, Comamonadaceae (52.5%), Alteromonadaceae (15.9%), and Thermaceae (7.5%) were mostly dominated in the ecosystem followed by Chromatiaceae, Microbacteriaceae, and Cyclobacteriaceae. Besides, members of Rhodobacteraceae, Moraxellaceae, Xanthomonadaceae, Aquificaceae, Enterobacteriaceae, Bacillaceae, Methylophilaceae, etc. were detected as a relatively lower abundance. In the present study we discuss the overall microbial community structure and their relevance to the ecology of the Soldhar hot spring environment.     

Molecular analyses of <Emphasis Type="Italic">Ficus erecta</Emphasis> and its allies within the subsection <Emphasis Type="Italic">Frutescentiae</Emphasis> (Moraceae)

Ficus (Moraceae) is a keystone group in tropical and subtropical forests with remarkable diversity of species and taxonomical challenges as a consequence of fig–pollinator coevolution. Ficus subsect. Frutescentiae includes about 30 species that are predominantly shrubs or small trees with Terminalia branching. Many of these species are difficult to delimit morphologically, and the group includes a tangle of uncertain taxa and incorrectly applied names. We conducted a phylogenetic analysis with internal and external transcribed spacer data (ITS and ETS) and data from 18 polymorphic microsatellite loci to evaluate the species status of the most perplexing members of this subsection. The results confirm the monophyly of subsect. Frutescentiae, with F. pedunculosa as sister to the rest. The F. erecta complex comprises approximately 17 taxa: F. erecta, F. abelii, F. boninsimae, F. nishimurae, F. iidaiana, F. gasparriniana var. laceratifolia, F. gasparriniana var. viridescens, F. pyriformis, F. stenophylla, F. fusuiensis, F. fengkaiensis, F. sinociliata, F. tannoensis, F. vaccinioides, F. formosana, F. pandurata, and F. periptera. The last five of these were supported as good species, while the others were not well supported by the present evidence. Evidence also supported the status of the non-F. erecta complex species including. F. pedunculosa, F. ischnopoda, F. heteromorpha, and F. variolosa. Ficus filicauda and F. neriifolia are possibly conspecific. The species status of F. potingensis should be restored and it should be treated as a member of section Eriosycea. Identification of the remaining taxa (F. gasparriniana var. esquirolii, F. ruyuanensis, F. daimingshanensis, F. chapaensis, F. changii, F. trivia, and F. tuphapensis) and their relationships to the F. erecta complex were not clarified. As a whole, only ten species in this subsection are confirmed, one is excluded, one is synonymous, and the others are either unresolved or short of samples. There appears to be a consistent genetic background among these unresolved groups, which suggests that repeated hybridization (as a result of pollinator host shifts) has filled up the interspecific gaps during the fig–pollinator coevolution process.