As the utilization and consumption of lignocellulosic biomass increases, so too will the need for an adequate supply of feedstock. To meet these needs, novel waste feedstock materials will need to be ...utilized. Exploitation of these novel feedstocks will require information both on the effects of solvent extraction on the succeeding analysis of potential novel feedstocks and how accurate current methodologies are in determining the composition of novel lignocellulosic feedstocks, particularly the carbohydrate and lignin fractions. In this study, the effects of solvent extraction on novel feedstocks, including tree foliage, tree bark and spent mushroom compost, with 95% ethanol, water and both sequentially were examined. Chemical analyses were carried out to determine the moisture content, ash, extractives, post-hydrolysis sugars, Klason lignin (KL) and acid-soluble lignin (ASL) within the selected feedstocks. The result of extraction could be seen most strongly for Klason lignin, with a strong association between higher levels of Klason lignin levels and greater amounts of non-removed extractives (tree foliage and bark). Higher Klason lignin levels are reported to be due the condensation of non-removed extractives during hydrolysis, hence the lower Klason lignin determinations following extraction are more exact. In addition, total sugar determinations were lower following extractions. This is because of the solubility of non-cell-wall carbohydrates; thus, the determinations following extraction are more accurate representations of structural cell-wall polysaccharides such as cellulose. Such determinations will assist in determining the best way to utilize novel feedstocks such as those analyzed in this work.
Ralstonia pickettii is a nosocomial infectious agent and a significant industrial contaminant. It has been found in many different environments including clinical situations, soil and industrial High ...Purity Water. This study compares the phenotypic and genotypic diversity of a selection of strains of Ralstonia collected from a variety of sources.
Ralstonia isolates (fifty-nine) from clinical, industrial and environmental origins were compared genotypically using i) Species-specific-PCR, ii) PCR and sequencing of the 16S-23S rRNA Interspatial region (ISR) iii) the fliC gene genes, iv) RAPD and BOX-PCR and v) phenotypically using biochemical testing. The species specific-PCR identified fifteen out of fifty-nine designated R. pickettii isolates as actually being the closely related species R. insidiosa. PCR-ribotyping of the 16S-23S rRNA ISR indicated few major differences between the isolates. Analysis of all isolates demonstrated different banding patterns for both the RAPD and BOX primers however these were found not to vary significantly.
R. pickettii species isolated from wide geographic and environmental sources appear to be reasonably homogenous based on genotypic and phenotypic characteristics. R. insidiosa can at present only be distinguished from R. pickettii using species specific PCR. R. pickettii and R. insidiosa isolates do not differ significantly phenotypically or genotypically based on environmental or geographical origin.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Autothermal thermophilic aerobic digestion (ATAD) is a microbial fermentation process characterized as a tertiary treatment of waste material carried out in jacketed reactors. The process can be ...carried out on a variety of waste sludge ranging from human, animal, food, or pharmaceutical waste where the addition of air initiates aerobic digestion of the secondary treated sludge material. Digestion of the sludge substrates generates heat, which is retained within the reactor resulting in elevation of the reactor temperature to 70-75 °C. During the process, deamination of proteinaceous materials also occurs resulting in liberation of ammonia and elevation of pH to typically pH 8.4. These conditions result in a unique microbial consortium, which undergoes considerable dynamic change during the heat-up and holding phases. The change in pH and substrate as digestion occurs also contributes to this dynamic change. Because the large reactors are not optimized for aeration, and because low oxygen solubility at elevated temperatures occurs, there are considerable numbers of anaerobes recovered which also contributes to the overall digestion. As the reactors are operated in a semi-continuous mode, the reactors are rarely washed, resulting in considerable biofilm formation. Equally, because of the fibrous nature of the sludge, fiber adhering organisms are frequently found which play a major role in the overall digestion process. Here, we review molecular tools needed to examine the ATAD sludge consortia, what has been determined through phylogenetic analysis of the consortia and the nature of the dynamics occurring within this unique fermentation environment.
The aim of this study was to analyse R997, the first integrative and conjugative element (ICE) isolated from the Indian Sub-Continent, and to determine its relationship to the SXT/R391 family of ...ICEs. WGS of Escherichia coli isolate AB1157 (which contains R997) was performed using Illumina sequencing technology. R997 context was assessed by de novo assembly, gene prediction and annotation tools, and compared to other SXT/R391 ICEs. R997 has a size of 85 Kb and harbours 85 ORFs. Within one of the variable regions a HMS-1 β-lactamase resistance gene is located. The Hotspot regions of the element contains restriction digestion systems and insertion sequences. R997 is very closely related to the SXT-like elements from widely dispersed geographic areas. The sequencing of R997 increases the knowledge of the earliest isolated SXT/R391 elements and may provide insight on the emergence of these elements on the Indian sub-continent.
SXT/R391 family of ICEs have been found to express an unusual function that enhances bacterial cell death post-UV irradiation. Previous analysis of ICE R391 found four core SXT/R391 ICE genes to be ...involved—orf96, orf90, orf91 and orf43. These genes functioned as part of a UV-inducible pathway, where upon exposure to UV, the levels of the Orf43 protein, a TraV homolog which we propose naming TraVR391, were upregulated, resulting in increased cell sensitization. Here, we examined the effect of orf43 overexpression and found it led to host cell permeabilization. The inducing agent for orf43, UV irradiation, is also known to increase the ICE R391 extrachromosomal form and apparent conjugative transfer rate. We demonstrated, via conjugative transfer deficient mutants, that orf43 overexpression alone restored a small level of ICE R391 transfer to recipient cells via an unknown mechanism other than conjugation. TraV homologs have been reported to function in conjugative transfer. However, TraVR391 is the first homolog to cause UV-associated cell sensitization. TraVR391 when overexpressed must contain a unique adaptation or function which results in cell lysis and decreased survival. A hypothesis for retaining such a detrimental effect may be in its role of enhancing ICE survival upon cell damage.
Cell sensitizing conjugation gene enhances ICE transfer rates.
Many SXT/R391-like enterobacterial Integrative Conjugative Elements (ICEs) have been found to express an atypical, recA-dependent, UV-inducible, cell-sensitising phenotype observed as a reduction in ...post-irradiation cell survival rates in host cells. Characterisation of a complete deletion library of the prototype ICE R391 identified the involvement of three core ICE genes, orfs90/91 encoding a putative transcriptional enhancer complex, and orf43, encoding a putative type IV secretion system, outer membrane-associated, conjugative transfer protein.
In this study, expression analysis of orf43 indicated that it was up-regulated as a result of UV irradiation in an orfs90/91-dependent manner. Induced expression was found to be controlled from a site preceding the gene which required functional orfs90/91. Expression of orfs90/91 was in turn found to be regulated by orf96, a λ cI-like regulator. Targeted construction of ICE R391 deletions, RT-PCR and qRT-PCR analysis confirmed a regulatory link between orfs90/91 and orf43 while site-directed mutagenesis of orf43 suggested an association with the cell membrane was a prerequisite for the cytotoxic effect.
Because of the recA-dependence of the effect, we hypothesise that UV induction of RecA results in cleavage of the cI-like ICE-encoded repressor protein, the product of orf96. This in turn allows expression of the transcriptional enhancer complex encoded by orfs90/91, which we conclude stimulates transcription of orf43, whose product is directly responsible for the effect.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
ICEpMERPH, the first integrative conjugative element (ICE) of the SXT/R391 family isolated in the United Kingdom and Europe, was analyzed to determine the nature of its adaptive functions, its ...genetic structure, and its homology to related elements normally found in pathogenic
or
species. Whole genome sequencing of
isolate K802 (which contains the ICEpMERPH) was carried out using Illumina sequencing technology. ICEpMERPH has a size of 110 Kb and 112 putative open reading frames (ORFs). The "hotspot regions" of the element were found to contain putative restriction digestion systems, insertion sequences, and heavy metal resistance genes that encoded resistance to mercury, as previously reported, but also surprisingly to arsenate. A novel arsenate resistance system was identified in hotspot 4 of the element, unrelated to other SXT/R391 elements. This arsenate resistance system was potentially linked to two genes:
, encoding an organoarsenical efflux major facilitator superfamily (MFS) transporter-like protein related to ArsJ, and
, encoding nicotinamide adenine dinucleotide (NAD)-dependent glyceraldehyde-3-phosphate dehydrogenase. Phenotypic analysis using isogenic strains of
strain AB1157 with and without the ICEpMERPH revealed resistance to low levels of arsenate in the range of 1-5 mM. This novel, low-level resistance may have an important adaptive function in polluted environments, which often contain low levels of arsenate contamination. A bioinformatic analysis on the novel determinant and the phylogeny of ICEpMERPH was presented.
To produce bioethanol from model cyanobacteria such as
, a two gene cassette consisting of genes encoding pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are required to transform ...pyruvate first to acetaldehyde and then to ethanol. However the partition of pyruvate to ethanol comes at a cost, a reduction in biomass and pyruvate availability for other metabolic processes. Hence strategies to divert flux to ethanol as a biofuel in
are of interest. PDC from
(ZpPDC) has been reported to have a lower Km then the
PDC (ZmPDC), which has traditionally been used in metabolic engineering constructs. The
gene was combined with the native
alcohol dehydrogenase gene (
A) in an attempt to increase ethanol production in the photoautotrophic cyanobacterium
sp. PCC 6803 over constructs created with the traditional Zmpdc. Native (Zppdc) and codon optimized (
) versions of the ZpPDC were cloned into a construct where
expression was controlled via the
A2 light inducible promoter from
sp. PCC 6803. These constructs were transformed into wildtype
sp. PCC 6803 for expression and ethanol production. Ethanol levels were then compared with identical constructs containing the
. While strains with the
(UL071) and
(UL072) constructs did produce ethanol, levels were lower compared to a control strain (UL070) expressing the pdc from
. All constructs demonstrated lower biomass productivity illustrating that the flux from pyruvate to ethanol has a major effect on biomass and ultimately overall biofuel productivity. Thus the utilization of a PDC with a lower Km from
unusually did not result in enhanced ethanol production in
sp. PCC 6803.
Autothermal thermophilic aerobic digestion (ATAD) is a biological wastewater treatment process used for stabilisation of domestic, animal, food and pharmaceutical sludges, and wastewater. It produces ...a high-quality effluent due to thermophilic processing conditions, however the stabilised sludge has poor settling characteristics, a high water content, low compaction capacity and is difficult to dewater by mechanical processes alone. These factors impact transport and disposal of processed ATAD sludge. We have carried out a detailed morphological characterisation of ATAD sludge at all stages of the ATAD process in an attempt to determine key characteristics of the sludge that might be responsible for its poor dewatering and settleability. A number of microscopic techniques including electron, optical, wide field and laser scanning confocal microscopy were applied to fresh, fixed or embedded sludge taken at various stages during a full scale ATAD process treating domestic sludge. The spatial distributions of structural sludge matrix components were determined and suggested a highly dynamic sludge morphology during the overall process. Large amounts of fibres were observed in the feed sludge, whereas thermophilic sludge liquor with low settleability was shown to have a lower protein to polysaccharide ratio (1:0.9) compared to the easily settled fraction where ratio values were in the range of (1:1.14–1:1.7) with a prevalence of protein constituents. ATAD sludge was also shown to contain colloids, slime, cellulose micro-particles and multiple hydrophobic droplets in the bulk liquor, factors that may markedly impact on sludge dewaterability characteristics. Laser scanning confocal microscopy demonstrated a superior ability to identify composition and spatial localisation of structural constituents in such a dispersed, high water content sludge.
► We utilised a number of microscopy and staining techniques to investigate for the first time the morphology of poorly settleable ATAD sludge. ► We identified a number of sludge characteristics that could influence the poor dewaterability of ATAD sludge. ► Such factors include the presence of colloids, slime, numerous cellulose micro-particles and multiple hydrophobic droplets in the bulk ATAD sludge liquor. ► This morphological characterisation points to ways that dewaterability might be addressed via process modifications.
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