Detailed analysis of metagenome datasets obtained from biogas-producing microbial communities residing in biogas reactors does not indicate the presence of putative pathogenic microorganisms

Background:
In recent years biogas plants in Germany have been supposed to be involved in amplification and dissemination of pathogenic bacteria causing severe infections in humans and animals. In particular, biogas plants are discussed to contribute to the spreading of Escherichia coli infections in humans or chronic botulism in cattle caused by Clostridium botulinum. Metagenome datasets of microbial communities from an agricultural biogas plant as well as from anaerobic lab-scale digesters operating at different temperatures and conditions were analyzed for the presence of putative pathogenic bacteria and virulence determinants by various bioinformatic approaches.
Results:
All datasets featured a low abundance of reads that were taxonomically assigned to the genus Escherichia or further selected genera comprising pathogenic species. Higher numbers of reads were taxonomically assigned to the genus Clostridium. However, only very few sequences were predicted to originate from pathogenic clostridial species. Moreover, mapping of metagenome reads to complete genome sequences of selected pathogenic bacteria revealed that not the pathogenic species itself, but only species that are more or less related to pathogenic ones are present in the fermentation samples analyzed. Likewise, known virulence determinants could hardly be detected. Only a marginal number of reads showed similarity to sequences described in the Microbial Virulence Database MvirDB such as those encoding protein toxins, virulence proteins or antibiotic resistance determinants.
Conclusions:
Findings of this first study of metagenomic sequence reads of biogas producing microbial communities suggest that the risk of dissemination of pathogenic bacteria by application of digestates from biogas fermentations as fertilizers is low, because obtained results do not indicate the presence of putative pathogenic microorganisms in the samples analyzed.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/49

Genome-scale analyses of butanol tolerance in Saccharomyces cerevisiae reveal an essential role of protein degradation

Background:
n-Butanol and isobutanol produced from biomass-derived sugars are promising renewable transport fuels and solvents. Saccharomyces cerevisiae has been engineered for butanol production, but its high butanol sensitivity poses an upper limit to product titers that can be reached by further pathway engineering. A better understanding of the molecular basis of butanol stress and tolerance of S. cerevisiae is important for achieving improved tolerance.
Results:
By combining a screening of the haploid S. cerevisiae knock-out library, gene overexpression, and genome analysis of evolutionary engineered n-butanol-tolerant strains, we established that protein degradation plays an essential role in tolerance. Strains deleted in genes involved in the ubiquitin-proteasome system and in vacuolar degradation of damaged proteins showed hypersensitivity to n-butanol. Overexpression of YLR224W, encoding the subunit responsible for the recognition of damaged proteins of an ubiquitin ligase complex, resulted in a strain with a higher n-butanol tolerance. Two independently evolved n-butanol-tolerant strains carried different mutations in both RPN4 and RTG1, which encode transcription factors involved in the expression of proteasome and peroxisomal genes, respectively. Introduction of these mutated alleles in the reference strain increased butanol tolerance, confirming their relevance in the higher tolerance phenotype. The evolved strains, in addition to n-butanol, were also more tolerant to 2-butanol, isobutanol and 1-propanol, indicating a common molecular basis for sensitivity and tolerance to C3 and C4 alcohols.
Conclusions:
This study shows that maintenance of protein integrity plays an essential role in butanol tolerance and demonstrates new promising targets to engineer S. cerevisiae for improved tolerance.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/48

Novel monosaccharide fermentation products in Caldicellulosiruptor saccharolyticus identified using NMR spectroscopy

Background:
Caldicellulosiruptor saccharolyticus is a thermophilic, Gram-positive, non-spore forming, strictly anaerobic bacterium of interest in potential industrial applications, including the production of biofuels such as hydrogen or ethanol from lignocellulosic biomass through fermentation. High-resolution, solution-state nuclear magnetic resonance (NMR) spectroscopy is a useful method for the identification and quantification of metabolites that result from growth on different substrates. NMR allows facile resolution of isomeric (identical mass) constituents and does not destroy the sample.
Results:
Profiles of metabolites produced by the thermophilic cellulose-degrading bacterium Caldicellulosiruptor saccharolyticus DSM 8903 strain following growth on different monosaccharides (D-glucose, D-mannose, L-arabinose, D-arabinose, D-xylose, L-fucose, and D-fucose) as carbon sources revealed several unexpected fermentation products, suggesting novel metabolic capacities and unexplored metabolic pathways in this organism. Both 1H and 13C nuclear magnetic resonance (NMR) spectroscopy were used to determine intracellular and extracellular metabolite profiles. One dimensional 1H NMR spectral analysis was performed by curve fitting against spectral libraries provided in the Chenomx software; 2-D homonuclear and heteronuclear NMR experiments were conducted to further reduce uncertainties due to unassigned, overlapping, or poorly-resolved peaks. In addition to expected metabolites such as acetate, lactate, glycerol, and ethanol, several novel fermentation products were identified: ethylene glycol (from growth on D-arabinose), acetoin and 2,3-butanediol (from growth on D-glucose, L-arabinose, and D-xylose), and hydroxyacetone (from growth on D-mannose, L-arabinose, and D-xylose). Production of ethylene glycol from D-arabinose was particularly notable, with around 10% of the substrate carbon converted into this uncommon fermentation product.
Conclusions:
The present research shows that C. saccharolyticus, already of substantial interest due to its capability for biological ethanol and hydrogen production, has further metabolic potential for production of higher molecular weight compounds, such as acetoin and 2,3-butanediol, as well as hydroxyacetone and the uncommon fermentation product ethylene glycol. In addition, application of nuclear magnetic resonance (NMR) spectroscopy facilitates identification of novel metabolites, which is instrumental for production of desirable bioproducts from biomass through microbial fermentation.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/47

Dynamic metabolic modeling of a microaerobic yeast co-culture: predicting and optimizing ethanol production from glucose/xylose mixtures

Background:
A key step in any process that converts lignocellulose to biofuels is the efficient fermentation of both hexose and pentose sugars. The co-culture of respiratory-deficient Saccharomyces cerevisiae and wild-type Scheffersomyces stipitis has been identified as a promising system for microaerobic ethanol production because S. cerevisiae only consumes glucose while S. stipitis efficiently converts xylose to ethanol.
Results:
To better predict how these two yeasts behave in batch co-culture and to optimize system performance, a dynamic flux balance model describing co-culture metabolism was developed from genome-scale metabolic reconstructions of the individual organisms. First a dynamic model was developed for each organism by estimating substrate uptake kinetic parameters from batch pure culture data and evaluating model extensibility to different microaerobic growth conditions. The co-culture model was constructed by combining the two individual models assuming a cellular objective of total growth rate maximization. To obtain accurate predictions of batch co-culture data collected at different microaerobic conditions, the S. cerevisiae maximum glucose uptake rate was reduced from its pure culture value to account for more efficient S. stipitis glucose uptake in co-culture. The dynamic co-culture model was used to predict the inoculum concentration and aeration level that maximized batch ethanol productivity. The model predictions were validated with batch co-culture experiments performed at the optimal conditions. Furthermore, the dynamic model was used to predict how engineered improvements to the S. stipitis xylose transport system could improve co-culture ethanol production.
Conclusions:
These results demonstrate the utility of the dynamic co-culture metabolic model for guiding process and metabolic engineering efforts aimed at increasing microaerobic ethanol production from glucose/xylose mixtures.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/44

Mapping the lignin distribution in pretreated sugarcane bagasse by confocal and fluorescence lifetime imaging microscopy

Background:
Delignification pretreatments of biomass and methods to assess their efficacy are crucial for biomass-to-biofuels research and technology. Here, we applied confocal and fluorescence lifetime imaging microscopy (FLIM) using one- and two-photon excitation to map the lignin distribution within bagasse fibers pretreated with acid and alkali. The evaluated spectra and decay times are correlated with previously calculated lignin fractions. We have also investigated the influence of the pretreatment on the lignin distribution in the cell wall by analyzing the changes in the fluorescence characteristics using two-photon excitation. Eucalyptus fibers were also analyzed for comparison.
Results:
Fluorescence spectra and variations of the decay time correlate well with the delignification yield and the lignin distribution. The decay dependences are considered two-exponential, one with a rapid (tau1) and the other with a slow (tau2) decay time. The fastest decay is associated to concentrated lignin in the bagasse and has a low sensitivity to the treatment. The fluorescence decay time became longer with the increase of the alkali concentration used in the treatment, which corresponds to lignin emission in a less concentrated environment. In addition, the two-photon fluorescence spectrum is very sensitive to lignin content and accumulation in the cell wall, broadening with the acid pretreatment and narrowing with the alkali one. Heterogeneity of the pretreated cell wall was observed.
Conclusions:
Our results reveal lignin domains with different concentration levels. The acid pretreatment caused a disorder in the arrangement of lignin and its accumulation in the external border of the cell wall. The alkali pretreatment efficiently removed lignin from the middle of the bagasse fibers, but was less effective in its removal from their surfaces. Our results evidenced a strong correlation between the decay times of the lignin fluorescence and its distribution within the cell wall. A new variety of lignin fluorescence states were accessed by two-photon excitation, which allowed an even broader, but complementary, optical characterization of lignocellulosic materials. These results suggest that the lignin arrangement in untreated bagasse fiber is based on a well-organized nanoenvironment that favors a very low level of interaction between the molecules.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/43

Dr.Anju.T.R,Young scient award winner(Dpt. of Biotechnology/centre for neuroscience ,CUSAT) – Video


Dr.Anju.T.R,Young scient award winner(Dpt. of Biotechnology/centre for neuroscience ,CUSAT)
Neonatal hypoxia a major cause of permanent neurological disabilities sustained during childhood has devastating consequences like cognitive dysfunctions, ne...

By: Ajayan Mannoor supran

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Dr.Anju.T.R,Young scient award winner(Dpt. of Biotechnology/centre for neuroscience ,CUSAT) - Video

Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes

Background:
Since its inception, the carbohydrate-active enzymes database (CAZy; http://www.cazy.org) has described the families of enzymes that cleave or build complex carbohydrates, namely the glycoside hydrolases (GH), the polysaccharide lyases (PL), the carbohydrate esterases (CE), the glycosyltransferases (GT) and their appended non-catalytic carbohydrate-binding modules (CBM). The recent discovery that members of families CBM33 and family GH61 are in fact lytic polysaccharide monooxygenases (LPMO), demands a reclassification of these families into a suitable category.
Results:
Because lignin is invariably found together with polysaccharides in the plant cell wall and because lignin fragments are likely to act in concert with (LPMO), we have decided to join the families of lignin degradation enzymes to the LPMO families and launch a new CAZy class that we name "Auxiliary Activities" in order to accommodate a range of enzyme mechanisms and substrates related to lignocellulose conversion. Comparative analyses of these auxiliary activities in 41 fungal genomes reveal a pertinent division of several fungal groups and subgroups combining their phylogenetic origin and their nutritional mode (white vs. brown rot).
Conclusions:
The new class introduced in the CAZy database extends the traditional CAZy families, and provides a better coverage of the full extent of the lignocellulose breakdown machinery.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/41

Structural evaluation and bioethanol production by simultaneous saccharification and fermentation with biodegraded triploid poplar

Background:
Pretreatment is a key step to decrease the recalcitrance of lignocelluloses and then increase the digestibility of cellulose in second-generation bioethanol production. In this study, wood chips from triploid poplar were biopretreated with white rot fungus Trametes velutina D10149. The effects of incubation duration on delignification efficiency and structural modification of cellulose were comparably studied, as well as the digestibility of cellulose by simultaneous saccharification and fermentation (SSF).
Results:
Although microbial pretreatments did not significantly introduce lignin degradation, the data from SSF exhibited higher cellulose conversion (21-75% for biopretreated samples for 4--16 weeks) as compared to the untreated poplar (18%). In spite of the essential maintain of crystallinity, the modification of lignin structure during fungal treatment undoubtedly played a key role in improving cellulose bioconversion rates. Finally, the ethanol concentration of 5.16 g/L was detected in the fermentation broth from the cellulosic sample biodegraded for 16 weeks after 24 h SSF, achieving 34.8% cellulose utilization in poplar.
Conclusion:
The potential fungal pretreatment with Trametes velutina D10149 was firstly explored in this study. It is found that the biopretreatment process had a significant effect on the digestibility of substrate probably due to the removal and unit variation of lignin, since the crystallinities of substrates were rarely changed. Additional investigation is still required especially to improve the selectivity for lignin degradation and optimize the digestibility of cellulose.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/42

Production and extraction of sugars from switchgrass hydrolyzed in ionic liquids

Background:
The use of Ionic liquids (ILs) as biomass solvents is considered to be an attractive alternative for the pretreatment of lignocellulosic biomass. Acid catalysts have been used previously to hydrolyze polysaccharides into fermentable sugars during IL pretreatment. This could potentially provide a means of liberating fermentable sugars from biomass without the use of costly enzymes. However, the separation of the sugars from the aqueous IL and recovery of IL is challenging and imperative to make this process viable.
Results:
Aqueous alkaline solutions are used to induce the formation of a biphasic system to recover sugars produced from the acid catalyzed hydrolysis of switchgrass in imidazolium-based ILs. The amount of sugar produced from this process was proportional to the extent of biomass solubilized. Pretreatment at high temperatures (e.g., 160[degree sign]C, 1.5 h) was more effective in producing glucose. Sugar extraction into the alkali phase was dependent on both the amount of sugar produced by acidolysis and the alkali concentration in the aqueous extractant phase. Maximum yields of 53% glucose and 88% xylose are recovered in the alkali phase, based on the amounts present in the initial biomass. The partition coefficients of glucose and xylose between the IL and alkali phases can be accurately predicted using molecular dynamics simulations.
Conclusions:
This biphasic system may enable the facile recycling of IL and rapid recovery of the sugars, and provides an alternative route to the production of monomeric sugars from biomass that eliminates the need for enzymatic saccharification and also reduces the amount of water required.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/39

Towards a metagenomic understanding on enhanced biomethane production from waste activated sludge after pH 10 pretreatment

Background:
Understanding the effects of pretreatment on anaerobic digestion of sludge waste from wastewater treatment plants is becoming increasingly important, as impetus moves towards the utilization of sludge for renewable energy production. Although the field of sludge pretreatment has progressed significantly over the past decade, critical questions concerning the underlying microbial interactions remain unanswered. In this study, a metagenomic approach was adopted to investigate the microbial composition and gene content contributing to enhanced biogas production from sludge subjected to a novel pretreatment method (maintaining pH at 10 for 8 days) compared to other documented methods (ultrasonic, thermal and thermal-alkaline).
Results:
Our results showed that pretreated sludge attained a maximum methane yield approximately 4-fold higher than that of the blank un-pretreated sludge set-up at day 17. Both the microbial and metabolic consortium shifted extensively towards enhanced biodegradation subsequent to pretreatment, providing insight for the enhanced methane yield. The prevalence of Methanosaeta thermophila and Methanothermobacter thermautotrophicus, together with the functional affiliation of enzymes-encoding genes suggested an acetoclastic and hydrogenotrophic methanogenesis pathway. Additionally, an alternative enzymology in Methanosaeta was observed.
Conclusions:
This study is the first to provide a microbiological understanding of improved biogas production subsequent to a novel waste sludge pretreatment method. The knowledge garnered will assist the design of more efficient pretreatment methods for biogas production in the future.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/38

Performance of AFEXTM pretreated rice straw as source of fermentable sugars: the influence of particle size

Background:
It is widely believed that reducing the lignocellulosic biomass particle size would improve the biomass digestibility by increasing the total surface area and eliminating mass and heat transfer limitation during hydrolysis reactions. However, past studies demonstrate that particle size influences biomass digestibility to a limited extent. Thus, this paper studies the effect of particle size (milled: 2 mm, 5 mm, cut: 2 cm and 5 cm) on rice straw conversion. Two different Ammonia Fiber Expansion (AFEX) pretreament conditions, AFEX C1 (low severity) and AFEX C2 (high severity) are used to pretreat the rice straw (named as AC1RS and AC2RS substrates respectively) at different particle size.
Results:
Hydrolysis of AC1RS substrates showed declining sugar conversion trends as the size of milled and cut substrates increased. Hydrolysis of AC2RS substrates demonstrated opposite conversion trends between milled and cut substrates. Increasing the glucan loading to 6% during hydrolysis reduced the sugar conversions significantly in most of AC1RS and AC2RS except for AC1RS-2 mm and AC2RS-5 cm. Both AC1RS-2 mm and AC2RS-5 cm indicated gradual decreasing trends in sugar conversion at high glucan loading. Analysis of SEM imaging for URS and AFEX pretreated rice straw also indicated qualitative agreement with the experimental data of hydrolysis. The largest particle size, AC2RS-5 cm produced the highest sugar yield of 486.12 g/kg of rice straw during hydrolysis at 6% glucan loading equivalent to 76.0% of total theoretical maximum sugar yield, with an average conversion of 85.9% from total glucan and xylan. In contrast, AC1RS-5 cm gave the lowest sugar yield with only 107.6 g/kg of rice straw, about 16.8% of total theoretical maximum sugar yield, and equivalent to one-quarter of AC2RS-5 cm sugar yield.
Conclusions:
The larger cut rice straw particles (5 cm) significantly demonstrated higher sugar conversion when compared to small particles during enzymatic hydrolysis when treated using high severity AFEX conditions. Analysis of SEM imaging positively supported the interpretation of the experimental hydrolysis trend and kinetic data.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/40

Ultrasonic disintegration of microalgal biomass and consequent improvement of bioaccessibility/bioavailability in microbial fermentation

Background:
Microalgal biomass contains a high level of carbohydrates which can be biochemically converted to biofuels using state-of-the-art strategies that are almost always needed to employ a robust pretreatment on the biomass for enhanced energy production. In this study, we used an ultrasonic pretreatment to convert microalgal biomass (Scenedesmus obliquus YSW15) into feasible feedstock for microbial fermentation to produce ethanol and hydrogen. The effect of sonication condition was quantitatively evaluated with emphases on the characterization of carbohydrate components in microalgal suspension and on subsequent production of fermentative bioenergy.MethodScenedesmus obliquus YSW15 was isolated from the effluent of a municipal wastewater treatment plant. The sonication durations of 0, 10, 15, and 60 min were examined under different temperatures at a fixed frequency and acoustic power resulted in morphologically different states of microalgal biomass lysis. Fermentation was performed to evaluate the bioenergy production from the non-sonicated and sonicated algal biomasses after pretreatment stage under both mesophilic (35[degree sign]C) and thermophilic (55[degree sign]C) conditions.
Results:
A 15 min sonication treatment significantly increased the concentration of dissolved carbohydrates (0.12 g g-1), which resulted in an increase of hydrogen/ethanol production through microbial fermentation. The bioconvertibility of microalgal biomass sonicated for 15 min or longer was comparable to starch as a control, indicating a high feasibility of using microalgae for fermentative bioenergy production. Increasing the sonication duration resulted in increases in both algal surface hydrophilicity and electrostatic repulsion among algal debris dispersed in aqueous solution. Scanning electron microscope images supported that ruptured algal cell allowed fermentative bacteria to access the inner space of the cell, evidencing an enhanced bioaccessibility. Sonication for 15 min was the best for fermentative bioenergy (hydrogen/ethanol) production from microalga, and the productivity was relatively higher for thermophilic (55[degree sign]C) than mesophilic (35[degree sign]C) condition.
Conclusion:
These results demonstrate that more bioavailable carbohydrate components are produced through the ultrasonic degradation of microalgal biomass, and thus the process can provide a high quality source for fermentative bioenergy production.Source:
http://www.biotechnologyforbiofuels.com/content/6/1/37