From mesophilic to thermophilic conditions: one-step temperature increase improves the methane production of a granular sludge treating agroindustrial effluents

Objectives

To assess the effect of one-step temperature increase, from 35 to 55 °C, on the methane production of a mesophilic granular sludge (MGS) treating wine vinasses and the effluent of a hydrogenogenic upflow anaerobic sludge blanket (UASB) reactor.

Results

One-step temperature increase from mesophilic to thermophilic conditions improved methane production regardless of the substrate tested. The biomethane potentials obtained under thermophilic conditions were 1.8–2.9 times higher than those obtained under mesophilic conditions. The MGS also performed better than an acclimated thermophilic digestate, producing 2.2–2.5 times more methane than the digestate under thermophilic conditions. Increasing the temperature from 35 to 55 °C also improved the methane production rate of the MGS (up to 9.4 times faster) and reduced the lag time (up to 1.9 times). Although the temperature increase mediated a decrease in the size of the sludge granules, no negative effects on the performance of the MGS was observed under thermophilic conditions.

Conclusions

More methane is obtained from real agroindustrial effluents at thermophilic conditions than under mesophilic conditions. One-step temperature increase (instead of progressive sequential increases) can be used to implement the thermophilic anaerobic digestion processes with MGS.

     

Contribution of glutamate decarboxylase in Lactobacillus reuteri to acid resistance and persistence in sourdough fermentation

Background

The gene encoding an atypical multi-modular glycoside hydrolase family 45 endoglucanase bearing five different family 1 carbohydrate binding modules (CBM1), designated PpCel45A, was identified in the Pichia pastoris GS115 genome.

Results

PpCel45A (full-length open reading frame), and three derived constructs comprising (i) the catalytic module with its proximal CBM1, (ii) the catalytic module only, and (iii) the five CBM1 modules without catalytic module, were successfully expressed to high yields (up to 2 grams per litre of culture) in P. pastoris X33. Although the constructs containing the catalytic module displayed similar activities towards a range of glucans, comparison of their biochemical characteristics revealed striking differences. We observed a high thermostability of PpCel45A (Half life time of 6 h at 80°C), which decreased with the removal of CBMs and glycosylated linkers. However, both binding to crystalline cellulose and hydrolysis of crystalline cellulose and cellohexaose were substantially boosted by the presence of one CBM rather than five.

Conclusions

The present study has revealed the specific features of the first characterized endo β-1,4 glucanase from yeast, whose thermostability is promising for biotechnological applications related to the saccharification of lignocellulosic biomass such as consolidated bioprocessing.     

Structural comparison of tRNA m1A58 methyltransferases revealed different molecular strategies to maintain their oligomeric architecture under extreme conditions

Background

tRNA m¹A58 methyltransferases (TrmI) catalyze the transfer of a methyl group from S-adenosyl-L-methionine to nitrogen 1 of adenine 58 in the T-loop of tRNAs from all three domains of life. The m¹A58 modification has been shown to be essential for cell growth in yeast and for adaptation to high temperatures in thermophilic organisms. These enzymes were shown to be active as tetramers. The crystal structures of five TrmIs from hyperthermophilic archaea and thermophilic or mesophilic bacteria have previously been determined, the optimal growth temperature of these organisms ranging from 37°C to 100°C. All TrmIs are assembled as tetramers formed by dimers of tightly assembled dimers.

Results

In this study, we present a comparative structural analysis of these TrmIs, which highlights factors that allow them to function over a large range of temperature. The monomers of the five enzymes are structurally highly similar, but the inter-monomer contacts differ strongly. Our analysis shows that bacterial enzymes from thermophilic organisms display additional intermolecular ionic interactions across the dimer interfaces, whereas hyperthermophilic enzymes present additional hydrophobic contacts. Moreover, as an alternative to two bidentate ionic interactions that stabilize the tetrameric interface in all other TrmI proteins, the tetramer of the archaeal P. abyssi enzyme is strengthened by four intersubunit disulfide bridges.

Conclusions

The availability of crystal structures of TrmIs from mesophilic, thermophilic or hyperthermophilic organisms allows a detailed analysis of the architecture of this protein family. Our structural comparisons provide insight into the different molecular strategies used to achieve the tetrameric organization in order to maintain the enzyme activity under extreme conditions.     

HU histone-like DNA-binding protein from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>: structural and evolutionary analyses

The histone-like DNA-binding proteins (HU) serve as model molecules for protein thermostability studies, as they function in different bacteria that grow in a wide range of temperatures and show sequence diversity under a common fold. In this work, we report the cloning of the hutth gene from Thermus thermophilus, the purification and crystallization of the recombinant HUTth protein, as well as its X-ray structure determination at 1.7 Å. Detailed structural and thermodynamic analyses were performed towards the understanding of the thermostability mechanism. The interaction of HUTth protein with plasmid DNA in solution has been determined for the first time with MST. Sequence conservation of an exclusively thermophilic order like Thermales, when compared to a predominantly mesophilic order (Deinococcales), should be subject, to some extent, to thermostability-related evolutionary pressure. This hypothesis was used to guide our bioinformatics and evolutionary studies. We discuss the impact of thermostability adaptation on the structure of HU proteins, based on the detailed evolutionary analysis of the Deinococcus–Thermus phylum, where HUTth belongs. Furthermore, we propose a novel method of engineering thermostable proteins, by combining consensus-based design with ancestral sequence reconstruction. Finally, through the structure of HUTth, we are able to examine the validity of these predictions. Our approach represents a significant advancement, as it explores for the first time the potential of ancestral sequence reconstruction in the divergence between a thermophilic and a mainly mesophilic taxon, combined with consensus-based engineering.     

Discrimination of thermophilic and mesophilic proteins

Background

There is a considerable literature on the source of the thermostability of proteins from thermophilic organisms. Understanding the mechanisms for this thermostability would provide insights into proteins generally and permit the design of synthetic hyperstable biocatalysts.

Results

We have systematically tested a large number of sequence and structure derived quantities for their ability to discriminate thermostable proteins from their non-thermostable orthologs using sets of mesophile-thermophile ortholog pairs. Most of the quantities tested correspond to properties previously reported to be associated with thermostability. Many of the structure related properties were derived from the Delaunay tessellation of protein structures.

Conclusions

Carefully selected sequence based indices discriminate better than purely structure based indices. Combined sequence and structure based indices improve performance somewhat further. Based on our analysis, the strongest contributors to thermostability are an increase in ion pairs on the protein surface and a more strongly hydrophobic interior.

     

The impacts of deacetylation prior to dilute acid pretreatment on the bioethanol process

Background

There is currently considerable interest in developing renewable sources of energy. One strategy is the biological conversion of plant biomass to liquid transportation fuel. Several technical hurdles impinge upon the economic feasibility of this strategy, including the development of energy crops amenable to facile deconstruction. Reliable assays to characterize feedstock quality are needed to measure the effects of pre-treatment and processing and of the plant and microbial genetic diversity that influence bioconversion efficiency.

Results

We used the anaerobic bacterium Clostridium phytofermentans to develop a robust assay for biomass digestibility and conversion to biofuels. The assay utilizes the ability of the microbe to convert biomass directly into ethanol with little or no pre-treatment. Plant samples were added to an anaerobic minimal medium and inoculated with C. phytofermentans, incubated for 3 days, after which the culture supernatant was analyzed for ethanol concentration. The assay detected significant differences in the supernatant ethanol from wild-type sorghum compared with brown midrib sorghum mutants previously shown to be highly digestible. Compositional analysis of the biomass before and after inoculation suggested that differences in xylan metabolism were partly responsible for the differences in ethanol yields. Additionally, we characterized the natural genetic variation for conversion efficiency in Brachypodium distachyon and shrub willow (Salix spp.).

Conclusion

Our results agree with those from previous studies of lignin mutants using enzymatic saccharification-based approaches. However, the use of C. phytofermentans takes into consideration specific organismal interactions, which will be crucial for simultaneous saccharification fermentation or consolidated bioprocessing. The ability to detect such phenotypic variation facilitates the genetic analysis of mechanisms underlying plant feedstock quality.     

A rigidifying salt-bridge favors the activity of thermophilic enzyme at high temperatures at the expense of low-temperature activity

Background

Thermophilic enzymes are often less active than their mesophilic homologues at low temperatures. One hypothesis to explain this observation is that the extra stabilizing interactions increase the rigidity of thermophilic enzymes and hence reduce their activity. Here we employed a thermophilic acylphosphatase from Pyrococcus horikoshii and its homologous mesophilic acylphosphatase from human as a model to study how local rigidity of an active-site residue affects the enzymatic activity.

Methods and Findings

Acylphosphatases have a unique structural feature that its conserved active-site arginine residue forms a salt-bridge with the C-terminal carboxyl group only in thermophilic acylphosphatases, but not in mesophilic acylphosphatases. We perturbed the local rigidity of this active-site residue by removing the salt-bridge in the thermophilic acylphosphatase and by introducing the salt-bridge in the mesophilic homologue. The mutagenesis design was confirmed by x-ray crystallography. Removing the salt-bridge in the thermophilic enzyme lowered the activation energy that decreased the activation enthalpy and entropy. Conversely, the introduction of the salt-bridge to the mesophilic homologue increased the activation energy and resulted in increases in both activation enthalpy and entropy. Revealed by molecular dynamics simulations, the unrestrained arginine residue can populate more rotamer conformations, and the loss of this conformational freedom upon the formation of transition state justified the observed reduction in activation entropy.

Conclusions

Our results support the conclusion that restricting the active-site flexibility entropically favors the enzymatic activity at high temperatures. However, the accompanying enthalpy-entropy compensation leads to a stronger temperature-dependency of the enzymatic activity, which explains the less active nature of the thermophilic enzymes at low temperatures.     

Preferred codons and amino acid couples in hyperthermophiles

Background

Most organisms grow at temperatures from 20 to 50°C but some prokaryotes, including Archaea and Bacteria, are capable of withstanding higher temperatures, from 60 to >100°C. What makes these cells so resistant to heat? Their biomolecules must be sufficiently stable, especially proteins, to work under these extreme conditions, but the bases for thermostability remains elusive.

Results

The preferential usage of certain couples of amino acids and codons in thermal adaptation was investigated, by comparative proteome analysis, using 28 complete genomes from 18 mesophiles, 4 thermophiles, and 6 hyperthermophiles. In the hyperthermophiles proteomes, whenever the percent of Glu (E) and Lys (K) Increased, the percent of Gln (Q) and His (H) decreased, so that the E+K/Q+H ratio was > 4,5; in the mesophiles proteomes, it was < 2,5 and in the thermophiles an intermediary value was observed. The E+K/Q+H ratios for chaperonins, potentially thermostable proteins, were higher than their proteome ratios whereas, for DNA ligases, not necessarily thermostable, they followed the proteome ones. Analysis of codon usage revealed that hyperthermophiles preferred AGR codons for Arg in detriment of CGN codons, which were preferred by mesophiles.

Conclusions

The results suggested that the E+K/Q+H ratio may provide a useful mark for distinguishing hyperthermophilic, thermophilic and mesophilic prokaryotes and that the high percent of the amino acid couple E+K, consistently associated to the low percent of the pair Q+H, could contribute to protein thermostability. Second, the preference for AGR codons for Arg was a signature of all hyperthermophilics so far analyzed.

     

Mycorrhizal responsiveness trends in annual crop plants and their wild relatives—a meta-analysis on studies from 1981 to 2010

Background and aims

Year of release of a cultivar reflects the agricultural and breeding practices of its time; we hypothesize that there are differences in mycorrhizal responsiveness of new high yielding and old crop plants and landraces. We evaluated the importance of the year of release on mycorrhizal responsiveness, arbuscular mycorrhizal (AM) fungal root colonization and P efficiency. We also analyzed the effect of experimental treatments, P efficiency (P acquisition and P utilization efficiency) and AM fungal root colonization on a potential mycorrhizal responsiveness trend for year of release.

Methods

We conducted a meta-analysis on 39 publications working on 320 different crop plant genotypes.

Results

New cultivars were less intensely colonized but were more mycorrhiza-responsive (and possibly dependent) compared to ancestral genotypes. This trend was potentially influenced by the moderator variables density, pre-germination, plant, plant type and AMF species. AM root colonization was also important for the mycorrhizal responsiveness trend for year of release, but P efficiency was not.

Conclusions

With the data available we could find no evidence that new crop plant genotypes lost their ability to respond to mycorrhiza due to agricultural and breeding practices.     

Ecology, genetic diversity and phylogeography of the Iberian endemic plant Jurinea pinnata (Lag.) DC. (Compositae) on two special edaphic substrates: dolomite and gypsum

Background and aims

Jurinea pinnata is an Iberian vascular plant which only grows on gypsum and dolomite, two types of rocks associated with their exclusive endemic floras. In addition, the plant has an island-like distribution which could affect the differentiation and the genetic variability of wild populations. Thus, the species provides a unique opportunity for comparing (bio)geographical and ecological (edaphic) differentiation by means of molecular markers.

Methods

For our investigation we took 24 soil samples paired with a similar number of foliar samples for nutritional analyses. Our molecular-marker approach (AFLPs) involved 16 populations.

Results

The edaphic parameters revealed significant dissimilarities between dolomitic and gypsum soils. These differences are also found in the mineral composition of the leaves. However, molecular data revealed that the differentiation between populations correlates better with geographical isolation than with the substrate character.

Conclusions

The populations showing the greatest genetic diversity are those of the East Baetic territory where the species grow on both substrates and its populations are closer together. The plant tolerance to gypsum and dolomite can be explained either as a result of common adaptive mechanisms or of a more general adaptation to arid environments.     

Co-occurring grass species differ in their associated microbial community composition in a temperate native grassland

Background and aims

Specific associations exist between plant species and the soil microbial community and these associations vary between habitat types and different plant groups. However, there is evidence that the associations are highly specific. Hence, we aimed to determine the specificity of plant-microbe relationships amongst co-occurring grass species in a temperate grassland.

Methods and results

We examined the broad microbial groups of bacteria and fungi as well as a specific fungal group, the arbuscular mycorrhizal community amongst two dominant C₃ and C₄ species and one sub-dominant C₃ species using terminal restriction fragment length polymorphism (T-RFLP) analysis. We found that the two dominant species were more similar to each other in their bacterial and arbuscular mycorrhizal community composition than either was to the sub-dominant species, but not in their fungal community composition. We also found no clear evidence that those differences were directly linked to soil chemical properties.

Conclusions

Our results demonstrate that co-occurring grass species have a distinct soil microbial community and T-RFLP analysis is able to detect plant species effect on the microbial community composition on an extremely local scale, providing an insight into the differences in the response of bacterial, fungal and arbuscular mycorrhizal communities to different, but similar and co-occurring, plant species.     

Nitrate-use traits of understory plants as potential regulators of vegetation distribution on a slope in a Japanese cedar plantation

Background and aims

Plant physiological traits and their relation to soil N availability was investigated as regulators of the distribution of understory shrub species along a slope in a Japanese cedar (Cryptomeria japonica) plantation in central Japan.

Methods

At the study site, previous studies demonstrated that both net and gross soil nitrification rates are high on the lower slope and there are dramatic declines in different sections of the slope gradient. We examined the distributions of understory plant species and their nitrate (NO ₃ ^? -N) use traits, and compared the results with the soil traits.

Results

Our results show that boundaries between different dominant understory species correspond to boundaries between different soil types. Leucosceptrum stellipilum occurs on soil with high net and gross nitrification rates. Hydrangea hirta is dominant on soil with high net and low gross nitrification rates. Pieris japonica occurs on soil with very low net and gross nitrification rates. Dominant understory species have species-specific physiological traits in their use of NO ₃ ^? -N. Pieris japonica lacks the capacity to use NO ₃ ^? -N as a N source, but other species do use NO ₃ ^? -N. Lindera triloba, whose distribution is unrelated to soil NO ₃ ^? -N availability, changes the extent to which it uses NO ₃ ^? -N in response to soil NO ₃ ^? -N availability.

Conclusions

Our results indicate that differences in the physiological capabilities and adaptabilities of plant species in using NO ₃ ^? -N as a N source regulate their distribution ranges. The identity of the major form of available soil N is therefore an environmental factor that influences plant distributions.     

A thermostable pyrimidine nucleoside phosphorylase from <Emphasis Type="Italic">Brevibacillus borstelensis</Emphasis> LK01 for synthesizing halogenated nucleosides

Objective

To isolate a thermostable pyrimidine nucleoside phosphorylase (PyNP) from mesophilic bacteria by gene mining.

Results

BbPyNP from Brevibacillus borstelensis LK01 was isolated by gene mining. BbPyNP had a highest 60% identity with that of reported PyNPs. BbPyNP could catalyze the phosphorolysis of thymidine, 2′-deoxyuridine, uridine and 5-methyuridine. BbPyNP had good thermostability and retained 73% of its original activity after 2 h incubation at 50 °C. BbPyNP had the highest activity at an optimum alkaline pH of 8.5. BbPyNP was stable from pH 7 to 9.8. Under preliminary optimized conditions, the biosynthesis of various 5-halogenated pyrimidine nucleosides by BbPyNP reached the yield of 61–84%.

Conclusion

An efficient approach was estimated in isolating thermostable PyNP from mesophilic bacteria.

     

Engineering a highly thermostable and stress tolerant superoxide dismutase by N-terminal modification and metal incorporation

Thermophilic or hyperthermophilic SODs (superoxide dismutase) usually offer substantial biotechnological advantages over mesophilic SODs. Previously a 244-amino acid N-terminal domain (NTD) from a heatresistant SOD of Geobacillus thermodenitrificans NG80-2 was discovered and demonstrated to be able to confer thermostability to homologous mesophilic SODs, which revealed a new type of heat resistance mechanism. To further improve the heat resistance and stress tolerance of thermophilic cambialistic superoxide dismutase (Fe/Mn- SOD _Ap ) from Aeropyrum pernix K1 through metal incorporation and fusion with the newly found peptide NTD for broadening its industrial application, the wildtype SOD _Ap and NTD-fused ntdSOD _Ap were expressed in E. coli BL21 and incorporated with metal cofactors by two ways. Recombinant fusion SOD obtained by in vitro reconstitution (Mn-rec ntdSOD _Ap ) exhibited improved optimum temperature at 70°C and dramatically enhanced thermostability especially at 110°C with enhanced pH stability from 4 to 10 and higher tolerance for denaturants and organic media than Mn-rec SOD _Ap . To the best of our knowledge, Mn-rec ntdSOD _Ap could be the most heat resistant SOD. In addition, metal incorporation of SOD _Ap and ntdSOD _Ap via in vivo modification have been developed and proved to be more practical for industrial use. These results indicate that fusion with NTD along with metal incorporation can generate superimposed effect and be applied to enhance the stability of cambialistic thermophilic SODs, thus providing a universal and convenient bioengineering method for generating extremely stable SODs.     

The root microbiota—a fingerprint in the soil?

Background

The root system of a plant is known to host a wide diversity of microbes that can be essential or detrimental to the plant. Microbial ecologists have long struggled to understand what factors structure the composition of these communities. An overlooked part of the microbial community succession in root systems has been the potential for individual variation among plants shaped by early colonisation events such as microbial exposure of the seed inside the parent plant and during dispersal.

Scope

In this review we outline life events of the plant that can affect the composition of its root microbiota and relate ecological theory of community assembly to the formation of the root microbiota.

Conclusion

All plants are exposed to environmental conditions and events throughout their lifetime that shape their phenotype. The microbial community associated with the plant is ultimately an extension of this phenotype. Therefore, only by following a plant from its origin inside the flower to senescence, can we fully understand how the associated microbial community was assembled and what determined its composition.     

Growth and physiological responses of trembling aspen (Populus tremuloides), white spruce (Picea glauca) and tamarack (Larix laricina) seedlings to root zone pH

Background and aims

Soil pH is among the major environmental factors affecting plant growth. Although the optimum range of soil pH for growth and the tolerance of pH extremes widely vary among plant species, the pH tolerance mechanisms in plants are still poorly understood. In this study, possible mechanisms were examined to explain the differences in tolerance of boreal plants to root zone pH.

Methods

In the controlled-environment solution culture experiments, we compared growth, physiological parameters and tissue nutrient concentrations in aspen, white spruce and tamarack seedlings that were subjected to 8 weeks of root zone pH treatments ranging from 5.0 to 9.0.

Results

The pH treatments had little effect on dry weights and net photosynthesis in white spruce seedlings despite reductions in transpiration rates at higher pH levels. In aspen and tamarack, both the growth and physiological parameters significantly decreased at pH higher than 6.0. The chlorosis of young tissues in aspen and tamarack was associated with the reductions in foliar concentrations of several of the examined essential nutrients including Fe and Mn. Although the plants varied in their ability to deliver essential nutrients to growing leaves, there was no direct correlation between tissue nutrient concentrations, chlorophyll concentrations and plant growth. The results also demonstrated strong inhibition of transpiration rates by high pH.

Conclusions

The results suggest that high root zone pH can upset water balance in pH sensitive species including aspen. Although the uptake and assimilation of essential elements such as Fe and Mn contribute to plant tolerance of high soil pH, we did not observe a direct relationship between growth and foliar nutrient concentrations to account for the observed differences in growth.