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Nanostructured cellulose (NC) represents an emerging sustainable biomaterial for diverse biotechnological applications; however, its production requires hazardous chemicals that render the process ecologically unfriendly. Using commercial plant-derived cellulose, an innovative strategy for NC production based on the combination of mechanical and enzymatic approaches was proposed as a sustainable alternative to conventional chemical procedures. After ball milling, the average length of the fibers was reduced by one order of magnitude (down to 10–20 μm) and the crystallinity index decreased from 0.54 to 0.07–0.18. Moreover, a 60 min ball milling pre-treatment followed by 3 h Cellic Ctec2 enzymatic hydrolysis led to NC production (15% yield). Analysis of the structural features of NC obtained by the mechano-enzymatic process revealed that the diameters of the obtained cellulose fibrils and particles were in the range of 200–500 nm and approximately 50 nm, respectively. Interestingly, the film-forming property on polyethylene (coating ≅ 2 μm thickness) was successfully demonstrated and a significant reduction (18%) of the oxygen transmission rate was obtained. Altogether, these findings demonstrated that nanostructured cellulose could be successfully produced using a novel, cheap, and rapid 2-step physico-enzymatic process that provides a potential green and sustainable route that could be exploitable in future biorefineries.

In the bio-based era, cellulolytic and hemicellulolytic enzymes are biocatalysts used in many industrial processes, playing a key role in the conversion of recalcitrant lignocellulosic waste biomasses. In this context, many thermophilic microorganisms are considered as convenient sources of carbohydrate-active enzymes (CAZymes). In this work, a functional genomic annotation of

Plant cell walls are interwoven structures recalcitrant to degradation. Both native and adapted microbiomes are particularly effective at plant cell wall deconstruction. Studying these deconstructive microbiomes provides an opportunity to assess microbiome performance and relate it to specific microbial populations and enzymes. To establish a system assessing comparative microbiome performance, parallel microbiomes were cultivated on sorghum (Sorghum bicolor L. Moench) from compost inocula. Biomass loss and biochemical assays indicated that these microbiomes diverged in their ability to deconstruct biomass. Network reconstructions from time-dependent gene expression identified key deconstructive groups within the adapted sorghum-degrading communities, including Actinotalea, Filomicrobium, and Gemmanimonadetes populations. Functional analysis of gene expression demonstrated that the microbiomes proceeded through successional stages that are linked to enzymes that deconstruct plant cell wall polymers. This combination of network and functional analysis highlighted the importance of celluloseactive Actinobacteria in differentiating the performance of these microbiomes.

The production of bio-chemicals requires the use of microbial strains with efficient substrate conversion and excellent environmental robustness, such as Bacillus coagulans spp. So far the genomes of about 50 strains have been sequenced. Herein, we report a comparative genomic analysis of nine strains on the full repertoire of CAZymes, secretion systems, and resistance mechanisms to environmental challenges. Moreover, B. coagulans Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) immune system along with CRISPR-associated Cas) genes, was also analysed. Overall, this study expands our understanding of the strains genomic diversity of B. coagulans to fully exploit its potential in biotechnological applications.

The production of biochemicals requires the use of microbial strains with efficient substrate conversion and excellent environmental robustness, such as

A thermo-acidophilic bacterium, Alicyclobacillus mali FL18, was isolated from a hot spring of Pisciarelli, near Naples, Italy; following genome analysis, a novel putative β-xylosidase, AmβXyl, belonging to the glycosyl hydrolase (GH) family 3 was identified. A synthetic gene was produced, cloned in pET-30a(+), and expressed in Escherichia coli BL21 (DE3) RIL. The purified recombinant protein, which showed a dimeric structure, had optimal catalytic activity at 80 °C and pH 5.6, exhibiting 60% of its activity after 2 h at 50 °C and displaying high stability (more than 80%) at pH 5.0–8.0 after 16 h. AmβXyl is mainly active on both para-nitrophenyl-β-D-xylopyranoside (KM 0.52 mM, kcat 1606 s−1, and kcat/KM 3088.46 mM−1·s−1) and para-nitrophenyl-α-L-arabinofuranoside (KM 10.56 mM, kcat 2395.8 s−1, and kcat/KM 226.87 mM−1·s−1). Thin-layer chromatography showed its ability to convert xylooligomers (xylobiose and xylotriose) into xylose, confirming that AmβXyl is a true β-xylosidase. Furthermore, no inhibitory effect on enzymatic activity by metal ions, detergents, or EDTA was observed except for 5 mM Cu2+. AmβXyl showed an excellent tolerance to organic solvents; in particular, the enzyme increased its activity at high concentrations (30%) of organic solvents such as ethanol, methanol, and DMSO. Lastly, the enzyme showed not only a good tolerance to inhibition by xylose, arabinose, and glucose, but was activated by 0.75 M xylose and up to 1.5 M by both arabinose and glucose. The high tolerance to organic solvents and monosaccharides together with other characteristics reported above suggests that AmβXyl may have several applications in many industrial fields.

Lignocellulosic biomass is composed of three major biopolymers: cellulose, hemicellulose and lignin. Analytical tools capable of quickly detecting both glycan and lignin deconstruction are needed to support the development and characterization of efficient enzymes/enzyme cocktails. Previously we have described nanostructure-initiator mass spectrometry-based assays for the analysis of glycosyl hydrolase and most recently an assay for lignin modifying enzymes. Here we integrate these two assays into a single multiplexed assay against both classes of enzymes and use it to characterize crude commercial enzyme mixtures. Application of our multiplexed platform based on nanostructure-initiator mass spectrometry enabled us to characterize crude mixtures of laccase enzymes from fungi Agaricus bisporus (Ab) and Myceliopthora thermophila (Mt) revealing activity on both carbohydrate and aromatic substrates. Using time-series analysis we determined that crude laccase from Ab has the higher GH activity and that laccase from Mt has the higher activity against our lignin model compound. Inhibitor studies showed a significant reduction in Mt GH activity under low oxygen conditions and increased activities in the presence of vanillin (common GH inhibitor). Ultimately, this assay can help to discover mixtures of enzymes that could be incorporated into biomass pretreatments to deconstruct diverse components of lignocellulosic biomass.

Geothermal springs are rich in various metal ions due to the interaction between rock and water that takes place in the deep aquifer. Moreover, due to seasonality variation in pH and temperature, fluctuation in element composition is periodically observed within these extreme environments, influencing the environmental microbial communities. Extremophilic microorganisms that thrive in volcanic thermal vents have developed resistance mechanisms to handle several metal ions present in the environment, thus taking part to complex metal biogeochemical cycles. Moreover, extremophiles and their products have found an extensive foothold in the market, and this holds true especially for their enzymes. In this context, their characterization is functional to the development of biosystems and bioprocesses for environmental monitoring and bioremediation. To date, the isolation and cultivation under laboratory conditions of extremophilic microorganisms still represent a bottleneck for fully exploiting their biotechnological potential. This work describes a streamlined protocol for the isolation of thermophilic microorganisms from hot springs as well as their genotypical and phenotypical identification through the following steps: (1) Sampling of microorganisms from geothermal sites ("Pisciarelli", a volcanic area of Campi Flegrei in Naples, Italy); (2) Isolation of heavy metal resistant microorganisms; (3) Identification of microbial isolates; (4) Phenotypical characterization of the isolates. The methodologies described in this work might be generally applied also for the isolation of microorganisms from other extreme environments.

The isolation of novel microbes from environmental samples continues to be a key strategy for the discovery of new metabolic capacities for the degradation and transformation of lignocellulose. We report the draft genome sequence of a new strain of Brevibacillus borstelensis isolated from a sorghum-adapted microbial community derived from a compost sample.

The production of biochemicals requires the use of microbial strains with efficient substrate conversion and excellent environmental robustness, such as Weizmannia coagulans species. So far, the genomes of 47 strains have been sequenced. Herein, we report a comparative genomic analysis of nine strains on the full repertoire of Carbohydrate-Active enZymes (CAZymes), secretion systems, and resistance mechanisms to environmental challenges. Moreover, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) immune system along with CRISPR-associated (Cas) genes, was also analyzed. Overall, this study expands our understanding of the strain’s genomic diversity of W. coagulans to fully exploit its potential in biotechnological applications.

The full utilization of hemicellulose sugars (pentose and exose) present in lignocellulosic material, is required for an efficient bio-based fuels and chemicals production. Two recombinant thermophilic enzymes, an endo-1,4-β-mannanase from Dictyoglomus turgidum (DturCelB) and an α-galactosidase from Thermus thermophilus (TtGalA), were assayed at 80 °C, to assess their heterosynergystic association on galactomannans degradation, particularly abundant in hemicellulose. The enzymes were tested under various combinations simultaneously and sequentially, in order to estimate the optimal conditions for the release of reducing sugars. The results showed that the most efficient degree of synergy was obtained in simultaneous assay with a protein ratio of 25% of DturCelB and 75% of TtGalA, using Locust bean gum as substrate. On the other hand, the mechanism of action was demonstrated through the sequential assays, i.e. when TtGalA acting as first to enhance the subsequent hydrolysis performed by DturCelB. The synergistic association between the thermophilic enzymes herein described has an high potential application to pre-hydrolyse the lignocellulosic biomasses right after the pretreatment, prior to the conventional saccharification step.

Sulfolobus spindle-shaped virus 1 is the only UV-inducible member of the virus family Fuselloviridae. Originally isolated from Saccharolobus shibatae B12, it can also infect Saccharolobus solfataricus. Like the CI repressor of the bacteriophage λ, the SSV1-encoded F55 transcription repressor acts as a key regulator for the maintenance of the SSV1 carrier state. In particular, F55 binds to tandem repeat sequences located within the promoters of the early and UV-inducible transcripts. Upon exposure to UV light, a temporally coordinated pattern of gene expression is triggered. In the case of the better characterized bacteriophage λ, the switch from lysogenic to lytic development is regulated by a crosstalk between the virus encoded CI repressor and the host RecA, which regulates also the SOS response. For SSV1, instead, the regulatory mechanisms governing the switch from the carrier to the induced state have not been completely unravelled. In this study we have applied an integrated biochemical approach based on a variant of the EMSA assay coupled to mass spectrometry analyses to identify the proteins associated with F55 when bound to its specific DNA promoter sequences. Among the putative F55 interactors, we identified RadA and showed that the archaeal molecular components F55 and RadA are functional homologs of bacteriophage λ (factor CI) and Escherichia coli (RecA) system.

Bacillus coagulans MA-13 is an efficient lactic acid producer which withstands high concentrations of the growth inhibitors formed during the pretreatment of lignocellulosic feedstock. This draft genome sequence is expected to pave the way toward the understanding of mechanisms responsible for the robustness of MA-13 during simultaneous saccharification and fermentation.

Background Lignocellulosic biomass is an abundant and sustainable feedstock, which represents a promising raw material for the production of lactic acid via microbial fermentation. However, toxic compounds that affect microbial growth and metabolism are released from the biomass upon thermochemical pre-treatment. So far, susceptibility of bacterial strains to biomass-derived inhibitors still represents a major barrier to lactic acid production from lignocellulose. Detoxification of the pre-treated lignocellulosic material by water washing is commonly performed to alleviate growth inhibition of the production microorganism and achieve higher production rates. Results In this study, we assessed the feasibility of replacing the washing step with integrated cellular adaptation during pre-culture of Bacillus coagulans MA-13 prior to simultaneous saccharification and lactic acid fermentation of steam exploded wheat straw. Using a seed culture pre-exposed to 30% hydrolysate led to 50% shorter process time, 50% higher average volumetric and 115% higher average specific productivity than when using cells from a hydrolysate-free seed culture. Conclusions Pre-exposure of B. coagulans MA-13 to hydrolysate supports adaptation to the actual production medium. This strategy leads to lower process water requirements and combines cost-effective seed cultivation with physiological pre-adaptation of the production strain, resulting in reduced lactic acid production costs. Electronic supplementary material The online version of this article (10.1186/s13068-019-1382-2) contains supplementary material, which is available to authorized users.

Extremophilic microorganisms are valuable sources of enzymes for various industrial applications. In fact, given their optimal catalytic activity and operational stability under harsh physical and chemical conditions, they represent a suitable alternative to their mesophilic counterparts. For instance, extremophilic enzymes are important to foster the switch from fossil-based to lignocellulose-based industrial processes. Indeed, more stable enzymes are needed, because the conversion of the lignocellulosic biomass to a wide palette of value-added products requires extreme chemo-physical pre-treatments. Galactomannans are part of the hemicellulose fraction in lignocellulosic biomass. They are heteropolymers constituted by a β-1,4-linked mannan backbone substituted with side chains of α-1,6-linked galactose residues. Therefore, the joint action of different hydrolytic enzymes (i.e. β-mannanase, β-mannosidase and α-galactosidase) is needed to accomplish their complete hydrolysis. So far, numerous galactomannan-degrading enzymes have been isolated and characterized from extremophilic microorganisms. Besides applications in biorefinery, these biocatalysts are also useful to improve the quality (i.e. digestibility and prebiotic properties) of food and feed as well as in paper industries to aid the pulp bleaching process. In this review, an overview about the structure, function and applications of galactomannans is provided. Moreover, a survey of (hyper)-thermophilic galactomannans-degrading enzymes, mainly characterized in the last decade, has been carried out. These extremozymes are described in the light of their biotechnological application in industrial processes requiring harsh conditions.

Background The peptide VLL-28, identified in the sequence of an archaeal protein, the transcription factor Stf76 from Sulfolobus islandicus, was previously identified and characterized as an antimicrobial peptide, possessing a broad-spectrum antibacterial activity. Methods Through a combined approach of NMR and Circular Dichroism spectroscopy, Dynamic Light Scattering, confocal microscopy and cell viability assays, the interaction of VLL-28 with the membranes of both parental and malignant cell lines has been characterized and peptide mechanism of action has been studied. Results It is here demonstrated that VLL-28 selectively exerts cytotoxic activity against murine and human tumor cells. By means of structural methodologies, VLL-28 interaction with the membranes has been proven and the binding residues have been identified. Confocal microscopy data show that VLL-28 is internalized only into tumor cells. Finally, it is shown that cell death is mainly caused by a time-dependent activation of apoptotic pathways. Conclusions VLL-28, deriving from the archaeal kingdom, is here found to be endowed with selective cytotoxic activity towards both murine and human cancer cells and consequently can be classified as an ACP. General significance VLL-28 represents the first ACP identified in an archaeal microorganism, exerting a trans-kingdom activity.

Background The genus Thermus, which has been considered for a long time as a fruitful source of biotechnological relevant enzymes, has emerged more recently as suitable host to overproduce thermozymes. Among these, α-galactosidases are widely used in several industrial bioprocesses that require high working temperatures and for which thermostable variants offer considerable advantages over their thermolabile counterparts. Results Thermus thermophilus HB27 strain was used for the homologous expression of the TTP0072 gene encoding for an α-galactosidase (TtGalA). Interestingly, a soluble and active histidine-tagged enzyme was produced in larger amounts (5 mg/L) in this thermophilic host than in Escherichia coli (0.5 mg/L). The purified recombinant enzyme showed an optimal activity at 90 °C and retained more than 40% of activity over a broad range of pH (from 5 to 8). Conclusions TtGalA is among the most thermoactive and thermostable α-galactosidases discovered so far, thus pointing to T. thermophilus as cell factory for the recombinant production of biocatalysts active at temperature values over 90 °C. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0638-4) contains supplementary material, which is available to authorized users.

The archaeon Sulfolobus solfataricus hosts a variety of viruses, among which the UV-inducible Sulfolobus spindle shaped virus 1 (SSV1) is the best characterized1. While studying the gene expression of SSV1 in the absence of UV irradiation2, a novel viral transcript (Tlys) was identified and found to encode for a transcription regulator, named F55, that folds into the ribbon-helix-helix DNA-binding motif. DNA band-shift assays demonstrated that F55 specifically recognises and binds to target sequences located in the promoters of the early induced SSV1 transcripts (i.e., T5, T6, and Tind) as well as in its own promoter (Tlys). The strongest affinity was observed towards T5 and T6 promoters and an apparent cooperativity in binding was observed for the Tind promoter2. Altogether, these in vitro evidences strongly indicated that F55 might be involved in the regulation of the SSV1 lysogeny and UV-induction. To shed light on the regulative role of F55, an in vivo survey of the molecular events occurring at the UV-inducible region of the SSV1 genome was carried out3. Chromatin immunoprecipitation (ChIP) assays followed by semi-quantitative PCR (sqPCR) analyses showed that F55 stably binds in vivo to the promoters of Tlys, T5, and T6 as well as to that of the UV-inducible Tind. Moreover, ChIP-sqPCR data indicated that F55 dissociates upon UV irradiation first from the promoter of Tind (2 hours post UV-irradiation) and subsequently from those of T5 and T6 (4 hours post UV-irradiation); thus, allowing their transcription to proceed3. The protein/DNA dissociation occurs because, upon UV-irradiation, the intracellular concentration of F55 becomes progressively suboptimal to saturate all the regulative binding sites, as shown by western blot analyses3. Further studies are underway to identify the molecular partners of F55 involved in the transition from the lysogenic to the UV-induced state of SSV1.

Second generation bioethanol production is becoming established in production plants across the world. The process can also be viewed as a model biorefinery concept for biotechnological conversion of recalcitrant lignocellulosic raw materials to a range of chemicals and other products. We have developed a Multi-Feed SSCF process, which is a systematic, model-driven design of feeding schemes of solid substrate, active and robust cell factories that are adapted to the present substrate, and enzymes, to fed-batch simultaneous saccharification and co-fermentation of steam-pretreated lignocellulosic materials in standard stirred tank reactors. The concept has been applied not only to ethanol production with S. cerevisiae, but also to lactic acid production from wheat straw by a thermophilic, cellulolytic strain of Bacillus coagulans(MA-13), isolated from bean processing waste. High Gravity operation, i.e. fermentation at high concentrations of water insoluble solids (WIS), pushes the process towards higher product concentrations and productivities, and improved energy and water economy. By using the multi-feed SSCF approach, the ethanol process was pushed towards final product concentrations above 60 g/L, at about 90% of the theoretical yields on consumed substrate, using 22% w/w accumulated WIS additions of acid- and steam explosion-pretreated wheat straw. Bacillus coagulansMA-13 was found to secrete cellulolytic enzymes and ferment lignocellulose-derived sugars to lactic acid; thus, it may be a potential platform for consolidated bioprocessing of lactic acid. We investigated its performance in multi-feed SSF and found that a pre-adaptation to the liquid fraction of steam-pretreated lignocellulosic materials improves lactate productivity and reduces the SSF time from 33 to 12 hours.

The Fuselloviridae prototype member Sulfolobus spindle-shaped virus 1 is a model of UV-inducible viruses infecting Crenarchaeota. Previous works on SSV1 UV induction were bases on empirically determined parameters that have not yet been standardized. Thus, in many peer reviewed literature, it is not clear how the fluence and irradiance have been determined. Here, we describe a protocol for the UV induction of SSV1 replication, which is based on the combination of the following instrumentally monitored parameters: (1) the fluence; (2) the irradiance; (3) the exposure time, and (4) the exposure distance. With the aim of finding a good balance between the viral replication induction and the host cells viability, UV-irradiated cultures were monitored for their ability to recover in the aftermath of the UV exposure. This UV irradiation procedure has been set up using the well-characterized Sulfolobus solfataricus P2 strain as model system to study host-virus interaction.