Clostridium Pasteurianum Research Articles

The final steps of [FeFe]-hydrogenase maturation

The active site (H-cluster) of [FeFe]-hydrogenases is a blueprint for the design of a biologically inspired H2-producing catalyst. The maturation process describes the preassembly and uptake of the unique [2FeH] cluster into apo-hydrogenase, which is to date not fully understood. In this study, we targeted individual amino acids by site-directed mutagenesis in the [FeFe]-hydrogenase CpI of Clostridium pasteurianum to reveal the final steps of H-cluster maturation occurring within apo-hydrogenase. We identified putative key positions for cofactor uptake and the subsequent structural reorganization that stabilizes the [2FeH] cofactor in its functional coordination sphere. Our results suggest that functional integration of the negatively charged [2FeH] precursor requires the positive charges and individual structural features of the 2 basic residues of arginine 449 and lysine 358, which mark the entrance and terminus of the maturation channel, respectively. The results obtained for 5 glycine-to-histidine exchange variants within a flexible loop region provide compelling evidence that the glycine residues function as hinge positions in the refolding process, which closes the secondary ligand sphere of the [2FeH] cofactor and the maturation channel. The conserved structural motifs investigated here shed light on the interplay between the secondary ligand sphere and catalytic cofactor.

A Gold Quartet Framework with Reversible Anisotropic Structural Transformation Accompanied by Luminescence Response

Summary Hierarchy and chirality are the fundamental characteristics of biomolecules. Chemistry on hierarchical self-assembly and spontaneous symmetry breaking helps to uncover the mechanism of living activities and the origin of chirality in nature. Herein, we describe a chiral gold quartet framework (GQF) with C4-symmetric structure hierarchically aggregated from an achiral anionic [Au10(dpptrz)4S4]2− (dpptrz-Au10), which was acquired via click-reaction-induced and Au(I)⋅⋅⋅Au(I)-interaction-directed cluster-to-cluster transformation (CCT) from the rationally designed [(dppa)6Au18S8]2+ (dppa-Au18). Inter-cluster hydrogen bonding and alkali ion coordination were observed to assist the hierarchical self-assembly and chiral self-organization of the GQF. Upon exposing the crystal to vacuum, the bottom-up constructed GQF showed an anisotropic structural transformation accompanied by a visual luminescence change in a single-crystal-to-single-crystal (SCSC) manner that became reversible upon removal of vacuum. This research largely advances the potential of polynuclear gold(I)-sulfido clusters in hierarchical self-assembly and luminescent materials.

Simultaneous intensification of direct acetate cleavage and CO2 reduction to generate methane by bioaugmentation and increased electron transfer

Direct interspecies electron transfer coupled to CO2 reduction, DIET-CO2 reduction, to generate methane is proposed and prosperous in 2010s. It is well known that bioaugmentation and increased electron transfer benefit DIET-CO2 reduction. Herein, we studied whether other methanogenic pathways, such as H2-mediated methanogenic progress and direct acetate cleavage (acetoclastic methanogenesis), are simultaneously favorable in the presence of conductive materials (CMs). If so, contribution of DIET-CO2 reduction may be overestimated because overwhelming studies just considered this pathway. Detailed researches on whether and how Clostridium pasteurianum coupled with CMs, granular activated carbon, biochar, nano-magnetite and grapheme, influenced methanogenic progresses were conducted. Overall, C. pasteurianum enhanced methane production rate, which was further improved by some CMs. Combined with metabolism, kinetic and electrochemical analysis, experimental results showed that hydrogenotrophic methanogenesis occurred and bioaugmentation strengthened this progress, which was further motivated by CMs, such as biochar and magnetite. 16S rRNA gene analysis suggested that Methanobacteriaceae was potentially responsible for methane production. Whereafter, DIET-CO2 reduction may become prosperous according to electrochemical and thermomechanical analysis. Acetoclastic methanogenesis was also triggered by bioaugmentation based on experiments by using inhibitor, CH3F, for acetoclastic methanogenesis and carbon isotope fractionation. More importantly, magnetite and graphene, which significantly accelerated electron transfer based on electrochemical analysis, further stimulated acetoclastic methanogenesis. Methanothrix dominated and may play an important role in this stage. This work suggests that diverse methanogenic pathways may be benefited from an increase of electron transfer other than DIET-CO2 reduction. Consequently, the long-standing view that only DIET-CO2 reduction was stimulated by conductive materials may need to be reevaluated. Our research provides potential guides to increase methane production during anaerobic digestion by the enhancement of acetoclastic methanogenesis.

Enhanced electron transfer of different mediators for strictly opposite shifting of metabolism in Clostridium pasteurianum grown on glycerol in a new electrochemical bioreactor.

Microbial electrosynthesis or electro-fermentation in bioelectrochemical systems (BES) have recently received much attention. Here, we demonstrate with the glycerol metabolism by Clostridium pasteurianum that H 2 from in situ water electrolysis, especially in combination with a redox mediator, provides a simple and flexible way for shifting product selectivity and enhancing product yield in the fermentation process. In particular, we report and quantify for the first time strictly different effects of Neutral Red (NR) and the barely studied redox mediator Brilliant Blue (BB) on the growth and product formation of C. pasteurianum grown on glycerol in a newly developed BES. We were able to switch the product formation pattern of C. pasteurianum with a concentration-dependent addition of NR and BB under varied iron availability. Interestingly, NR and BB influenced the glycerol metabolism in a strictly opposite manner concerning the formation of the major products 1,3-propanediol (1,3-PDO) and n-butanol (BuOH). Whereas, NR and iron generally enhance the formation of BuOH, BB favors the formation of 1,3-PDO. In BES the metabolic shifts were enhanced, leading to a further increased yield by as high as 33% for BuOH in NR fermentations and 21% for 1,3-PDO in BB fermentations compared with the respective controls. For the first time, the electron transfer mediated by these mediators and their recycle (recharge) were unambiguously quantified by excluding the overlapping effect of iron. BB has a higher capacity than NR and iron. The extra electron transfer by BB can account for as high as 30-75% of the total NAD + regeneration under certain conditions, contributing significantly to the product formation.

Stimulation of ferrihydrite nanorods on fermentative hydrogen production by Clostridium pasteurianum

Conversion of organic matter to biohydrogen possesses promising application potential. In this study, low-cost ferrihydrite nanorods were used to enhance hydrogen production by Clostridium pasteurianum. The maximum cumulative hydrogen production and the hydrogen yield were 1.03 mmol and 3.55 mol H2/mol glucose, respectively, which were 68.9% and 15.6% higher than those of the batch groups without ferrihydrite addition. Moreover, in comparison with magnetite and hematite nanoparticles, ferrihydrite presented the best stimulation for hydrogen evolution. The enhancement mechanisms were explored based on metabolic distribution, gene expression, enzymatic activity, and metabolite determination, such as Fe(II) concentration and pH value. The potential stimulation mechanisms are summarized as follows: ferrihydrite improves glucose conversion efficiency and promotes cell growth; ferrihydrite elevates the transcripts and activity of hydrogenase; and ferrihydrite reduction via its buffer function could ease acidification. This study demonstrates that ferrihydrite addition is an effective and green strategy to enhance fermentative hydrogen production.

Homologous overexpression of hydrogenase and glycerol dehydrogenase in Clostridium pasteurianum to enhance hydrogen production from crude glycerol

This study reports engineering of a hypertransformable variant of C. pasteurianum for bioconversion of glycerol into hydrogen (H2). A functional glycerol-triggered hydrogen pathway was engineered based on two approaches: (1) increasing product yield by overexpression of immediate enzyme catalyzing H2 production, (2) increasing substrate uptake by overexpression of enzymes involved in glycerol utilization. The first strategy aimed at overexpression of hydA gene encoding hydrogenase, and the second one, through combination of overexpression of dhaD1 and dhaK genes encoding glycerol dehydrogenase and dihydroxyacetone kinase. These genetic manipulations resulted in two recombinant strains (hydA++/dhaD1K++) capable of producing 97% H2 (v/v), with yields of 1.1 mol H2/mol glycerol in hydA overexpressed strain, and 0.93 mol H2/mol glycerol in dhaD1K overexpressed strain, which was 1.5 fold higher than wild type. Among two strains, dhaD1K++ consumed more glycerol than hydA++ which proves that overexpression of glycerol enzymes has enhanced glycerol intake rate.

Metabolic engineering of Clostridium beijerinckii to improve glycerol metabolism and furfural tolerance

BackgroundInefficient utilization of glycerol by Clostridium beijerinckii (Cb) is a major impediment to adopting glycerol metabolism as a strategy for increasing NAD(P)H regeneration, which would in turn, alleviate the toxicity of lignocellulose-derived microbial inhibitory compounds (LDMICs, e.g., furfural), and improve the fermentation of lignocellulosic biomass hydrolysates (LBH) to butanol. To address this problem, we employed a metabolic engineering strategy to enhance glycerol utilization by Cb.ResultsBy overexpressing two glycerol dehydrogenase (Gldh) genes (dhaD1 and gldA1) from the glycerol hyper-utilizing Clostridium pasteurianum (Cp) as a fused protein in Cb, we achieved approximately 43% increase in glycerol consumption, when compared to the plasmid control. Further, Cb_dhaD1 + gldA1 achieved a 59% increase in growth, while butanol and acetone–butanol–ethanol (ABE) concentrations and productivities increased 14.0%, 17.3%, and 55.6%, respectively, relative to the control. Co-expression of dhaD1 + gldA1 and gldA1 + dihydroxyacetone kinase (dhaK) resulted in significant payoffs in cell growth and ABE production compared to expression of one Gldh. In the presence of 4–6 g/L furfural, increased glycerol consumption by the dhaD1 + gldA1 strain increased cell growth (> 50%), the rate of furfural detoxification (up to 68%), and ABE production (up to 40%), relative to the plasmid control. Likewise, over-expression of [(dhaD1 + gldA1) dhaK] improved butanol and ABE production by 70% and 50%, respectively, in the presence of 5 and 6 g/L furfural relative to the plasmid control.ConclusionsOverexpression of Cp gldhs and dhaK in Cb significantly enhanced glycerol utilization, ABE production, and furfural tolerance by Cb. Future research will address the inability of recombinant Cb to metabolize glycerol as a sole substrate.

Determining butanol inhibition kinetics on the growth of Clostridium pasteurianum based on continuous operation and pulse substrate additions

AbstractBACKGROUNDClostridium pasteurianum is a well described strain for the conversion of glycerol into butanol. In general, cell growth kinetics depends on the type of glycerol used and the concentration of butanol in the fermentation broth. However, despite the numerous studies existing on the subject, there is limited information in the literature regarding growth inhibition kinetics due to the cytotoxic effect of butanol on cell growth. This can be attributed to the difficulty of growing cells at high butanol concentration which renders the determination of inhibition kinetics a rather challenging task.RESULTSDuring this study a new approach for the determination of butanol inhibition kinetics on the growth of C. pasteurianum was tested. Specifically, pulses of crude glycerol were applied when steady state was reached during continuous fermentation experiments with increasing butanol concentration in the feed. Combining pulse experiments with batch fermentation at low substrate concentration allowed for accurate determination of the kinetic constants for inhibited growth. This approach also minimized the correlation of the growth constants which often leads to poor identifiability.CONCLUSIONThe proposed experimental approach showed good identifiability of the kinetic parameters for butanol inhibition of the microbial growth and can be proven valuable for the determination of inhibitory effects of highly toxic compounds. © 2019 Society of Chemical Industry

Ramalan Sistem Endogen CRISPR/Cas: Menuju ke Arah Memanfaatkan CRISPR/Cas Untuk Kejuruteraan Genetik

Genetic engineering field has become an imperative approach for enhancement of various bioproducts yield and productivity; and found extended applications in gene therapy, nanotechnology, as well as industrial microbiology. Modern genetic engineering tool CRISPR/Cas system, specifically the Type II system from Streptococcus pyogenes, is gaining traction in recent years and being utilized to engineer novel strains to overproduce primary fermentation product of interest. Employing this technology for non-model microorganism such as Clostridium spp is still restricted due to several limitations such as inadequate genome information, resistance against transformation, low plasmid replication, and the ability for gene expression. The prediction of CRISPR/Cas systems in microbial genomes is fundamentally the initial step towards exploitation of this technology to engineer Clostridium spp. In this study, we demonstrate a simple yet effective method to predict component of endogenous CRISPR/Cas systems, using Clostridium spp genomes as a proof-of-concept. We identified the “real” CRISPR array together with the cas gene operon consist of Type I B signature proteins in Clostridium pasteurianum which is in agreement with the previous report, implying that this strategy generates reliable CRISPR/Cas systems prediction. Thus, this provides a glimpse on how bioinformatics and biocomputational tools can be utilized to overcome barriers in genetic engineering.

Improved electrocompetence and metabolic engineering of Clostridium pasteurianum reveals a new regulation pattern of glycerol fermentation.

Clostridium pasteurianum produces industrially valuable chemicals such as n-butanol and 1,3-propanediol from fermentations of glycerol and glucose. Metabolic engineering for increased yields of selective compounds is not well established in this microorganism. In order to study carbon fluxes and to selectively increase butanol yields, we integrated the latest advances in genome editing to obtain an electrocompetent Clostridium pasteurianum strain for further engineering. Deletion of the glycerol dehydratase large subunit (dhaB) using an adapted S. pyogenes Type II CRISPR/Cas9 nickase system resulted in a 1,3-propanediol-deficient mutant producing butanol as the main product. Surprisingly, the mutant was able to grow on glycerol as the sole carbon source. In spite of reduced growth, butanol yields were highly increased. Metabolic flux analysis revealed an important role of the newly identified electron bifurcation pathway for crotonyl-CoA to butyryl-CoA conversion in the regulation of redox balance. Compared to the parental strain, the electron bifurcation pathway flux of the dhaB mutant increased from 8 to 46% of the overall flux from crotonyl-CoA to butyryl-CoA and butanol, indicating a new, 1,3-propanediol-independent pattern of glycerol fermentation in Clostridium pasteurianum.

Diffusion network of CO in FeFe-Hydrogenase.

FeFe-hydrogenase is an efficient enzyme to produce H2 under optimal conditions. However, the activity of this enzyme is highly sensitive to the presence of inhibitory gases CO and O2 that cause irreversible damage to the active site. Therefore, a detailed knowledge of the diffusion pathways of these inhibitory gases is necessary to develop strategies for designing novel enzymes that are tolerant to these gases. In this work, we studied the diffusion pathways of CO in the CpI FeFe-hydrogenase from Clostridium pasteurianum. Specifically, we used several enhanced sampling and free-energy simulation methods to reconstruct a three-dimensional free-energy surface for CO diffusion which revealed 45 free-energy minima forming an interconnected network of pathways. We discovered multiple pathways of minimal free-energy as diffusion portals for CO and found that previously suggested hydrophobic pathways are not thermodynamically favorable for CO diffusion. We also observed that the global minimum in the free-energy surface is located in the vicinity of the active-site metal cluster, the H-cluster, which suggests a high-affinity for CO near the active site. Among 19 potential residues that we propose as candidates for future mutagenesis studies, 11 residues are shared with residues that have been previously proposed to increase the tolerance of this enzyme for O2. We hypothesize that these shared candidate residues are potentially useful for designing new variants of this enzyme that are tolerant to both inhibitory gases.

A novel strategy to enhance biohydrogen production using graphene oxide treated thermostable crude cellulase and sugarcane bagasse hydrolyzate under co-culture system

Graphene oxide (GO) treated thermostable crude cellulase has been obtained via fungal co-cultivation of strain Cladosporium cladosporioides NS2 and Emericella variecolor NS3 using mix substrate of orange peel and rice straw under solid state fermentation (SSF). Enzyme activity of 60 IU/gds FP, 300 IU/gds EG and 400 IU/gds BGL are recorded in the presence of 1.0% GO in 96 h. This crude enzyme showed 50 °C as optimum incubation temperature, thermally stable at 55 °C for 600 min and stability in the pH range 4.5–8.0. Further, 70.04 g/L of sugar hydrolyzate is obtained from enzymatic conversion of 3.0% alkali pre-treated baggase using GO treated crude cellulase. Finally, 2870 ml/L cumulative biohydrogen production having bacterial biomass ∼2.2 g/L and the complimentary initial pH 7.0 is recorded from sugar hydrolyzate via dark fermentation using co-culture of Clostridium pasteurianum (MTCC116) and a newly isolated Bacillus subtilis PF_1.

Anaerobic biotransformation of hexachlorocyclohexane isomers by Dehalococcoides species and an enrichment culture.

The biotransformation of hexachlorocyclohexane isomers (HCH) by two Dehalococcoides mccartyi strains (195 and BTF08) and an enrichment culture was investigated and compared to conversion by the obligate anaerobic strain Clostridium pasteurianum strain DSMZ 525. The D. mccartyi strains preferentially transformed γ-HCH over α-HCH and δ-HCH isomers while β-HCH biotransformation was not significant. In case of the enrichment culture, γ-HCH was preferentially transformed over the δ-HCH, β-HCH and α-HCH isomers. Major observed metabolites in both cases were tetrachlorocyclohexene and as end products monochlorobenzene (MCB) and benzene. Dechlorination of the γ-HCH isomer was linked to an increase in cell numbers for strain 195. γ-HCH transformation was linked to considerable carbon stable isotope fractionation with the enrichment factor εc = - 5.5 ± 0.8‰ for D. mccartyi strain 195, εc = - 3.1 ± 0.4‰ for the enrichment culture and εc = - 4.1 ± 0.6‰ for co-metabolic transformation by C. pasteurianum.

A novel All-in-One electrolysis electrode and bioreactor enable better study of electrochemical effects and electricity-aided bioprocesses.

An autoclavable All-in-One electrolysis electrode in a rod shape assembly is developed as a new tool for bioelectrochemical systems and electricity-aided bioprocesses. It can replace the classic two-chamber bioelectrochemical system for electrolysis reactions, be inserted into conventional bioreactors and is easily adaptable as electrocatalytic surface or generator of super-fine bubbles (H2 and O2) for bioconversion processes. Whereas the bioreactor itself functions as the working electrode chamber, a well-integrated inner counter electrode chamber enables water electrolysis without the normally encountered undesired ion-transfer effect. The efficiencies of the electrode are characterized and its advantages and usefulness compared to the classic H-Cell bioelectrochemical system (BES) are demonstrated with glycerol fermentations by Clostridium pasteurianum DSM 525.

Analysis of the Continuous Bioconversion of Glycerol by Promotion of Highly Glycerol-Resistant Glycerol-Degrading Bacteria

We identified component microorganisms in a fed-batch operation by modulating the mixed flora via addition of glucose to achieve continuous bioconversion of hardly degradable glycerol. To study the microbial community structure of the flora accumulated by the addition of glucose, 16S ribosomal RNA (rRNA) gene was sequenced using PCR with denaturing gradient gel electrophoresis (DGGE). Burkholderia vietnamiensis, Burkholderia phenoliruptrix, Staphylococcus aureus, Bacillus licheniformis, and Clostridium pasteurianum were identified as component strains. Using the colony containing C. pasteurianum, the hydrogen yield was 0.34 mol/(mol glycerol). C. pasteurianum, B. licheniformis, B. vietnamiensis, and B. phenoliruptrix utilized both glycerol and glucose as substrates and could tolerate high glycerol loads. In early fermentation, predominance of the hydrogen-producing C. pasteurianum resulted in the conversion of glycerol into hydrogen and 1,3-propanediol. In contrast, in late fermentation, the auxiliary degradation of B. licheniformis and the two Burkholderia strains enabled continuous conversion of the glycerol to valuable compounds. Glucose addition results in a stable flora by optimizing the ratio of highly glycerol-resistant glycerol-degrading bacteria, thereby establishing an anaerobic digestion process that allows continuous conversion of high loads of glycerol.

Application of forward osmosis technology in crude glycerol fermentation biorefinery-potential and challenges

Forward osmosis (FO) is a low energy-intensive process since the driving force for water transport is the osmotic pressure difference, Δπ, between the feed and draw solutions, separated by the FO membrane, where πdraw > πfeed. The potential of FO in wastewater treatment and desalination have been extensively studied; however, regeneration of the draw solution (thereby generating clean water) requires application of an energy-intensive process step like reverse osmosis (RO). In this study, the potential of applying FO for direct water recirculation from diluted fermentation effluent to concentrated feedstock, without the need for an energy-intensive regeneration step (e.g. RO), has been investigated. Butanol production during crude glycerol fermentation by Clostridium pasteurianum, has been selected as a model process and the effect of cross-flow velocity and the dilution of draw solution on the water flux during short-term experiments (200min), were investigated. Statistical analysis revealed that the dilution of the draw solution is the most influential factor for the water flux. Subsequent modelling of an integrated FO-fermentation process, showed that water recoveries could lead to substantial financial benefits, although the integrated FO-fermentation process demonstrated lower water flux than expected. FTIR analyses of the membrane surface implied that the decrease in water flux was due to the presence of proteins, polysaccharides and other extracellular polymeric substances on the membrane active layer, indicating the presence of a fouling layer. Based on these findings, possible fouling alleviation strategies and future research directions are discussed and proposed.

Effects of low-strength ultrasonication on dark fermentative hydrogen production: Start-up performance and microbial community analysis

Our previous studies have demonstrated the positive impacts of low-strength (LS)-ultrasonication on biological methane production via anaerobic digestion. In this study, the effects of LS-ultrasonication and effluent recirculation on dark fermentative hydrogen production using upflow anaerobic sludge blanket reactor were investigated. Significant increase in hydrogen (H2) yield (up to 0.89 mol H2/mol hexose) was observed under LS-ultrasonication conditions. This enhanced H2 yield seems to be associated with the decrease in dissolved H2 concentration via a degassing effect, as evident from increased H2 content (up to 20%). Effluent recirculation alone led to positive effects. However, applying both LS-ultrasonication and effluent recirculation decreased H2 yield compared to the trial with only LS-ultrasonication (0.89–0.72 mol H2/mol hexose), possibly due to the cumulative shear stress and sludge bed expansion above the ultrasonication zone. Microbial community analysis revealed that the presence/absence of LS-ultrasonication and/or effluent recirculation significantly affected the microbial communities. Ethanoligenes cloacae, Aeromonas hydrophila and Clostridium pasteurianum were identified as principal H2 producers under the LS-ultrasonic conditions, while C. butyricum, the most commonly reported H2 producer, was more abundant in the controls. The energy balance of the LS-ultrasonication system was evaluated and future research directions were suggested.

Intensified crude glycerol conversion to butanol by immobilized Clostridium pasteurianum

Butanol production from glycerol was investigated through Clostridium pasteurianum entrapped into polyvinyl alcohol particles. Using an optimized system, batch and repeated batch fermentations with free and entrapped cells were performed, respectively. In both systems, glycerol samples of different purity were tested. In repeated batch fermentations, process time decreased from 19.5 to 2.7 h and butanol productivity increased 6.3 times (3.08 g L−1 h−1) compared with the free-cell process, using pure glycerol. In the case of glycerol from biodiesel production, butanol productivity of 2.90 and 1.76 g L−1 h−1 was achieved from glycerol 01 and glycerol 02, respectively. No cell growth or solvent production was observed when the same glycerols were used in free-cell fermentations.

Microbial synthesis of metallic molybdenum nanoparticles

The production of nanoparticles through biosynthesis is a reliable, non-toxic, and sustainable alternative to conventional chemical and physical methods of production. While noble metals, such as palladium, gold, and silver, have been formed via bioreduction, biologically-induced reduction of electroactive elements to a metallic state has not been reported previously. Herein, we report the reduction of an electroactive element, molybdenum, via microbial reduction using Clostridium pasteurianum. C. pasteurianum was able to reduce 88% of the added Mo6+ ions. The bioreduced molybdenum was shown to be metallically bonded in a prototypical crystal structure with an average particle size of 15 nm. C. pasteurianum was previously shown to degrade azo dyes using in situ formed Pd nanoparticles, but this study shows that in situ formed Mo particles also act as catalysts for degradation of azo dyes. C. pasteurianum cultures with the bioformed Mo nanoparticles were able completely degrade 155 μM methyl orange within 6 min, while controls with no Mo took 36 min. This research demonstrates, for the first time, that the bioreduction of active elements and formation of catalytic particles is achievable.

Catalytic Activity of Microbially‐formed Palladium Nanoparticles

AbstractThe catalytic behavior of microbially‐formed palladium nanoparticles using Clostridium pasteurianum BC1 is reported. To our knowledge, this is the first study that evaluates the electrochemical catalytic activity of microbially‐formed palladium nanoparticles. Pd nanoparticles formed using immobilized microbes were found to exhibit an average mass activity of 177 mA mg−1 which was almost twice the activity of the abiotically formed controls (94 mA mg−1). The results of this study aim to support the use of nanoparticles formed using facile and environmentally‐friendly microbial synthesis methods as a suitable alternative as opposed to standard physical and chemical synthesis methods.