A thermostable #x003B2;-ketothiolase of polyhydroxyalkanoates (PHAs) in <Emphasis Type="Italic">Thermus thermophilus</Emphasis>: Purification and biochemical properties

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesised by numerous bacteria as intracellular carbon and energy storage compounds which accumulate as granules in the cytoplasm of the cells. The biosynthesis of PHAs, in the thermophilic bacterium T. thermophilus grown in a mineral medium supplemented with sodium gluconate as sole carbon source has been recently reported. Here, we report the purification at apparent homogeneity of a #x003B2;-ketoacyl-CoA thiolase from T. thermophilus, the first enzyme of the most common biosynthetic pathway for PHAs. B-Ketoacyl-CoA thiolase appeared as a single band of 45.5-kDa molecular mass on SDS/PAGE. The enzyme was purified 390-fold with 7% recovery. The native enzyme is a multimeric protein of a molecular mass of approximately of 182 kDa consisting of four identical subunits of 45.5 kDa, as identified by an in situ renaturation experiment on SDS-PAGE. The enzyme exhibited an optimal pH of approximately 8.0 and highest activity at 65 °C for both direction of the reaction. The thiolysis reaction showed a substrate inhibition at high concentrations; when one of the substrates (acetoacetyl CoA or CoA) is varied, while the concentrations of the second substrates (CoA or acetoacetyl CoA respectively) remain constant. The initial velocity kinetics showed a pattern of a family of parallel lines, which is in accordance with a ping-pong mechanism. #x003B2;-Ketothiolase had a relative low Km of 0.25 mM for acetyl-CoA and 11 M and 25 M for CoA and acetoacetyl-CoA, respectively. The enzyme was inhibited by treatment with 1 mM N-ethylmaleimide either in the presence or in the absence of 0.5 mM of acetyl-CoA suggesting that possibly a cysteine is located at/or near the active site of #x003B2;-ketothiolase. (Mol Cell Biochem 269: 27–36, 2005)     

Synthesis of <Emphasis Type="Italic">scl</Emphasis>-poly (3-hydroxyalkanoates) by <Emphasis Type="Italic">Bacillus cereus</Emphasis> found in freshwater,from monosaccharides and disaccharides

Background

Polyhydroxyalkanoates are a good substitute for synthetic plastic because they are highly biocompatible, ecofriendly, and biodegradable. Bacteria in freshwater bodies such as rivers, tube wells, and canals are exposed to alternating high and low concentrations of substrates that induce PHA production.

Methods

Fresh water samples were collected for isolation of bacterial strains. Screening of PHA in bacterial cells was performed with Sudan and Nile Red staining. Extracted PHA was characterized by FTIR.

Results

In this study, nine bacterial isolates were selected for PHA production on the basis of phenotypic screening. Their ability to accumulate PHAs was determined using different monosaccharides and disaccharides. Two bacterial isolates Bacillus cereus T1 (KY746353) and Bacillus cereus R3 (KY746354) produced PHAs. Optimal growth of the bacterial strain (T1) was observed in the presence of glucose, followed by maximum production of PHAs (63% PHAs) during the logarithmic phase of growth. B. cereus R3 (KY746354) accumulated 60% PHAs by dry cell weight.

Conclusion

PHA accumulation was relatively less with fructose, but both strains showed increased production (up to 50%) with sucrose. The polymer produced was characterized by Fourier-transform infrared spectroscopy (FTIR), which showed that the compound contains short-chain PHAs.

     

Biosynthesis of medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) by Comamonas testosteroni during cultivation on vegetable oils

Comamonas testosteroni has been studied for its ability to synthesize and accumulate medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) during cultivation on vegetable oils available in the local market. Castor seed oil, coconut oil, mustard oil, cotton seed oil, groundnut oil, olive oil and sesame oil were supplemented in the mineral medium as a sole source of carbon for growth and PHAs accumulation. The composition of PHAs was analysed by a coupled gas chromatography/mass spectroscopy (GC/MS). PHAs contained C6 to C14 3-hydroxy acids, with a strong presence of 3-hydroxyoctanoate when coconut oil, mustard oil, cotton seed oil and groundnut oil were supplied. 3-hydroxydecanoate was incorporated at higher concentrations when castor seed oil, olive oil and sesame oil were the substrates. Purified PHAs samples were characterized by Fourier Transform Infrared (FTIR) and 13C NMR analysis. During cultivation on various vegetable oils, C. testosteroni accumulated PHAs up to 78.5-87.5% of the cellular dry material (CDM). The efficiency of the culture to convert oil to PHAs ranged from 53.1% to 58.3% for different vegetable oils. Further more, the composition of the PHAs formed was not found to be substrate dependent as PHAs obtained from C. testosteroni during growth on variety of vegetable oils showed similar compositions; 3-hydroxyoctanoic acid and/or 3-hydroxydecanoic acid being always predominant. The polymerizing system of C. testosteroni showed higher preference for C8 and C10 monomers as longer and smaller monomers were incorporated less efficiently.     

Microscopic examination in vivo and in vitro of natural and cross-linked polyunsaturated mclPHA

Pseudomonas aeruginosa 42A2 produces a polyunsaturated polyhydroxyalkanoates (PHA-L) when grown on linseed oil as a substrate. Its high unsaturation content (36.5%) provides highly reactive PHA-L, generating a cross-linked biopolymer after ultraviolet (UV) irradiation. Both PHAs (PHA-L and uvPHA-L) were characterized by nuclear magnetic resonance, Fourier transform infrared spectroscopy, gel permeation chromatography, gas chromatography–mass spectrometry and differential scanning calorimetry–thermogravimetric analysis. The structural analysis of the new polymer revealed a dramatic decrease in unsaturated monomer content (8.5%), due to the complete disappearance of the polyunsaturated monomers (C_12:2, C_14:2, and C_14:3). The cross-linking reaction was also confirmed by atomic force microscopy (AFM) and transmission electron microscopy. AFM showed morphological changes in bacteria cells with and without PHA granules. The microscope techniques provided us with micrographs of the native and cross-linked polymers, showing the formation of a reticular structure as the consequence of the cross-linking reaction.     

Reduction of cell lysate viscosity during processing of poly(3-hydroxyalkanoates) by chromosomal integration of the staphylococcal nuclease gene in Pseudomonas putida

Poly(3-hydroxyalkanoates) (PHAs) are biodegradable thermoplastics which are accumulated by many bacterial species in the form of intracellular granules and which are thought to serve as reserves of carbon and energy. Pseudomonas putida accumulates a polyester, composed of medium-side-chain 3-hydroxyalkanoic acids, which has excellent film-forming properties. Industrial processing of PHA involves purification of the PHA granules from high-cell-density cultures. After the fermentation process, cells are lysed by homogenization and PHA granules are purified by chemical treatment and repeated washings to yield a PHA latex. Unfortunately, the liberation of chromosomal DNA during lysis causes a dramatic increase in viscosity, which is problematic in the subsequent purification steps. Reduction of the viscosity is generally achieved by the supplementation of commercially available nuclease preparations or by heat treatment; however, both procedures add substantial costs to the process. As a solution to this problem, a nuclease-encoding gene from Staphylococcus aureus was integrated into the genomes of several PHA producers. Staphylococcal nuclease is readily expressed in PHA-producing Pseudomonas strains and is directed to the periplasm, and occasionally to the culture medium, without affecting PHA production or strain stability. During downstream processing, the viscosity of the lysate from a nuclease-integrated Pseudomonas strain was reduced to a level similar to that observed for the wild-type strain after treatment with commercial nuclease. The nuclease gene was also functionally integrated into the chromosomes of other PHA producers, including Ralstonia eutropha.     

Embryonic integument and "molts" in Manduca sexta (Insecta,Lepidoptera)

In Manduca sexta the germ band is formed 12 h post-oviposition (p.o.) (=10% development completed) and is located above the yolk at the egg surface. The cells show a polar organization. They are engaged in the uptake and degradation of yolk globules, pinched off from the yolk cells. This process can be observed in the integumental cells during the first growth phase of the embryo that lasts until "katatrepsis," an embryonic movement that takes place at 40% development completed. At 37% development completed, the ectoderm deposits a thin membrane at its apical surface, the first embryonic membrane, which detaches immediately before katatrepsis. The second period of embryonic growth--from katatrepsis to 84 h p.o. (70% development completed)--starts with the deposition of a second embryonic membrane that is somewhat thicker than the first one and shows a trilaminar, cuticulin-like structure. Whereas the apical cell surface is largely smooth during the deposition of the first embryonic membrane, it forms microvilli during deposition of the second one. At the same time, uptake of formed yolk material ceases and the epidermal cells now contain clusters of mitochondria below the apical surface. Rough endoplasmic reticulum (RER) increases in the perinuclear region. The second embryonic membrane detaches about 63 h p.o. At 69 h p.o., a new generation of microvilli forms and islands of a typical cuticulin layer indicate the onset of the deposition of the larval cuticle. The third growth phase is characterized by a steady increase in the embryo length, the deposition of the larval procuticle, and by cuticular tanning at about 100 h p.o. Beginning at that stage, electron-lucent vesicles aggregate below the epidermal surface and are apparently released below the larval cuticle. Manduca sexta is the first holometabolous insect in which the deposition of embryonic membranes and cuticles has been examined by electron microscopy. In correspondence with hemimetabolous insects, the embryo of M. sexta secretes three covers at approximately the same developmental stage. A marked difference: the second embryonic cover, which in Hemimetabola clearly exhibits a cuticular organization, has instead a membranous, cuticulin-like structure. We see the difference as the result of an evolutionary reductional process promoted by the redundancy of embryonic covers in the egg shell. Embryonic "molts" also occur in noninsect arthropods; their phylogenetical aspects are discussed.     

Production of functionalized polyhydroxyalkanoates by genetically modified <Emphasis Type="Italic">Methylobacterium extorquens</Emphasis> strains

Background  

Methylotrophic (methanol-utilizing) bacteria offer great potential as cell factories in the production of numerous products from biomass-derived methanol. Bio-methanol is essentially a non-food substrate, an advantage over sugar-utilizing cell factories. Low-value products as well as fine chemicals and advanced materials are envisageable from methanol. For example, several methylotrophic bacteria, including Methylobacterium extorquens, can produce large quantities of the biodegradable polyester polyhydroxybutyric acid (PHB), the best known polyhydroxyalkanoate (PHA). With the purpose of producing second-generation PHAs with increased value, we have explored the feasibility of using M. extorquens for producing functionalized PHAs containing C-C double bonds, thus, making them amenable to future chemical/biochemical modifications for high value applications.     

Polyhydroxyalkanoate (PHA) biosynthesis in Thermus thermophilus: Purification and biochemical properties of PHA synthase

The biosynthesis of polyhydroxyalkanoates (PHAs) was studied, for the first time, in the thermophilic bacterium Thermus thermophilus. Using sodium gluconate (1.5% w/v) or sodium octanoate (10 mM) as sole carbon sources, PHAs were accumulated to approximately 35 or 40% of the cellular dry weight, respectively. Gas chromatographic analysis of PHA isolated from gluconate-grown cells showed that the polyester (M_w: 480,000 g.mol^–1) was mainly composed of 3-hydroxydecanoate (3HD) with a molar fraction of 64%. In addition, 3-hydroxyoctanoate (3HO), 3-hydroxyvalerate (3HV) and 3-hydroxybutyrate (3HB) occurred as constituents. In contrast, the polyester (M_w: 391,000 g mol^–1) from octanoate-grown cells was composed of 24.5 mol% 3HB, 5.4 mol% 3HO, 12.3 mol% 3-hydroxynonanoate (3HN), 14.6 mol% 3HD, 35.4 mol% 3-hydroxyundecanoate (3HUD) and 7.8 mol% 3-hydroxydodecanoate (3HDD). Activities of PHA synthase, a -ketothiolase and an NADPH-dependent reductase were detected in the soluble cytosolic fraction obtained from gluconate-grown cells of T. thermophilus. The soluble PHA synthase was purified 4271-fold with 8.5% recovery from gluconate-grown cells, presenting a K_m of 0.25 mM for 3HB-CoA. The optimal temperature of PHA synthase activity was about 70°C and acts optimally at pH near 7.3. PHA synthase activity was inhibited 50% with 25 M CoA and lost all of its activity when it was treated with alkaline phosphatase. PHA synthase, in contrary to other reported PHA synthases did not exhibit a lag phase on its kinetics, when low concentration of the enzyme was used. Incubation of PHA synthase with 1 mM N-ethyl-maleimide inhibits the enzyme 56%, indicating that cysteine might be involved in the catalytic site of the enzyme. Acetyl phosphate (10 mM) activated both the native and the dephosphorylated enzyme. A major protein (55 kDa) was detected by SDS-PAGE. When a partially purified preparation was analyzed on native PAGE the major band exhibiting PHA synthase activity was eluted from the gel and analyzed further on SDS-PAGE, presenting the first purification of a PHA synthase from a thermophilic microorganism.     

Production of polyhydroxyalkanoates (PHAs) with canola oil as carbon source

Wautersia eutropha was able to synthesize medium chain length polyhydroxyalkanoates (PHAs) when canola oil was used as carbon source. W. eutropha was cultivated using fructose and ammonium sulphate as carbon and nitrogen sources, respectively, for growth and inoculum development. The experiments were done in a laboratory scale bioreactor in three stages. Initially, the biomass was adapted in a batch culture. Secondly, a fed-batch was used to increase the cell dry weight and PHA concentration to 4.36 g L(-1) and 0.36 g L(-1), respectively. Finally, after the addition of canola oil as carbon source a final concentration of 18.27 g L(-1) PHA was obtained after 40 h of fermentation. With canola oil as carbon source, the polymer content of the cell dry matter was 90%. The polymer was purified from dried cells and analyzed by FTIR, NMR and DSC using PHB as reference. The polymer produced by W. eutropha from canola oil had four carbon monomers in the structure of the PHA and identified by 1H and 13C NMR analysis as 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxyoctanoate (3HO), and 3-hydroxydodecanoate (3HDD).     

Starter culture of <Emphasis Type="Italic">Pseudomonas veronii</Emphasis> strain B for aerobic granulation

Aggregation of bacterial cells is used in formation of microbial granules. Aerobically grown microbial granules can be used as the bio-agents in the treatment of wastewater. However, there are problems with start up of microbial granulation and biosafety of this process. Aim of this research was selection and testing of safe microbial strain with high cell aggregation ability to shorten period of microbial granules formation. Five bacterial strains with cell aggregation index higher than 50% have been isolated from the granules. Strain of Pseudomonas veronii species was considered as most probably safe starter culture for granulation because other strains belonged to the species known as human pathogens. The microbial granules were formed after 3 days of cultivation in case when P. veronii strain B was applied to start-up aerobic granulation process using model wastewater. The granules were produced from activated sludge after 9 days of cultivation. Microbial aggregates produced from starter culture of P. veronii strain B were more compact (sludge volume index was 70 ml/g) than those produced from activated sludge (sludge volume index was 106 ml/g). It is a first proof that application of selected safe starter pure culture with high cell aggregation ability can accelerate and enhance formation of microbial granules.     

Poly(3-hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli harbouring Streptomyces aureofaciens PHB biosynthesis genes: effect of various carbon and nitrogen sources

Recombinant Escherichia coli (ATCC:PTA-1579) harbouring poly(3-hydroxybutyrate) (PHB) synthesising genes from Streptomyces aureofaciens NRRL 2209 accumulates PHB. Effects of different carbon and nitrogen sources on PHB accumulation by recombinant E. coli were studied. Among the carbon sources used glycerol, glucose, palm oil and ethanol supported PHB accumulation. No PHB accumulated in recombinant cells when sucrose or molasses were used as carbon source. Yeast extract, peptone, a combination of yeast extract and peptone, and corn steep liquor were used as nitrogen sources. The maximum PHB accumulation (60% of cell dry weight) was measured after 48 h of cell growth at 37 degrees C in a medium with glycerol as the sole carbon source, and yeast extract and peptone as nitrogen sources. Scanning electron microscopy of the PHB granules isolated from recombinant E. coli revealed these to be spherical in shape with a diameter ranging from 0.11 to 0.35 pm with the mean value of 0.23 +/- 0.06 pm.     

Reduction of Cell Lysate Viscosity during Processing of Poly(3-Hydroxyalkanoates) by Chromosomal Integration of the Staphylococcal Nuclease Gene in Pseudomonas putida

Poly(3-hydroxyalkanoates) (PHAs) are biodegradable thermoplastics which are accumulated by many bacterial species in the form of intracellular granules and which are thought to serve as reserves of carbon and energy. Pseudomonas putida accumulates a polyester, composed of medium-side-chain 3-hydroxyalkanoic acids, which has excellent film-forming properties. Industrial processing of PHA involves purification of the PHA granules from high-cell-density cultures. After the fermentation process, cells are lysed by homogenization and PHA granules are purified by chemical treatment and repeated washings to yield a PHA latex. Unfortunately, the liberation of chromosomal DNA during lysis causes a dramatic increase in viscosity, which is problematic in the subsequent purification steps. Reduction of the viscosity is generally achieved by the supplementation of commercially available nuclease preparations or by heat treatment; however, both procedures add substantial costs to the process. As a solution to this problem, a nuclease-encoding gene from Staphylococcus aureus was integrated into the genomes of several PHA producers. Staphylococcal nuclease is readily expressed in PHA-producing Pseudomonas strains and is directed to the periplasm, and occasionally to the culture medium, without affecting PHA production or strain stability. During downstream processing, the viscosity of the lysate from a nuclease-integrated Pseudomonas strain was reduced to a level similar to that observed for the wild-type strain after treatment with commercial nuclease. The nuclease gene was also functionally integrated into the chromosomes of other PHA producers, including Ralstonia eutropha.     

Biosynthesis and characterization of polyhydroxyalkanoates in the polysaccharide-degrading marine bacterium <Emphasis Type="Italic">Saccharophagus degradans</Emphasis> ATCC 43961

The marine bacterium Saccharophagus degradans was investigated for the synthesis of polyhydroxyalkanoates (PHAs), using glucose as the sole source of carbon in a two-step batch culture. In the first step the microorganism grew under nutrient balanced conditions; in the second step the cells were cultivated under limitation of nitrogen source. The biopolymer accumulated in S. degradans cells was detected by Nile red staining and FT-IR analysis. From GC-MS analysis, it was found that this strain produced a homopolymer of 3-hydroxybutyric acid. The cellular polymer concentration, its molecular mass, glass transition temperature, melting point and heat of fusion were 17.2+/-2.7% of dry cell weight, 54.2+/-0.6 kDa, 37.4+/-6.0 degrees C, 165.6+/-5.5 degrees C and 59.6+/-2.2 J g(-1), respectively. This work is the first report determining the capacity of S. degradans to synthesize PHAs.     

Exploitation of inexpensive substrates for production of a novel SCL–LCL-PHA co-polymer by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> MTCC 7925

Studies conducted with various inexpensive carbon sources such as whey, vegetable oils (palm, mustard, soybean and coconut), a low-cost source of glucose-D, rice and wheat bran, and mustard and palm oil cakes demonstrated palm oil as the best substrate for accumulation of a novel short-chain-length–long-chain-length polyhydroxyalkanoate (SCL–LCL-PHA) co-polymer containing SCL 3HAs [3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV)] and LCL 3HAs of 3-hydroxyhexadecanoic acid (3HHD) and 3-hydroxyoctadecanoic acid (3HOD) units as constituents by a sludge-isolated Pseudomonas aeruginosa MTCC 7925. The co-polymer content reached up to 60% of dry cell weight (dcw) at 48 h of incubation in 0.5% (v/v) palm oil and the extract of 0.5% (v/v) palm oil cake supplemented vessels. The PHAs pool was further enhanced up to 69 and 75% (dcw), when the above culture was subjected to P- and N-limitation, respectively. The mol fraction of 3HB:3HV:3HHD:3HOD units were, respectively, 83.1:7.7:3.8:5.4 and 87.3:5.1:3.6:4.0 in P- and N-limited cultures. Consequently, a co-polymer yield of 5 g l⁻¹ (approx.) was achieved, which was about 80-fold higher as compared to 69 mg l⁻¹ of the control culture. On substrate basis, the accumulation reached up to 0.62 g PHAs per g substrate, which was significantly higher as compared to the yield obtained from starch by Haloferax mediterranei and Azotobacter chroococum, from molasses by A. vinelandii UWD, and from lactose and xylose by Pseudomonas cepacia. This novel P(3HB-co-3HV-co-3HHD-co-3HOD) co-polymer exhibited better thermal and mechanical properties as revealed from the differential scanning calorimetry and mechanical property studies, thus opens up new possibilities for various industrial applications.     

Recovery of medium-chain-length polyhydroxyalkanoates (PHAs) through enzymatic digestion treatments and ultrafiltration

Medium-chain-length (mcl) polyhydroxyalkanoates (PHAs) are biodegradable polyesters accumulated intracellularly as energy resources by bacterial species such as Pseudomonas putida. The most popular method for PHA recovery in the downstream processing is solvent extraction using chloroform and methanol. An alternate method is bioseparation using enzymatic digestion process which eliminates the need for hazardous solvents. This research focuses on an attempt to optimize the recovery of PHAs by solubilisation of non-PHA granules through enzymatic treatments such as; Alcalase (to digest the denatured proteins), sodium dodecyl sulfate (SDS) to assist solubilisation, ethylene diamine tetra acetic acid (EDTA) to complex divalent cations and lysozyme to digest the peptidoglycan wall enveloping the cell. The experiment was designed through Taguchi's design of experiment (DOE) using Qualitek-4 software. The results show that Alcalase enzyme used had the most significant effect on the treatment process and contributed to about 71.5% in terms of process factor importance among the different factors on treatment performance for PHA recovery. It is desired to recover the PHA granules in water suspension after the enzymatic treatment by removing the solubilised non-PHA cell material through crossflow ultrafiltration system and purified through continuous diafiltration process. Final purity of PHA in water suspension obtained using GC analysis is 92.6%, with a nearly 90% recovery, thus concluding that this method is indeed a suitable alternative.     

Cassava wastewater as a substrate for the simultaneous production of rhamnolipids and polyhydroxyalkanoates by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Glycerol, cassava wastewater (CW), waste cooking oil and CW with waste frying oils were evaluated as alternative low-cost carbon substrates for the production of rhamnolipids and polyhydroxyalkanoates (PHAs) by various Pseudomonas aeruginosa strains. The polymers and surfactants produced were characterized by gas chromatography–mass spectrophotometry (MS) and by high-performance liquid chromatography–MS, and their composition was found to vary with the carbon source and the strain used in the fermentation. The best overall production of rhamnolipids and PHAs was obtained with CW with frying oil as the carbon source, with PHA production corresponding to 39% of the cell dry weight and rhamnolipid production being 660 mg l⁻¹. Under these conditions, the surface tension of the culture decreased to 30 mN m⁻¹, and the critical micelle concentration was 26.5 mg l⁻¹. It would appear that CW with frying oil has the highest potential as an alternative substrate, and its use may contribute to a reduction in the overall environmental impact generated by discarding such residues.     

Chemical-physical characterization of polyhydroxyalkanoates recovered by means of a simplified method from cultures of <Emphasis Type="Italic">Halomonas campaniensis</Emphasis>

In this study we suggest a simplified and effective method to directly recover polyhydroxyalkanoates (PHAs) from humid biomass of Halomonas campaniensis with no pre-treatment steps. Sodium dodecyl sulphate (SDS) was directly added to dispersed biomass of cultured micro-organism (w/w ratio: 1) in distilled water followed by shaking, heat treatment, and washing steps. The purity of the recovered PHAs synthesized by H. campaniensis was over 95%, regardless of the cell concentrations and the best yield was 12% (w/w) of the cell wet weight when the micro-organism was cultivated in a glucose-based medium or a glucose/propionate-based medium. MS spectroscopy and ¹H, ¹³C-NMR analysis were used to chemically characterize the PHAs; their thermal characteristics were obtained using a differential scanning calorimeter and the average viscosity molecular weight was assessed through specific viscosity measurements. Due to its ease and velocity, our simplified method is suitable for the detection and recovery of PHAs from humid biomasses with high yield and purity. The method, which is quick and at low environmental impact, is very valuable for the simultaneous testing of cultures grown with different inducers for PHAs having particular chemical/physical characteristics.