Heavy Metal Resistant Bacteria Research Articles

Heavy metal resistant bacteria from coal dumping site with plant growth promoting potentials

Heavy metal resistant bacteria from coal dumping site with plant growth promoting potentials

The potential of PGPR in bioremediation of soils with heavy metal contamination

Utilising genetically engineered PGPRs to remediate highly contaminated soil could help to reduce food and fibre production's negative environmental impact. Since the discovery of rhizobia, commercially produced rhizobia inoculants have been available and the usage of PGPR has increased significantly in India recently as a result of improved knowledge about farming techniques. Many substances that are considered hazardous by regulations can be converted into non-hazardous products. The completion of bioremediation can be impacted by a few factors in which abiotic and biotic factors are both included. The most hazardous and chronic contaminants in the soil include heavy metals, metalloids and radionuclides. PGPR was discovered to be effective in combination with certain contaminant-degrading bacteria and another prominent technique for microbially assisted soil remediation is biological reduction. By transferring heavy metal (loids) resistant bacteria to other microbial species, the efficacy of biomedicine can be improved. The development of biofilm helps to detoxify the heavy metals, which is done by enhancement of ability of tolerance of the microbes.

Heavy Metals and Antibiotic Co-Resistance in Bacterial Isolates of Industrial Effluents

Heavy metal and antibiotic co-resistance is a global issue. The goal of this research was to explore the heavy metal also antibiotic resistance patterns of effluent bacterial isolates. Heavy metal resistant bacteria were isolated from effluents and their Minimum Inhibitory Concentration (MIC) was determined. The Multi-Metal resistance (MMR) pattern and antibiotic resistance trait of isolates were defined. The MIC of Cu2+, Pb2+, Cd2+ and Zn2+ was 4, 8, 12 and 24 mM/L, respectively. Most of the isolates indicated the Cd2+, Pb2+ and Zn2+ resistance and high resistance to the most tested antibiotics. The 16S rDNA gene sequences of resistant isolates were handed over to NCBI-GenBank as Staphylococcus sp. ATHA2(JX120151) and Klebsiella oxytoca ATHA1(JQ928574). Between metal tolerances, heavy metal concentration also antibiotic resistance in bacteria existed a correlation. Thus, we’d better not only be alert of antibiotics misapplication, but also responsive of over discharge of effluent containing heavy metals to the environment.

Stabilization of lead and cadmium in soil by sulfur-iron functionalized biochar: Performance, mechanisms and microbial community evolution

Sulfur-iron functionalized biochar (BC-Fe-S) was designed by simultaneously supporting Fe2O3 nanoparticles and grafting sulfur-containing functional groups onto biochar to stabilize Pb and Cd in soil. The BC-Fe-S exhibited excellent stabilization performance for Pb and Cd with fast kinetic equilibrium within 5 days associating with pseudo-second-order model. The bioavailable-Pb and -Cd contents decreased by 59.22% and 70.28% with 3% BC-Fe-S treatment after 20 days of remediation. Speciation transformation analysis revealed that the increase of stabilization time and BC-Fe-S dosage with appropriate soil moisture and pH promoted toxicities decrease of Pb and Cd with transformation of labile fractions to more steady fractions. The labile fractions of Pb and Cd decreased by 12.22% and 16.21% with 3% BC-Fe-S treatment, and transformed to the residual speciation. Meanwhile, wetting-drying and freezing-thawing aging did not markedly alter the bioavailability of Pb and Cd, proving that the BC-Fe-S holds promise for stabilization of Pb and Cd in varying environmental conditions. 16S rRNA sequencing analysis demonstrated that the BC-Fe-S significantly improved diversity and composition of microbial community, especially increasing the relative abundance of heavy metal-resistant bacteria. Overall, these results suggested BC-Fe-S as a high-performance and environmental-friendly amendment with stability to remediate heavy metals polluted soil.

Heavy metal (As, Cd, Cr, Hg, Pb) analysis and identification of heavy metal resistant bacteria in sediments from Manado Bay

Environmental pollution from heavy metals is becoming a growing concern due to the adverse effects it is causing throughout the world. This study aims to analyze heavy metal concentrations and identify heavy metal resistant bacteria in the bay of Manado. Sediment samples were collected from five bays in Manado. The concentrations of heavy metals As, Cd, Cr, Hg and Pb were analyzed using ICP-OES, and Hg using CV-AFS. Bacteria from the sediment were grown in nutrient broth media containing heavy metals As, Cd, Cr, Hg and Pb respectively. Microbiology and 16SrRNA gene analysis were used to identify the bacteria that grown on media containing varying concentrations of heavy metals. The results showed that the sediments from the five bays in Manado contained heavy metals with an average concentration of As <1mg/kg, Cd 1.8mg/kg), Cr 6.2mg/kg, Hg <0.07mg/kg). and Pb 11.2mg/kg. The results of microbiological and molecular analysis showed that 5 species of heavy metal resistant bacteria were Pseudomonas aeruginosa, Bacillus cereus, Staphylococcus arlettae, Acinobacter sp., and Brevibacterium sp. The five bacteria found to be resistant to heavy metals can be used to detoxify As, Cd, Cr, Hg, and Pb.

Nisaea sediminum sp. nov., a heavy metal resistant bacterium isolated from marine sediment in the East China Sea.

A Gram-negative, rod-shaped, motile and strictly aerobic bacterium, designated NBU1469T, was isolated from marine sediment sampled on Meishan Island located in the East China Sea. Strain NBU1469T grew optimally at temperature of 40°C, NaCl concentration of 2.0% (w/v) and pH 7.5. Catalase and oxidase activities, H2S production, nitrate reduction and hydrolysis of Tween 20 were positive. Indole, methyl red reaction, urease, hydrolysis of gelatin, starch, casein, Tweens 40, 60 and 80 were negative. The major cellular fatty acids were C16:0, C19:0 cyclo ω8c and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). The only respiratory quinone was ubiquinone-10 (Q-10). The major polar lipids were phosphatidylglycerol, two unidentified amino-phospholipids and two unidentified phospholipids. Comparative analysis of the 16S rRNA gene sequence showed highest similarities to the species with validated name Nisaea nitritireducens DR41_18T (98.1%) and Nisaea denitrificans DR41_21T (97.6%). Phylogenetic analyses indicated that strain NBU1469T formed a distinct lineage with strains Nisaea nitritireducens DR41_18T and Nisaea denitrificans DR41_21T within the genus Nisaea. The average nucleotide identity and digital DNA-DNA hybridization values between strain NBU1469T and related species of genus Nisaea were well below the threshold limit for prokaryotic species delineation. The DNA G + C content was 63.6%. Based on its phenotypic, chemotaxonomic and genotypic data, strain NBU1469T is considered to be a representative of a novel species in the genus Nisaea, for which the name Nisaea sediminum sp. nov. is proposed. The type strain is NBU1469T (=KCTC 82224T =MCCC 1K04763T).

Effective immobilization of heavy metals via reactive barrier by rhizosphere bacteria and their biofilms

As the portal of plants, rhizosphere microorganisms play an essential role in controlling the species, transformation, and bioavailability of heavy metals, yet the potential passivation mechanism is still unclear. In this study, two heavy metal resistant and growth-promoting rhizosphere bacteria were screened, and their mechanisms in dealing with external stress and immobilizing heavy metal were explored. The results showed that heavy metals inhibited the ability of Pseudomonas sp. H13 and Brevundomonas sp. H16 to promote plant growth, but stimulated the production of extracellular polysaccharides and inorganic labile sulfide, and enhanced biofilm formation, thereby significantly improved the removal efficiency of Cu2+, Zn2+, Cd2+, and Pb2+. Compared with H16, the biofilm of H13 disintegrated rapidly in the later stage, so more metal ions were adsorbed on the planktonic cells. The C–OH and PO groups related to polysaccharides play a crucial role in heavy metal adsorption, and the immobilization mechanism of the planktonic cell is mainly ion exchange and group complex, but for H16, intracellular enrichment cannot be ignored. Functional group complexes played a dominant role in biofilm, and the immobilized heavy metals were more difficult to release into the environment. This study highlighted the potential application prospects of biofilm bacteria in heavy metal remediation and explained the reactive barrier of rhizosphere bacteria to heavy metals.

Application of acid and heavy metal resistant bacteria from rat-hole coal mines in bioremediation strategy.

The Gram-negative bacteria isolated from acid mine drainage (AMD) of rat-hole coal mines were found to be resistant to acidic conditions as well as Fe, Cd, and Cr. The minimum inhibitory concentration (MIC) and maximum bactericidal concentration (MBC) of iron, cadmium, and chromium against the isolates of Enterobacter huaxiensis KHED8 were 4000, 4096, and 256 mg/L, respectively, while the MIC and MBC of Fe, Cd, and Cr against two isolates (KH5M10 and KHCL12) of Serratia marcescens subsp. sakuensis isolates were 4000, 8192, and 256 mg/L, respectively. It was also found that E. huaxiensis KHED8 was able to remove 89%, 90%, and 82.45% of Fe, Cd, and Cr, respectively, and the two isolates of S. marcescens subsp. sakuensis KH5M10 and KHCL12 were able to remove 90%, 95%, and 85.62% of Fe, Cd, and Cr, respectively. The findings of the study provide lead to the use of these heavy metals resistant bacterial isolates for bioremediation of heavy metals laden AMD prevalent in unique rat-hole coal mines.

Responses of heavy metals mobility and resistant bacteria to adding time of activated carbon during chicken manure composting

With the wide application of compost in agriculture, heavy metals (HMs) continue to accumulate in the soil environment, which poses a great threat to the health of the soil environment. Therefore, it is critical for effectively reduce the mobility of HMs. In this study, the influence of activated carbon (AC) addition time on mobility of HMs (Cu, Zn and Pb) and HMs resistant bacteria structure were evaluated during chicken manure composting. The result showed that the addition of AC in the thermophilic period could effectively reduce the mobility of HMs. Subsequently, high-throughput sequencing results showed that the dominant phyla were Proteobacteria, Firmicutes, Actinbacteria, Deinococcus-Thermus, Chloroflexi, Gemmatimonadetes and Bacteroidetes within the sample, which were ubiquitous and abundant in composting. The Redundancy analysis (RDA) results indicated that the mobility of HMs (Cu, Zn and Pb) by superior bacteria fate varied in AC amendment composting. Ultimately, a regulation method is proposed to influence the mobility of HMs by regulating the bacteria community in the AC compost. Our current studies suggest that the addition of AC during compost preparation (thermophilic period) is an effective strategy in regulating the mobility (bioavailability) of HMs, thereby significantly reducing environmental pollution problems.

Patterns of heavy metal resistant bacterial community succession influenced by biochar amendment during poultry manure composting

The purpose of this study was to investigate the heavy metal resistant bacteria (HMRB) community succession and bacterial activity in poultry manure (PM) composting. Five different concentration of chicken manure biochar (CMB) at 0%, 2%, 4%, 6%, and 10% on a dry weight basis was applied with initial feedstock (poultry manure + wheat straw) and indicated with T1, T2, T3, T4, and T5. In addition, high-throughput sequencing, principal coordinate analysis, and correlation analysis were used to analyze the evolution of HMRB communities during composting. The study indicated that crucial phyla were Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. The bacterial diversity in the CMB amendment treatment was higher than in the control treatment, and T4 treatment has the highest among all CMB applied treatments. Moreover, results from CCA indicated that T4 and T5 treatments quickly enters the high-temperature period which is maintained for 5 days, and is significantly positively correlated with Proteobacteria, and Actinobacteria. These findings offer insight into potential strategies to understand the succession of HMRBs during PM reuse. Overall, the above results show the addition of 6% biochar (T4) was potentially beneficial to enrich the abundance of bacterial community to improve composting environment quality and composting efficiency. In addition, effective to immobilized the heavy metals and HMRB in the end product.

Application potential of Comamonas testosteroni ZG2 for vegetable cultivation in nickel and cadmium polluted soil

Microbial-induced carbonate precipitation plays an important role in the remediation of heavy metal contaminated soils, but the research on microorganisms that can both induce carbonate precipitation and promote plant growth is insufficient. In this study, a nickel-cadmium-resistant bacterial strain, which can not only produce urease activity to induce carbonate precipitation but also indole-3-acetic acid (IAA) and siderophores, was isolated from heavy metal contaminated soil. It was identified as Comamonas testosteroni and named ZG2. The bacterial cells of strain ZG2 can resist high concentrations of Ni and Cd (500 mg/L and 20 mg/L, respectively) by becoming longer and thinner. The urease activity and IAA concentration in the culture broth decreased with the increase of heavy metal concentrations, while the siderophore content increased. The strain ZG2 could remove more amount of cadmium than nickel and can induce the formation of carbonate precipitation. The Cd-containing carbonate precipitate was irregularly shaped of cadmium carbonate crystals, while the Ni-containing carbonate precipitate has a regular cubic shape, but there is no crystal form. Furthermore, in soil treatment experiments, the strain ZG2 could increase the soil pH, reduce the content of available Ni and Cd in the soil and the edible parts of pakchoi, increase the biomass of pakchoi, and also promote the root system growth and development. The above results show that the strain ZG2 is valuable in the Ni–Cd contaminated soil remediation application for the safe production of vegetables. • A heavy metal-resistant bacteria, Comamonas testosteroni strain ZG2, was isolated, with the ability to produce urease enzyme, indole-3-acetic acid (IAA), and siderophores. • The bacterial strain ZG2 could induce the Cd-containing carbonate mineral and remove more than 60% of Ni 2+ and Cd 2+ concentrations in the fermentation broth. • Strain ZG2 can immobilize heavy metals (Ni and Cd) from the polluted soil, reduce the Ni and Cd available contents in the soil and the edible parts of Pakchoi, and promote the growth of Pakchoi.

Complete Genome Sequence of Bacillus safensis Strain 3A, a Heavy Metal-Resistant Bacterium Isolated from Contaminated Estuarine Sediment in Brazil.

Bacillus safensis 3A was isolated from a contaminated estuarine sediment sample with mine tailing from the Samarco dam disaster, which occurred in 2015 in Minas Gerais State, Brazil. We report here a draft genome sequence (3.6 Mb) of this bacterial strain. B. safensis exhibited strong resistance to heavy metals.

Removal of cadmium by heavy metal–resistant bacteria isolated from Hussain Sagar Lake—Hyderabad

Hussain Sagar Lake (17.4239° N, 78.4738° E) located at the heart of the capital of Telangana state, Hyderabad, is well known by many as a tourist spot. However, during the recent years, this lake has been contaminated by various industries surrounding the lake, especially from the Kukatpally channel and NTR Marg. Such inflow and heavy dumping of wastes have led to huge amount of toxic silt at the bottom and have taken a toll on the holding capacity of the lake. This paper focuses on the effect of bacteria on the heavy metals in the lake soil samples. Cadmium sulfate was used to prepare the heavy metal sample and a cadmium-resistant bacterium was obtained after 48 h of treatment. Among the eight bacterial isolates, one showed cadmium absorption capacity with a minimal inhibitory concentration of 500 mg/l of synthetically prepared solution. The sample also showed resistance to other heavy metals like copper and zinc. The bacterium from this sample was identified to be Pseudomonas fluorescens which was used for optimization whose growth was assessed based on UV absorbance at 600 nm. The optimum solution was obtained at a pH of 6.01 and a temperature of 32.7 °C, and the removal rate for 50 mg/l of cadmium was observed to be 90% and that of 100 mg/l was found to be 72%.

Copper removal from semiconductor CMP wastewater in the presence of nano-SiO2 through biosorption

Abstract Copper-bearing wastewater from chemical mechanical planarization (CMP) is a typical semiconductor development byproduct. How to effectively treat Cu2+ in the CMP wastewater is a great concern in the microchip manufacturing industry. In this study, we investigated the potential for the microbial removal of Cu2+ by a multiple heavy metal-resistant bacterium Cupriavidus gilardii CR3. The environmental factors, including pH, nano-SiO2, ionic strengths, and initial concentrations of Cu2+, and adsorption times on the bioremoval of Cu2+ in CMP wastewater were optimized. Under optimal condition, the maximum biosorption capacity for Cu2+ was 18.25 mg g−1 and the bioremoval rate was 95.2%. The Freundlich model is described well for the biosorption of Cu2+ in CMP wastewater in the presence of nano-SiO2 (R2 = 0.99). The biosorption process obeyed the pseudo-second-order kinetic equation (R2 &amp;gt; 0.99). In the column experiment, the advection–dispersion–retention model fitted the breakthrough curve of all experiments well (R2 &amp;gt; 0.95). The attachment coefficient in the sand matrix coated by CR3 biofilm was 2.24–2.80 times as that in clean sand. Overall, C. gilardii CR3 is a promising candidate to remove Cu2+ from CMP wastewater. Nano-SiO2 in CMP wastewater did not inhibit the bioremoval of Cu2+ but showed a slight promotion effect instead.

Roseomonas coralli sp. nov., a heavy metal resistant bacterium isolated from coral.

A Gram-stain-negative, aerobic, mesophilic, non-motile bacterium, designated M0104T, was isolated from a gorgonian coral collected from Xieyang island, Guangxi Province, PR China. Colonies of the strain were non-motile cocci and pink. The strain grew at 15-34 °C (optimum, 28 °C), pH 4.5-8.0 (optimum, pH 7.0) and with 0-4% (w/v) NaCl (optimum, 0-2 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain M0104T was closely related to Roseomonas deserti JCM 31275T (96.2 %), Roseomonas vastitatis KCTC 62043T (96.0 %), Roseomonas aerofrigidensis JCM 31878T (95.9 %) and Roseomonas oryzae KCTC 42542T (95.7 %). The strain had an assembly size of 5.0 Mb and a G+C content of 71.0mol%. Genes involved in copper, cadmium, lead, arsenic and zinc resistance were identified in the genome of strain M0104T. The digital DNA-DNA hybridization and average nucleotide identity values between the genome sequence of strain M0104T and those of closely related type strains were 19.4-24.9 % and 74.3-81.8 %, respectively. Strain M0104T contained C18:1 ω7c, C18:3 ω3c, anteiso C11:0 and C16:0 as the major fatty acids (>7 %) and ubiquinone Q-10 as the sole isoprenoid quinone. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine were its major polar lipids. Based on its phenotypic, phylogenetic and chemotaxonomic properties, strain M0104T is proposed to represent a novel species within the genus Roseomonas, for which the name Roseomonas coralli sp. nov. is proposed. The type strain is M0104T (=KCTC 62359T=MCCC 1K03632T).

Heavy Metal Resistant Bacteria from Soil as Potential Bioremediation Targets: Isolation, Screening andamp; Biochemical Identification

This study investigates the role of bacterial species capable of mitigating metal-induced toxicity by bioaccumulation and biotransformation. Study focuses on five metals including Pb+2, Ni+2, Cd+2, Cr+2, and Cu+2 in a range of 50-300and#181;g/ml of concentration initially in qualitative (metal-specific) and subsequently quantitative (dose-specific) approach, but results turned out to be much in favor of a quantitative impact of the study. Thirty 30 bacterial strains from soil were isolated and biochemically identified that showed promising metal-resisting behavior. Identification of bacterial strain was based upon biochemical (phenotypic) method. The isolate number 1, 12, 29 and 30 showed high degree of resistance against all metals of concentration up to 300and#181;g/ml suggesting the potential possibility of these species of bacteria to provide some beneficial tools for bio-mining, and bioremediation as well as removing of metal contaminations from heavy metal contaminated sites.

Heavy Metal Resistant Bacteria from Soil as Potential Bioremediation Targets: Isolation, Screening andamp; Biochemical Identification

This study investigates the role of bacterial species capable of mitigating metal-induced toxicity by bioaccumulation and biotransformation. Study focuses on five metals including Pb+2, Ni+2, Cd+2, Cr+2, and Cu+2 in a range of 50-300and#181;g/ml of concentration initially in qualitative (metal-specific) and subsequently quantitative (dose-specific) approach, but results turned out to be much in favor of a quantitative impact of the study. Thirty 30 bacterial strains from soil were isolated and biochemically identified that showed promising metal-resisting behavior. Identification of bacterial strain was based upon biochemical (phenotypic) method. The isolate number 1, 12, 29 and 30 showed high degree of resistance against all metals of concentration up to 300and#181;g/ml suggesting the potential possibility of these species of bacteria to provide some beneficial tools for bio-mining, and bioremediation as well as removing of metal contaminations from heavy metal contaminated sites.

An insight into the mechanism of ‘symbiotic-bioremoval’ of heavy metal ions from synthetic and industrial samples using bacterial consortium

Abstract Almost all the compartments of the environment have been severely polluted by heavy metals anthropologically, rendering its intrinsic values. In this approach, the present work has been aimed at developing a consortium of heavy metal resistant bacteria isolated from various wastewater sites. The designed bioaugmentation was aimed to specifically remove lead (Pb 2 + ) and nickel (Ni 2 + ) metal ions from the aqueous solutions. From the total 17 different industrial collection sites, 2 strains viz. Bacillus cereus and Bacillus pumilus have shown remarkable removal of Pb 2 + (95.93%) and Ni 2 + (95.54%) ions from the wastewater samples. The influential parameters like pH, incubation time, inoculum size, and initial metal concentration have been monitored for better insight into the interactional mechanism. The mechanism of heavy metals removal involves uptake of metal ions (Pb 2 + and Ni 2 + ) by means of complex formation, crystallization, and surface adsorption; invigorated with intracellular uptake and stabilization. The obtained results witness the hauling potential of the B. cereus and B. pumilus against the removal of bioavailable fractions of heavy metals thus projects of their practice on a commercial level.

Can biochar regulate the fate of heavy metals (Cu and Zn) resistant bacteria community during the poultry manure composting?

In this study, the influence of coconut shell biochar addition (CSB) on heavy metals (Cu and Zn) resistance bacterial fate and there correlation with physicochemical parameters were evaluated during poultry manure composting. High-throughput sequencing was carried out on five treatments, namely T1−T5, where T2 to T5 were supplemented with 2.5%, 5%, 7.5% and 10% CSB, while T1 was used as control for the comparison. The results of HMRB indicated that the relative abundance of major potential bacterial host altered were Firmicutes (52.88–14.32%), Actinobacteria (35.20–4.99%), Bacteroidetes (0.05–15.07%) and Proteobacteria (0.01–20.28%) with elevated biochar concentration (0%−10%). Beta and alpha diversity as well as network analysis illustrated composting micro-environmental ecology with exogenous additive biochar to remarkably affect the dominant resistant bacterial community distribution by adjusting the interacting between driving environmental parameters with potential host bacterial in composting. Ultimately, the amendment of 7.5% CSB into poultry manure composting was able to significantly reduce the HMRB abundance, improve the composting efficiency and end product quality.

Microbacterium sp. MRS-1, a potential bacterium for cobalt reduction and synthesis of less/non-toxic cobalt oxide nanoparticles (Co3O4)

BackgroundDetoxification of heavy metal pollutants in wastewater has become a serious problem to surrounding environment. This research was conducted to utilize a potential heavy metal-resistant bacterium for the remediation of cobalt metal and simultaneous synthesis of cobalt oxide nanoparticles in the form of powder for various industrial applications. Metal oxide nanoparticles have great applications in electrochemical devices such as supercapacitors, biosensors, and batteries.MethodA heavy metal-resistant bacterium Microbacterium sp. MRS-1 isolated from electroplating industrial effluent reduced cobalt ions from an initial concentration of 200 mg/L to 26 mg/L were analyzed by atomic absorption spectroscopy. Instrumental analysis of bacterially synthesized Co3O4 has been characterized. Cytotoxicity of synthesized nanoparticles was assessed by MTT assay.ResultsMicrobacterium sp. MRS-1 isolated from electroplating industrial effluent was found to be suitable for cobalt oxide nanoparticles as it showed tolerance towards high concentration of metal. The nutrient broth containing metal solution and Microbacterium sp. MRS-1 showed color change from light pink to dark pink indicated the formation of extracellular nanoparticles. It also converted soluble cobalt salts into less soluble cobalt oxide nanoparticles outside the cell which allows easy recovery of nanoparticles without the destruction of cells and simultaneous detoxification of toxic metal ions. Electron microscopic imaging verified that nanoparticles were predominantly surrounding the bacterial cells and SEM imaging revealed that the produced particles were in the range of 10–100 nm in size. XRD spectrum exhibited 2θ values were corresponding to cubic face-centered cobalt oxide (Co3O4) nanoparticles.ConclusionThe present study investigated new prospective for eco-friendly detoxification of toxic heavy metal Co from metal-polluted sites and the production of cobalt oxide nanoparticles in powder form for clinical and other industrial applications.