Interactions among microbial organisms often cannot be observed directly, but they can be inferred genetically using new molecular techniques. The analysis of secondary metabolite gene expression produced by co-cultured marine microbial species allows us to see how these organisms interact with one another when kept in the same environment. Co-cultures of three different strains of marine bacteria, P. aeruginosa PAO1, Roseobacter denitrificans OCH114, and Salinispora arenicola CNS-205 were grown in a laboratory setting, and using the Real-Time qPCR method gene expression levels of two different secondary metabolite producing genes from each organism was accessed across three time points. P. aeruginosa PAO1’s secondary metabolite genes RdhA and PhzH stayed repressed through all co-cultures and time points in this study, and Roseobacter denitrificans OCH-114’s secondary metabolite genes metallo-beta-lactamase and DMSP lyase were up-regulated after the 30 minute time point in the P. aeruginosa-R. denitrificans co-culture and at the 0 minute time point in the R. denitrificans-S. arenicola co-culture.

Phages are the most abundant components of the marine environments and can control host abundances. The severity of viral infections may depend on whether phages are lytic, lysogenic, or chronic, which can be influenced by host activity and by environmental conditions. Lysogeny remains the least understood process. Knowledge of virioplankton dynamics and their life strategies in the Red Sea remain unexplored. In this Ph.D. research we aimed to quantify virioplankton abundance, the variability on viral and bacterial dynamics, and to investigate the occurrence of lytic and lysogenic phages in the Red Sea. Accordingly, we used the flow cytometric technique to enumerate viral and bacterial abundances in the coastal pelagic area during two years of sampling and in the coastal lagoon waters for one year, together with water column distribution in open Red Sea waters. We conducted incubations of natural microbial communities in the laboratory to induce lysogenic bacteria by using the chemical mutagenic mitomycin C. We also explored the influence of host abundance, temperature, and ultraviolet radiation on viral dynamics and lysogeny. Our results showed that abundances of virses in the Red Sea ranged from 106 to 107 virus-like particles per mL, and bacteria ranged from 104 to 105 cells per mL. We observed a large variability i the values of virus-to-bacterium ratios, and lower values of viral production to those for temperate coastal waters and relatively close to values reported in other oligotrophic areas. Although the lytic phase was prevalent, lysogeny was detected when bacterial abundances decreased. We determined inducible lysogenic bacteria from undetectable to ~56% in the coastal Red Sea, although we found a lower maximum of 29.1% at a eutrophic coastal lagoon. The decay rates of viruses were influenced by UVB exposure, suggesting their susceptibility to solar radiation. Exposure to UVB radiation-induced prophage varied between 4 and 34%. Our findings identified the significant role of viral infections in controlling bacterial abundance and the importance of both lytic and lysogenic phases in the Red Sea waters. This study contributes to the understanding of lysogeny in marine phages.

Seventeen bacterial strains were isolated from 9 marine mud samples from the inshore environments of the East Sea. Four bacterial strains showed an inhibition against all tested microorganisms Staphylococcus aureus ATCC10832, Escherichia coli JM109, and Fusarium oxysporum. 16S rRNA sequences of four bacterial strains were obtained by PCR using specific primers. PCR products were cloned into E. coli DH5a using pJET1.2 blunt vector. The recombinant plasmids were sequenced and the lengths of these 16S rRNA sequences were ~930bp. The 16S rRNA sequence from the four bacterial DB1.2, DB1.2.3, DB4.2 and DB5.2 strain showed a high identity of 97 to 99% with the 16S rRNA sequence from Photobacterium sp., Oceanisphaera sp., Shigella sp., Stenotrophomonas sp, respectively. / Mười bảy chủng vi khuẩn đã được phân lập từ 9 mẫu bùn biển từ các vùng ven bờ biển Việt Nam. Bốn chủng vi khuẩn được ghi nhận có khả năng ức chế mạnh sự sinh trưởng và phát triển của các chủng vi khuẩn Staphylococcus aureus ATCC10832, Escherichia coli JM109, và thậm chí cả nấm Fusarium oxysporum. Trình tự gene 16S rRNA của bốn chủng vi khuẩn này đã được khuếch đại bằng PCR sử dụng cặp mồi đặc hiệu. Sản phẩm PCR được nối ghép vào vector pJET1.2 blunt sử dụng T4 ligase, hình thành plasmid tái tổ hợp và biến nạp vào E. coli DH5α. Khuẩn lạc có plasmid mang phân đoạn DNA chèn được nuôi cấy và tách plasmid. Trình tự 16S rRNA từ 4 chủng DB1.2, DB1.2.3, DB4.2 and DB5.2 chỉ ra có sự tương đồng 97 ÷ 99% so với trình tự 16S rRNA tương ứng của các chủng vi sinh vật biển trên ngân hàng gene thế giới là Photobacterium sp., Oceanisphaera sp., Shigella sp., và Stenotrophomonas sp.

Graduation date: 2004

In aquatic environments the availability of nitrogen (N) generally limits primary production. N2-fixing prokaryotes (diazotrophs) can convert N2 gas into ammonium and provide significant input of N into the oceans. Cyanobacteria are thought to be the main N2-fixers but diazotrophs also include a wide range of heterotrophic bacteria. However, their activity and regulation in the water column is largely unknown. In this thesis the distribution, diversity, abundance, and activity of marine and estuarine heterotrophic diazotrophs was investigated. With molecular methods targeting the nifH gene, encoding the nitrogenase enzyme for N2 fixation, it was shown that diverse nifH genes affiliating with heterotrophic bacteria were ubiquitous in surface waters from ten marine locations world-wide and the estuarine Baltic Sea. Through enrichment cultures of Baltic Sea surface water in anaerobic N-free medium, heterotrophic N2 fixation was induced showing that there was a functional N2-fixing community present and isolates of heterotrophic diazotrophs were obtained. In Sargasso Sea surface waters, transcripts of nifH related to heterotrophic bacteria were detected indicating heterotrophic N2-fixing activity. Nitrogenase expression is thought to be highly regulated by the availability of inorganic N and the presence of oxygen. Low oxygen zones within the water column can be found in association with plankton. The presence of diazotrophs as symbionts of heterotrophic dinoflagellates was investigated and nifH genes related to heterotrophic diazotrophs rather than the cyanobacterial symbionts were found, suggesting that a symbiotic co-existence prevailed. Oxic-anoxic interfaces could also be potential sites for heterotrophic N2 fixation. The Baltic Sea contains large areas of anoxic bottom water. At the chemocline and in anoxic deep water heterotrophic diazotrophs were diverse, abundant and active. These findings extend the currently known regime of N2 fixation to also include ammonium-rich anaerobic waters. The results of this thesis suggest that heterotrophic diazotrophs are diverse and widely distributed in marine and estuarine waters and that they can also be active. However, limits in the knowledge on their physiology and factors which regulate their N2 fixation activity currently prevent an evaluation of their importance in the global marine N budget.

Os surfatantes, moléculas anfipáticas compostas por uma porção polar e outra apolar, constituem um grupo heterogêneo de compostos de superfície ativa. Sua porção polar pode ser formada por peptídeos, ânions ou cátions, por mono, di ou polissacarídeos, enquanto sua porção apolar pode ser formada por estruturas saturadas, insaturadas ou ácidos graxos hidroxilados, ou ainda peptídeos hidrofóbicos. Essas substâncias podem ser sintéticas, obtidas a partir de síntese química, ou produzidas por micro-organismos, principalmente, bactérias e leveduras, passando a ser denominadas biossurfatantes (BS). O BS representa uma alternativa aos tensoativos sintéticos utilizados em diversos seguimentos da indústria devido a sua baixa toxicidade e alta biodegradabilidade, além de aplicabilidade na descontaminação ambiental. Bactérias isoladas em diversos biomas têm sido muito exploradas para produção de BS, enquanto ecossistemas marinhos ainda são pouco explorados, apesar do grande potencial existente. Os micro-organismos marinhos quando expostos a condições extremas de pressão, salinidade e temperatura produzem compostos estáveis e, portanto, úteis em aplicações industriais. O presente trabalho objetivou investigar novos BS produzidos por bactérias de origem marinha capazes de produzir tal substância a partir de fontes de carbono de baixo custo (glicerol, óleo de soja, vaselina e sacarose). Foram analisados 59 isolados bacterianos marinhos, as bactérias selecionadas foram identificadas e a produção do BS foi estudada em escala laboratorial. Após a extração do BS, suas propriedades físico-química como tensão superficial (TS), tensão interfacial (IT) e concentração micelar crítica (CMC) foram determinadas; além disso, foi realizada a caracterização estrutural preliminar e o seu potencial de aplicação para biorremediação de petróleo e como agente emulsificador. Os resultados identificaram a produção de BS por três bactérias marinhas: Arthrobacter defluvii, Brevibacterium luteolum e Gordonia sp. A partir das análises químicas foi possível identificar dois BS lipopepítidicos (A. Defluvii e B. luteolum) e um BS com grupos glicosídicos (Gordonia sp.). O BS produzido por A. defluvii apresentou tensão superficial TS = 34,5 mN m-1; TI = 15 mN m-1; e CMC = 129 mg L-1 crescendo em óleo de soja, B. luteolum TS = 27 mN m-1; TI = 0,84 mN m-1; e CMC = 40 mg L-1 crescendo em vaselina e Gordonia sp. TS = 33 mN m-1; TI = 1,4 mN m-1; e CMC = 85 mg L-1 crescendo em óleo de soja. Os BS estudados apresentaram capacidade de remoção de petróleo de areia contaminada, assim como atividade emulsificante frente a diferentes substâncias ( óleo de soja, vaselina, decano, querosene, hexadecano e gordura vegetal). Os testes de estabilidade realizados para BS produzido por B. luteolum indicaram que: o BS é estável quando submetido a temperaturas até 60oC por 24 h e até 121oC por 20 min; a maior atividade tensoativa para o BS é encontrada em solução com pH entre 6 e 8; e a força iônica não afeta a atividade tensoativa até concentrações salinas abaixo de 16%. Portanto, os três micro-organismos marinhos selecionados foram capazes de produzir compostos com relevante atividade tensoativa utilizando substratos de baixo custo. / The surfactants, amphipathic molecules containing both polar and nonpolar portions, are a heterogeneous group of surface-active compounds. Its polar portion may be composed by peptides, anions or cations, mono, di, or polysaccharides, while its polar portion may contain saturated, unsaturated or hydroxylated fatty acids or hydrophobic peptides. These molecules may be synthetic, derived from chemical synthesis, or produced by microorganisms, mainly bacteria and yeast, and also known as biosurfactants (BS). The BS represent an alternative to synthetic surfactants used in various segments of industry due to their low toxicity and high biodegradability. The major classes of BS include glycolipids, lipopeptides, lipoproteins, phospholipids, fatty acids and polymeric surfactants. Bacteria isolated from different biomes have been heavily exploited for BS production, while marine ecosystems are still poorly explored, despite its great potential. The marine microorganisms when exposed to extremes of pressure, temperature and salinity, produce stable compounds and, therefore, useful in industrial applications. The aim of this study was to investigate new BS produced by marine bacteria capable of producing such molecules growing in low-cost carbon sources (mineral oil, sucrose, soybean oil and glycerol). The selected bacterial isolates were identified and the biosurfactant production was studied in laboratory scale. After extraction of BS their physicochemical properties as surface tension (ST), interfacial tension (IT) and critical micelle concentration (CMC) were determined; preliminary structural characterization evaluated by FTIR and TLC and their potential application in bioremediation of crude oil and as emulsifier was also investigated. The results identified the BS production by three marine bacteria Arthrobacter defluvii, Brevibacterium luteolum and Gordonia sp. Based on the chemical analysis it was possible to identify two lipopeptides BS (A. defluvii and B. luteolum) and a BS with glycosides groups (Gordonia sp.). The BS produced by A. defluvii growing in soybean oil showed ST = 34,5 mNm-1; IT = 15 mNm-1 and CMC = 129 mgL-1; B. luteolum growing in mineral oil showed a ST = 27 mNm-1, IT = 0,84 mNm-1 and CMC = 40 mgL-1 and Gordonia sp. growing in soybean oil showed ST = 33 mNm-1, IT = 1,4 mNm-1 and CMC = 85 mgL-1. The BS obtained exhibited capacity to remove crude oil from contaminated sand as well as emulsifying activity against different hydrophobic substances (soybean oil, mineral oil, decane, kerosene, animal fat and hexadecane). Stability tests carried out to BS produced by B. luteolum indicated that: the BS is stable when submitted to temperatures down to 60° C for 24 h to 121° C for 20 min; the higher activity to the BS is found in solution with pH between 6 and 8; and the ionic strength does not affect the surfactant activity until salt concentrations under 16%. Therefore, the three selected marine micro-organisms were able to produce compounds with significant surfactant activity using low cost substrates.

Os surfatantes, moléculas anfipáticas compostas por uma porção polar e outra apolar, constituem um grupo heterogêneo de compostos de superfície ativa. Sua porção polar pode ser formada por peptídeos, ânions ou cátions, por mono, di ou polissacarídeos, enquanto sua porção apolar pode ser formada por estruturas saturadas, insaturadas ou ácidos graxos hidroxilados, ou ainda peptídeos hidrofóbicos. Essas substâncias podem ser sintéticas, obtidas a partir de síntese química, ou produzidas por micro-organismos, principalmente, bactérias e leveduras, passando a ser denominadas biossurfatantes (BS). O BS representa uma alternativa aos tensoativos sintéticos utilizados em diversos seguimentos da indústria devido a sua baixa toxicidade e alta biodegradabilidade, além de aplicabilidade na descontaminação ambiental. Bactérias isoladas em diversos biomas têm sido muito exploradas para produção de BS, enquanto ecossistemas marinhos ainda são pouco explorados, apesar do grande potencial existente. Os micro-organismos marinhos quando expostos a condições extremas de pressão, salinidade e temperatura produzem compostos estáveis e, portanto, úteis em aplicações industriais. O presente trabalho objetivou investigar novos BS produzidos por bactérias de origem marinha capazes de produzir tal substância a partir de fontes de carbono de baixo custo (glicerol, óleo de soja, vaselina e sacarose). Foram analisados 59 isolados bacterianos marinhos, as bactérias selecionadas foram identificadas e a produção do BS foi estudada em escala laboratorial. Após a extração do BS, suas propriedades físico-química como tensão superficial (TS), tensão interfacial (IT) e concentração micelar crítica (CMC) foram determinadas; além disso, foi realizada a caracterização estrutural preliminar e o seu potencial de aplicação para biorremediação de petróleo e como agente emulsificador. Os resultados identificaram a produção de BS por três bactérias marinhas: Arthrobacter defluvii, Brevibacterium luteolum e Gordonia sp. A partir das análises químicas foi possível identificar dois BS lipopepítidicos (A. Defluvii e B. luteolum) e um BS com grupos glicosídicos (Gordonia sp.). O BS produzido por A. defluvii apresentou tensão superficial TS = 34,5 mN m-1; TI = 15 mN m-1; e CMC = 129 mg L-1 crescendo em óleo de soja, B. luteolum TS = 27 mN m-1; TI = 0,84 mN m-1; e CMC = 40 mg L-1 crescendo em vaselina e Gordonia sp. TS = 33 mN m-1; TI = 1,4 mN m-1; e CMC = 85 mg L-1 crescendo em óleo de soja. Os BS estudados apresentaram capacidade de remoção de petróleo de areia contaminada, assim como atividade emulsificante frente a diferentes substâncias ( óleo de soja, vaselina, decano, querosene, hexadecano e gordura vegetal). Os testes de estabilidade realizados para BS produzido por B. luteolum indicaram que: o BS é estável quando submetido a temperaturas até 60oC por 24 h e até 121oC por 20 min; a maior atividade tensoativa para o BS é encontrada em solução com pH entre 6 e 8; e a força iônica não afeta a atividade tensoativa até concentrações salinas abaixo de 16%. Portanto, os três micro-organismos marinhos selecionados foram capazes de produzir compostos com relevante atividade tensoativa utilizando substratos de baixo custo. / The surfactants, amphipathic molecules containing both polar and nonpolar portions, are a heterogeneous group of surface-active compounds. Its polar portion may be composed by peptides, anions or cations, mono, di, or polysaccharides, while its polar portion may contain saturated, unsaturated or hydroxylated fatty acids or hydrophobic peptides. These molecules may be synthetic, derived from chemical synthesis, or produced by microorganisms, mainly bacteria and yeast, and also known as biosurfactants (BS). The BS represent an alternative to synthetic surfactants used in various segments of industry due to their low toxicity and high biodegradability. The major classes of BS include glycolipids, lipopeptides, lipoproteins, phospholipids, fatty acids and polymeric surfactants. Bacteria isolated from different biomes have been heavily exploited for BS production, while marine ecosystems are still poorly explored, despite its great potential. The marine microorganisms when exposed to extremes of pressure, temperature and salinity, produce stable compounds and, therefore, useful in industrial applications. The aim of this study was to investigate new BS produced by marine bacteria capable of producing such molecules growing in low-cost carbon sources (mineral oil, sucrose, soybean oil and glycerol). The selected bacterial isolates were identified and the biosurfactant production was studied in laboratory scale. After extraction of BS their physicochemical properties as surface tension (ST), interfacial tension (IT) and critical micelle concentration (CMC) were determined; preliminary structural characterization evaluated by FTIR and TLC and their potential application in bioremediation of crude oil and as emulsifier was also investigated. The results identified the BS production by three marine bacteria Arthrobacter defluvii, Brevibacterium luteolum and Gordonia sp. Based on the chemical analysis it was possible to identify two lipopeptides BS (A. defluvii and B. luteolum) and a BS with glycosides groups (Gordonia sp.). The BS produced by A. defluvii growing in soybean oil showed ST = 34,5 mNm-1; IT = 15 mNm-1 and CMC = 129 mgL-1; B. luteolum growing in mineral oil showed a ST = 27 mNm-1, IT = 0,84 mNm-1 and CMC = 40 mgL-1 and Gordonia sp. growing in soybean oil showed ST = 33 mNm-1, IT = 1,4 mNm-1 and CMC = 85 mgL-1. The BS obtained exhibited capacity to remove crude oil from contaminated sand as well as emulsifying activity against different hydrophobic substances (soybean oil, mineral oil, decane, kerosene, animal fat and hexadecane). Stability tests carried out to BS produced by B. luteolum indicated that: the BS is stable when submitted to temperatures down to 60° C for 24 h to 121° C for 20 min; the higher activity to the BS is found in solution with pH between 6 and 8; and the ionic strength does not affect the surfactant activity until salt concentrations under 16%. Therefore, the three selected marine micro-organisms were able to produce compounds with significant surfactant activity using low cost substrates.

The marine surface-associated bacterium Pseudoaltermonas tunicata, produces a range of compounds that inhibit fouling organisms, including invertebrate larvae, bacteria, algal spores and fungi. In addition to these antifouling compounds P. tunicata cells produce both a yellow and a purple pigment. The aim of this study was to further characterise the antifouling activities, their regulation and relationship with pigmentation, and the ecological significance of P. tunicata and related organisms. It was discovered that the anti-algal compound was extracellular, heat sensitive, polar and between 3 and 10 kDa in size. The anti-fungal compound was found to be the yellow pigment and active against a wide range of fungal and yeast isolates. Chemical analysis suggests that this compound consists of a carbon ring bound to a fatty-acid side chain. Genetic analysis supports the chemical data for the active compound as a mutant in a gene encoding for a long-chain fatty-acid CoA ligase was deficient for anti-fungal activity. To address the regulation of antifouling compounds and their relationship to pigmentation transposon mutagenesis of P. tunicata was performed. Mutants lacking the yellow pigment displayed a reduced ability to inhibit fouling organisms. Further analysis of these mutants identified genes involved with the synthesis and regulation of synthesis of pigment and antifouling compounds. One of these mutants was disrupted in a gene (wmpR) with similarity to the transcriptional regulators ToxR from Vibrio cholerae and CadC from Escherichia coli. Analysis of global protein expression using two-dimensional gel electrophoresis showed that WmpR is essential for the expression of at least fifteen proteins important for the synthesis of fouling inhibitors. The ecological significance of antifouling bacteria was addressed by assessing the antifouling capabilities of a collection of bacteria isolated from different marine surfaces. Overall, isolates from living surfaces displayed more antifouling traits then strains isolated from non-living surfaces. Five dark-pigmented strains originating from the alga Ulva lactuca were further studied. Phylogenetic and phenotypic analysis revealed that they were all members of the genus Pseudoalteromonas and were closely related to P. tunicata. Two strains represented a novel species within the genus and were taxonomically defined as P. ulvae sp. nov.

Typescript. Includes vita and abstract. Bibliography: Includes bibliographical references (leaves 60-63). Description: xii, 63 leaves ; 29 cm.

Le milieu marin constitue la plus grande partie de la biosphère et contient les formes les plus anciennes et les plus variées de la vie. Les espèces microbiennes, relativement négligées jusqu’à présent, pourraient bien être le principal gisement de nouvelles molécules des prochaines décennies grâce à leurs adaptations à un environnement marin atypique et/ou à des conditions extrêmes (zones hydrothermales, sédiments des fonds sous-marins, lagunes hyper-salines, continent arctique et antarctique, tapis microbiens…). La Nouvelle-Calédonie est dotée de milieux naturels littoraux, côtiers et marins au sein desquels existent des gradients thermiques, d’hypersalure/dessalure, de chocs UV, de pH, d’évaporation, d’inondation/exondation… qui déterminent des habitats atypiques dans lesquels les micro-organismes doivent développer des stratégies adaptatives et de défense potentiellement uniques. Un travail de bioprospection de ces milieux atypiques a ainsi permis la création d’une souchothèque riche à ce jour de 771 isolats bactériens néo-calédoniens. Sur la base de cette collection, des études ont été engagées pour la recherche et la caractérisation de biopolymères de type exopolysaccharides (EPS) et polyhydroxyalcanoates (PHA). Les EPS sont des complexes osidiques de grande taille dont la composition est variable selon l’organisme producteur. Les PHAs sont des macromolécules formées de liaisons ester présentant de fortes similitudes avec les plastiques issues de la pétrochimie mais sont totalement biodégradables et biocompatibles. Les débouchés pour la valorisation des biopolymères marins sont très variés et touche plusieurs secteurs comme la cosmétique (gélifiant épaississant, antirides, etc.) la santé (immunostimulation, anticoagulant, cicatrisant), l’agroalimentaire, la bioremédiation les emballages, l’enrobage… Les criblages réalisés sur l’ensemble de la collection pour les deux types de polymères ont permis de mettre en évidence qu’une part importante des souches de la collection étaient potentiellement capables de sécréter ces polymères. À l’heure actuelle, 10 types d’EPS et 5 types de PHAs ont été produits et caractérisés afin de cibler des domaines d’applications. L’étude des bactéries marines pour leur faculté à produire des polymères est donc un secteur en pleine expansion et les premiers résultats montrent que ce type de biotechnologies pourrait constituer une perspective de développement intéressante pour la Nouvelle-Calédonie. / Previous works on marine bacteria led to the discovery of molecules of great biotechnological interest. Under unusual physical and chemical conditions some microorganisms have developed various survival strategies including exopolysaccharides (EPS) and Poly-3-hydroxyalkanoates (PHAs) production. Due to their many interesting biological, physical and chemical properties, those polymers have found applications in many industrial sectors. Due to interesting physical and chemical properties, EPS can find applications in many industrial sectors including the food industry, cosmetics, for oil and metal recovery from industrial waste and in the mining industry as well. During the last decades EPS have also been demonstrated as interesting bioactive molecules with many applications for human health. PHAs are biopolyesters accumulated as granules in bacteria in order to endure long starving periods. Those biodegradable biopolymers can be used as an alternative to petroleum derived polymers and can be produced from renewable carbon sources. PHAs exhibit a wide variety of properties and structures depending of the carbon source available and the microorganism used for the production. New Caledonia (NC) is frequently referred as a hotspot biodiversity. During a prospection campaign performed in different marine costal ecosystems of NC, a great number (770) of bacteria were isolated from different locations. Screening showed that 55% of the isolates were able to produce under lab conditions EPS and 53% to produce PHA. Partial chemical characterization was performed on purified samples using colorimetric methods, infrared spectrometry (FTIR), gas chromatography (GC) and nuclear magnetic resonance (NMR). Marine bacteria from New Caledonian ecosystems were shown to produce EPS with unusual chemical composition with potential applications in cosmetics. Preliminary experiments also showed high metal-binding capacity with applications in bioremediation. Different PHAs were also produced using different types of sugars and oil as renewable resources. Blue biotechnologies can have various applications in many industrial sectors (Health, food industry, environment, cosmetics etc…) and there is a great international demand for new molecules issue from marine areas. New Caledonian marine bacteria have proved their capacity for producing innovative biopolymers with a wide range of application that can be valuating in on short time period (environment, cosmetics) or at long time (pharmaceutics, surgeries). These applications are promising in order to develop