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Article

Idiomarina sp. Isolates from Cold and Temperate Environments as Biosurfactant Producers

by
Carmen Rizzo
1,2,*,
Maria Papale
2 and
Angelina Lo Giudice
2
1
Stazione Zoologica Anton Dohrn, National Institute of Biology, Sicily Marine Centre, Department Ecosustainable Marine Biotechnology, Villa Pace, Contrada Porticatello 29, 98167 Messina, Italy
2
Institute of Polar Sciences, National Research Council (CNR-ISP), Spianata S. Raineri, 86, 98122 Messina, Italy
*
Author to whom correspondence should be addressed.
J. Mar. Sci. Eng. 2022, 10(8), 1135; https://doi.org/10.3390/jmse10081135
Submission received: 11 July 2022 / Revised: 13 August 2022 / Accepted: 15 August 2022 / Published: 17 August 2022
(This article belongs to the Section Marine Biology)
Browse Figures
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Abstract

:
Background: The cold-adapted Idiomarina sp. 185 from Antarctic shoreline sediment and the mesophilic Idiomarina sp. A19 from the brackish Lake Faro (Italy) were screened for their efficiency in biosurfactant production by a temperature-mediated approach, when grown in rich culture medium and mineral medium supplemented with biphenyl. Methods: oxidation of polychlorobiphenyls and standard screening tests were performed, i.e., E24 index detection, surface tension measurement, blood agar plate and C-TAB agar plate. Results: During incubation in rich medium, the strain Idiomarina sp. A19 produced an excellent stable emulsion, recording an E24 of 73.5%. During growth in mineral medium, isolates showed good efficiency in at least one performed condition by showing species-specific differences related to optimum temperature. In the presence of biphenyl, both Idiomarina isolates created stable emulsions (E24 ≈ 47.5 and 35%, respectively), as well as surface tension reductions of 30.05 and 35.5 mN/m, respectively. Further differences between isolates were observed by phenotypic characterization. The genome mining approach on available deposited genome sequences for closest relatives offered further insights about the presence of genes for biphenyl degradation, especially for microorganisms derived from different extreme environments. Conclusions: Our results allowed for an interesting comparison which underlined differences in metabolic patterns and in the kinetics of BS production, probably due to the different origins of the strains.

1. Introduction

Research in the field of biosurfactants (BSs) represents one of the key strands of bioprospecting. Generally, BSs are constituted by a hydrophilic component of polysaccharides, proteins or peptides, and a hydrophobic group mainly constituted by fatty acids of alcohols, and have an amphipathic structure, which makes them suitable for numerous applications. Marine bioprospecting, in particular, has gained increasing interest as a possible pool of novelties. However, after an initial scramble for research, which for years filled the literature with publications on BS bacterial producers and evidenced the importance of these eco-friendly alternative molecules, the current state of the literature highlights a stalemate. There are two critical issues that seem to not allow for the achievement of a turning point: repetitiveness and too high application costs. The pursuit of novel sources in the field of BS bioprospecting, producers and molecular structures has led to a focus on untapped or less explored environments that meet the criteria of high biodiversity and specialization levels. As recently reviewed [1,2], extreme cold environments have been rarely considered as a source of BSs, despite the pivotal role of such molecules that has been proven in the cold adaptation and survival of microorganisms. Indeed, bacterial BS production is involved in the secretion of special cell envelopes, useful for preserving microorganisms from harsh conditions of salinity, temperature, and osmotic pressure [2].
Additionally, the use of biotic matrices as a source of bacterial BS producers has only been explored in a restricted context, mainly focused on temperate environments. Nevertheless, studies reporting on the use of benthic filter-feeding organisms have pointed out their implication in hosting bacterial communities specialized in the removal of contaminants accumulated during filtration activity, also by involving BS production [3,4,5,6,7,8,9]. With the development of new analytical approaches and research strategies, interest in BSs has gained a further boost, as they are considered promising potential biomolecules which can address urgent needs in the medical and environmental fields. Extensive efforts have been recently focused on the study of marine microorganisms, which may be key for the resolution of bioremediation problems, especially those related to hydrocarburic pollutants [10]. Indeed, BS-producing microorganisms may have a distinct advantage over competitors in contaminated areas, and therefore samples from such sites are often rich in bacterial strains with desired characteristics for in situ and ex situ bioremediation processes. The use of bacterial producers of BSs has gained attention because of their easy manipulation in the laboratory, the increasing possibility of obtaining maximum optimization, and the new cultivable techniques coupled with heterologous production methodologies, which are considered some of the main successful approaches to enhance BS productivity [1].
The cold-adapted bacterial BS producers reported to date have been mainly isolated from Antarctic abiotic matrices. They are members of Actinobacteria, Gammaproteobacteria and Firmicutes [2] in the genera Rhodococcus, Bacillus, Pseudomonas and Pseudoalteromonas. Even so, if we refer to the total number of available studies, it is clear how few draw possible conclusions in regards to the research area. The urgent and already stressed need for progress in this research area requires an ever-deeper knowledge of the ideal conditions of biosynthesis, especially when it runs into little-known or poorly reported producers. Investigation of cold extremophilic bacteria is useful as a potential for novelty in this research area, and is particularly interesting also in terms of energy-saving issues. The use of new molecules functioning at cold temperatures could help in reductions in the high energy consumption normally needed due to the high temperatures involved in scale-up production processes [1].
The genus Idiomarina was proposed by Ivanova et al. [11] to include two marine species, namely I. abyssalis and I. zobellii. The genus comprises Gram-negative, aerobic, flagellar species, generally isolated from saline habitats with a wide range of salinities, including oceanic water, coastal sediments, submarine hyperthermal fluids, solar salt-making works, and inland hypersaline wetlands [12]. Recently, the draft genome sequence of Idiomarina sp. strain 28-8 isolated from Korean ark shells [13] and of Idiomarina sp. strain A28L isolated from the alkaline brackish water of the Pangong Lake [14] were reported. The biotechnological potential of Idiomarina members is still less explored. I. fontislapidosi F32T and I. ramblicola R22T were proven as producers of anionic exopolysaccharides composed mainly of glucose, mannose, and galactose [15]. Idiomarina members were also reported as possible source of extremozymes [16] and siderophores [17]. Less reports are available on the involvement of the genus in hydrocarbon degradation and BS production by members isolated from cold environments or biotic matrices [8,9,18,19].
The present study was aimed at comparing two Idiomarina BS producers isolated from totally different environments, namely Antarctica and the Mediterranean Sea, previously processed with different strategies and selected on the basis of their good surface-active properties in the presence of hydrocarburic substrates. The study’s main aims were to test the adaptability and the performances of the two strains, by verifying reactions and productivity at conditions different from those occurring in their origin environments, and to contribute to the exploration of BS-mediated PCB oxidation by underexplored strains.

2. Materials and Methods

2.1. Bacterial Strains

BS-producing Idiomarina isolates, namely the psychrotolerant Idiomarina sp. 185 (sequence deposited in the NCBI GenBank database under the accession number KF683135) and the mesophilic Idiomarina sp. A19 (sequence deposited in the NCBI GenBank database under the accession number JX298543), were previously isolated from Antarctic shoreline sediments (Byers Peninsula South Shetlands Islands) [18] and from the Mediterranean polychaete Megalomma claparedei from the brackish Lake Faro (Messina, Italy) [8,9], respectively. Idiomarina sp. 185 belongs to the Italian Collection of Antarctic Bacteria of the National Antarctic Museum (CIBAN-MNA; Museal Code: MNA-CIBAN-0437]. Previous data on Idiomarina spp. 185 and A19 are summarized in Table 1, which reports information on their isolation, optimal conditions for BS production and phenotypic characterization [8,9,18]. The cold-adapted strain Idiomarina sp. 185 was previously tested only in mineral medium (Bushnell Haas Broth, Difco) in the presence of hydrocarbon (tetradecane, final concentration 2%, v/v), and was incubated at 4 °C. The strain produced a maximum stable emulsion of 30% and reduced the surface tension to a final value of 29.9 mN/m. Conversely, the strain Idiomarina sp. A19 was previously tested in rich medium at 25 °C, with the production of a stable emulsion for a maximum E24 of 20% after 120 h of incubation, and a reduction in surface tension to 56.5 and 60 mN/m after 48 and 120 h of incubation observed. Moreover, the same strain was screened on hydrocarbon-supplemented mineral medium (tetradecane, final concentration 2%, v/v) at 28 °C, and produced a maximum emulsion index of 36.6% and a surface tension reduction to a value of 42.6% mN/m after 336 h of incubation.
Here, API API 20E and API 20NE tests (BioMerieux) were performed on Idiomarina sp. A19 according to manufacturer’s instructions to integrate data available for Idiomarina sp. 185. API strips were examined after incubation at 25 °C for 24 h.

2.2. Oxydation of Polychlorobiphenyls

The ability of the isolates to oxidize polychlorobiphenyls (PCBs) was explored by testing their growth on Aroclor 1242 (Sigma-Aldrich, St. Louis, MO, USA), a mixture of PCB congeners containing 42% chlorine by weight [20]. The ability to use PCBs as carbon source was evaluated according to the turbidity level or cellular floc occurrence in liquid culture assessed in BH supplemented with Aroclor 1242 (100 ppm in dichloromethane) (final concentration 0.1%, w/vol). Uninoculated medium was incubated in parallel as a negative control. Moreover, each isolate was also seeded in mineral medium with any carbon source. The isolates were furtherly screened for the presence of the catabolic gene bphA, which is involved in PCB degradation, as reported by Papale et al. [21]. PCR reactions were assessed using 0.4 µL of Taq polymerase 5 PRIME (5 U µL−1) in sterile Milli-Q water to a final volume of 20 µL containing 1 µL DNA, 1 µL of each of the two primers (10 µM) (2BPHFWD1; 5′ ADVCCSCGBGCCGCBTCHTCG 3′; 2BPHREV1; 5′ ADVCCSCGBGCCGCBTCHTCG 3′), 0.4 µL of each dNTP (10 mM), 2 µL of reaction buffer 10×, 0.4 µL of BSA (2.5%), and 0.4 µL Taq polymerase 5 PRIME (5 U μL−1), and sterileMilli-Q water to a final volume of 20 μL. DNA from B. xenovorans (DSM 17367) was used as a positive control. PCR amplicons were visualized by electrophoresis on a 2% agarose gel.

2.3. Biosurfactant Production

Whereas previous data on BS production during growth in rich medium were available only for the Idiomarina sp. A19, a first screening was performed here on both strains in Marine Broth (MB 2216, Difco) by adopting a comparative approach. An incubation temperature different from those previously investigated was used, namely 15 °C. The strains were furtherly compared by screening them for BS production during growth in a mineral medium and incubation at 15 °C and 25 °C. In such a way, information on the strain Idiomarina sp. 185 was integrated with data on BS production in rich medium, previously absent, and the mesophilic strain Idiomarina sp. A19 was instead tested at a lower temperature than those already treated. In parallel, BS production by Idiomarina strains was tested during growth in Bushnell Haas Broth supplemented with biphenyl (Difco; final concentration 2%, v/v) and incubated at 15 and 25 °C under continuous shaking at 130 rpm (HT Infors Multitron II, INFORS GmbH, Einsbach, Germany). BS production was evaluated at regular 48 h intervals by standard screening tests, as previously described [4,9], until the end of the incubation (seven days). Briefly, the emulsifying activity, expressed as the emulsification index (E24), was monitored by shaking vigorously for 1 min a 2-mL aliquot of each cell culture with 2 mL of kerosene (Petroleum ether, Panreac). After 24 h, the E24 index was calculated as suggested by Satpute et al. [22,23]. The surface tension (ST) was measured on cell-free supernatants (obtained after centrifugation of cell cultures at 4700 rpm for 20 min at 4 °C) by using a digital tensiometer K10T (Krϋss, Hamburg, Germany). Two supplementary tests, i.e., blood agar and C-TAB agar plate assays, were performed as previously described by Youssef et al. [24] and Walter et al. [25]. All tests were performed in triplicates.

2.4. Phylogenetic Analysis

As a complement of analysis, a phylogenetic tree of the two strains was constructed using the MEGA X (Molecular Evolutionary Genetics Analysis) software (Version 10.2.6, Philadelphia, PA, USA) [26]. The robustness of the inferred tree was evaluated by 400 bootstrap re-samplings. Moreover, PATRIC database was used for the search of closest bacterial relatives with potential features involved in biphenyl degradation [27].

2.5. Statistical Analysis

The results are presented as arithmetic averages of three replicates, and the error bars in the pictures indicate the standard deviations. The means were compared using one-way ANOVA, followed by Tukey’s test with a confidence level of 95%.

3. Results

3.1. Phenotypic Characterization

The isolate Idiomarina sp. A19 exhibited the following biochemical characteristics according to the results from the API 20NE and API 20E galleries: nitrate and nitrite were reduced. Adipic acid and malate utilization were shown by Idiomarina sp. A19. As previously reported [18], Idiomarina sp. 185 was positive to other biochemical tests, i.e., urease, arginine dihydrolase, gelatinase, citrate utilization and glucose fermentation.

3.2. Biosurfactant Production

After incubation in Marine Broth at 15 °C, the mesophilic strain Idiomarina sp. A19 showed a higher emulsifying activity than the psychrotolerant strain 185, achieving an E24 index of 73.5% after 192 h of incubation. Idiomarina sp. 185 produced a stable emulsion, recording percentage values lower than 10% (Figure 1). Neither strain 185 nor A19 reduced the ST appreciably when grown in Marine Broth. Conversely, as shown in Figure 2, Idiomarina sp. 185 produced a maximum E24 of 35% after 240 h of incubation and reduced the ST to a value of 27 mN/m, with a global reduction of 35.5 mN/m recorded, during incubation at 15 °C in Bushnell Haas Broth supplemented with biphenyl. During incubation at 25 °C, the cold-adapted strain 185 emulsified kerosene up to 10% after 240 h of incubation and reduced the ST by 7 units. The mesophilic Idiomarina sp. A19 produced a stable emulsion up to 47.5% after 192 h of incubation, and reduced the ST by about 30 mN/m during incubation at 25 °C in Bushnell Haas Broth supplemented with biphenyl, while during incubation at 15 °C, it produced a stable emulsion of 15% after 240 h of incubation and reduced the ST by about 9 units. Details on the time-course of BS production are reported in Supplementary Figure S1.
With respect to supplementary assays, neither strain 185 nor A19 produced blue halos on C-TAB agar plates. Conversely, the cold-adapted Idiomarina sp. 185 showed hemolytic activity by producing a green halo around its colonies when grown on blood agar plates.

3.3. Oxidation of Polychlorobiphenyls and Phylogenetic Analysis

Idiomarina sp. A19 and Idiomarina sp. 185 were able to grow in the presence of Aroclor 1242 as the sole carbon source at 15 °C and 25 °C. Both isolates harbored the bphA gene fragment, as a 947 bp amplicon was obtained; this was confirmed by agarose gel electrophoresis. Figure 3 shows the phylogenetic tree inferred with the 16S rRNA gene sequences of the Idiomarina strains used in this study and their next relatives retrieved from the GenBank database. The strains, despite being clustered separately, seem not to be very distant from an evolutionary point of view.

4. Discussion

In this period of intense scientific activity focused on biotechnology, the interest towards BSs is due to their environmentally friendly features and wide range of applications across different fields, including in environmental remediation. Increasing global environmental awareness has stimulated the study of natural compounds that can be used in the recovery of environments contaminated by toxic and persistent compounds. BSs have been shown to be efficient compounds for the removal of residual oils [30,31] and the chelation of heavy metals [32], promoting degradation of toxic chemicals.
In this study, experiments were performed with the intent of testing the different adaptability of mesophilic and cold-adapted BS-producing Idiomarina strains, verifying their ability to modulate BS biosynthesis under conditions different from those considered as optimal for them. A number of studies exploring BSs of bacterial origin have demonstrated both qualitatively and quantitatively that several parameters modulate BS production, i.e., temperature, pH level, salinity, and ion concentration [4,5,18]. For example, temperature may affect the chemical structures of BSs, while salinity may act on cellular activity by varying BS production [32]. Furthermore, the type and concentration of the carbon source can induce bacteria to use different metabolic pathways for the synthesis of BSs [4,33]. Members of the genus Idiomarina were previously detected in hydrocarbon-enriched samples, but only recently were they reported as BS producers [8,9,18,19]. To the best of our knowledge, the literature is scant on BS production in the presence of PCBs. Previous reports deal with Idiomarina spp. strains that are able to grow in the presence of aromatic hydrocarbons, i.e., naphthalene, phenanthrene, pyrene, and benzopyreno [19], but not in the presence of PCBs.
In this study, by comparing the different efficiencies of the two strains under optimal and non-optimal conditions, we intended to observe the different behaviors at different temperature conditions and to screen the possibility of future scale-up production. The BS production ability of the mesophilic Idiomarina sp. A19 was previously tested during incubation in Marine Broth and Bushnell Haas Broth supplemented with biphenyl [8,9], but with incubation at a higher temperature, while here BS production ability was tested at an incubation temperature of 15 °C to assess its efficiency under the coldest conditions. On the other hand, the cold-adapted Idiomarina sp. 185 was not previously screened under higher temperature growth conditions, and here was tested at an incubation temperature higher than 20 °C.
The use of rich medium was confirmed as a good substrate for BS production only for the isolate of mesophilic origin, which in terms of the carbon source condition was proven to have a higher ductility. Indeed, Idiomarina sp. A19 achieved E24 index values higher than those previously reported with incubation at 25 °C [8]. During growth in mineral medium, Idiomarina sp. A19 showed the strongest emulsifying activity, while in terms of interface action, the strain acted by achieving a comparable surface tension reduction. Stable emulsions with index > 50% have been reported for Idiomarina spp. strains during growth in mineral medium supplemented with sucrose [19]. In our case, Idiomarina sp. A19 achieved a value > 70% during growth in rich medium, and both strains were able to emulsify kerosene after incubation in mineral medium in the presence of recalcitrant compounds.
It is clear that both strains are correlated to their optimum temperatures, as evidenced by their better performance during growth at temperature values more similar to those experienced in their natural environments. The approach assumed here was the analysis of results in i—optimal and ii—non optimal temperature conditions. Results obtained at optimal growth conditions for both strains in terms of temperature (15 °C and 25 °C for Idiomarina sp. 185 and Idiomarina sp. A19, respectively) suggested the production of tensioactive agents with different properties. Interface activity was more evident for BSs from the psychrotrophic strain 185, with reductions in surface tension to values lower than previously detected recorded [19], whereas emulsifying activity was more efficient for BSs from the mesophilic strain A19. Conversely, results obtained during incubation at non-optimal conditions (25 °C for Idiomarina sp. 185 and 15 °C for Idiomarina sp. A19) showed that both strains produced BSs, with their best performance being in terms of emulsification.
Surface tension measurement showed an absence of reduction during incubation in rich medium, while a strong reduction occurred during screening on mineral salt medium with biphenyl. These results may confirm that surface-active compounds exhibit their function by reducing interfacial tension between immiscible liquids or at the solid–liquid interface.
In this study, only Idiomarina sp. 185 showed hemolysis on BA plates, indicating the probable production of anionic surfactants. Such a method should be considered as a useful integration of more efficient screening procedures. This finding is in line with results previously obtained by other authors [23,25], who reported the poor specificity of the method.
The choice of screening tests to be used is pivotal in studies on the production of bioactive molecules in general, including BSs. In the specific case of BSs, there are many existing screening tests, as recently reported by Twigg et al. [34], but several authors agree that the combination of several tests provides reliable and interesting observations [35,36,37].
The ability to produce BSs in the presence of biphenyl observed even in non-optimal conditions is an important element that confirms the potential of members of this genus, and has promising applications in the bioremediation field. The results reported here represent the first evidence of BS production by Idiomarina spp. strains in the presence of PCBs, which are more difficult to remove than other linear aliphatic hydrocarbons previously tested. The two strains reported here show different potential and adaptability, with there being special regard for the cold-adapted Idiomarina sp. 185. The potential of extremophilic microorganisms due to their uniqueness and specificity compared to organisms from other environments was recently highlighted by various researchers [1]. The use of low temperatures to reduce production costs is promising for both strains, with there being higher expectations for Idiomarina sp. 185.
Based on the obtained results, it is possible to assume that in the presence of recalcitrant compounds as a stressful condition, an optimum temperature is necessary for the strains to produce BSs, but their different responses could be due also to their different origins. The pairwise alignment performed on the 16S rRNA sequences of the two strains showed they have a similarity of 96%, which clustered in two different clades as shown in the phylogenetic tree. After consultation of the open-source sequences on GenBank, we found that a total of 77 complete genomes are available for the genus Idiomarina. Despite this, the phylogenetic alignment of sequences from our isolates performed on the NCBI GenBank database showed more than 100 phylogenetically closest relatives for each isolate, and among them, only two genomes are available as next relatives for the strain Idiomarina sp. 185, namely Idiomarina loihiensis GSL 199 (CP005964.1, genome ID 1321370.3) isolated from the hypersaline north arm of the Great Salt Lake, Utah, and Idiomarina loihiensis L2TR (AE017340.1, genome ID 283942.6) from a hydrothermal vent at a depth of 1300 m from the Lòihi Seamount, southeast of Hawaii. It is noteworthy that both closest relatives come from extreme environments, such as the strain studied in this paper. To complement our results, the PATRIC database [27] was used to carry out a search for phenotypes related to PCB degradation or BS production. Results showed the absence of genes involves in hydrocarbon and PCB degradation in the first closest relative genome. Differently, the genome of the second closest relative presented genes involved in xenobiotic degradation, including the gene ubiX, encoding for a 3-polyprenyl-4-hydroxybenzoate carboxy-lyase UbiX, which is engaged in the pathway of biphenyl degradation (AN NC_006512). Interestingly, the bphA gene searched for and detected in the strains used in this study was still not revealed in other Idiomarina spp. strains. Although this study was not aimed at comparison between strains at the genome level, these observations are useful as they suggest that the strains, albeit congeners, adopt different strategies to deal with stressful conditions. This is probably because of their different origins and therefore different environmental drivers, which may play a key role in biological responses.

5. Conclusions

The identification of new potential candidates for BS production is one of the main objectives in the field. A useful approach is to focus on new taxonomic groups, including microorganisms with special metabolic abilities or from uncommon environments. This increases the probability of identifying new molecular structures and therefore new functionalities, but above all biosynthesis performances different from those already extensively studied. This study provides a valid contribution that responds to the growing interest in surfactant molecules of natural origin, focusing on their possible use in bioremediation. The results highlighted an interesting comparison between strains which, despite their similar taxonomic affiliations, showed different metabolic patterns.
Future steps could involve low temperature scale-up production with less stressful conditions and low-cost carbon sources, and an estimate of the low temperature BS-mediated biphenyl biodegradation rates for both strains. Finally, the results constitute a valid basis to further investigate the chemical characterization of BSs and the possible applications of the heterologous production approach and scale-up optimization.

Supplementary Materials

The following supporting information can be downloaded at: www.mdpi.com/article/10.3390/jmse10081135/s1, Figure S1: Production of stable emulsion and surface tension reduction by Idiomarina sp. A19 (a,b) and Idiomarina sp. 185 (c,d) during incubation in BHbp at 15 °C and 25 °C.

Author Contributions

Conceptualization, C.R. and A.L.G.; methodology, C.R. and M.P.; software, M.P.; formal analysis, C.R.; investigation, C.R.; data curation, C.R. and A.L.G.; writing—original draft preparation, C.R.; writing—review and editing, A.L.G.; visualization, M.P.; supervision, C.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Programma Nazionale di Ricerche in Antartide (grant No. PNRA2004/1.6).

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Jmse 10 01135 g001 550
Figure 1. Production of stable emulsion by Idiomarina sp. A19 and Idiomarina sp. 185 during incubation in Marine Broth at 15 °C and pH 7.6.
Figure 1. Production of stable emulsion by Idiomarina sp. A19 and Idiomarina sp. 185 during incubation in Marine Broth at 15 °C and pH 7.6.
Jmse 10 01135 g001
Jmse 10 01135 g002 550
Figure 2. Production of stable emulsion (left side) and surface tension reduction (reported as difference between initial and final value) by Idiomarina sp. A19 and Idiomarina sp. 185 during incubation in BHbp.
Figure 2. Production of stable emulsion (left side) and surface tension reduction (reported as difference between initial and final value) by Idiomarina sp. A19 and Idiomarina sp. 185 during incubation in BHbp.
Jmse 10 01135 g002
Jmse 10 01135 g003 550
Figure 3. Rooted phylogenetic tree calculated by Tajima–Nei method distance estimation algorithm [28] showing affiliation of most representative bacterial isolates to closest-related sequences from either cultivated or cloned bacteria. The evolutionary history was inferred using the Neighbor-Joining method [29]. The tree was outgrouped with 16S rRNA gene sequence of Methanocaldococcus jannaschii DSM2661. Representative isolates are indicated in bold.
Figure 3. Rooted phylogenetic tree calculated by Tajima–Nei method distance estimation algorithm [28] showing affiliation of most representative bacterial isolates to closest-related sequences from either cultivated or cloned bacteria. The evolutionary history was inferred using the Neighbor-Joining method [29]. The tree was outgrouped with 16S rRNA gene sequence of Methanocaldococcus jannaschii DSM2661. Representative isolates are indicated in bold.
Jmse 10 01135 g003
Table
Table 1. Previous results on BS-producing Idiomarina isolates used in this study.
Table 1. Previous results on BS-producing Idiomarina isolates used in this study.
StrainSource *Isolation
Medium *		E24
(%)		ST **
(mN/m)		Growth ConditionsRef
Strain 185Enrichment cultures of Antarctic shoreline sediments in seawater plus COBH
plus
CO		3029.9BH, 4 °C, pH 7.6,
NaCl 3%,
Tetradecane 2%		[18]
Strain A19Enrichment culture of polychaete homogenate in ONR7a plus COONR7a
plus
BP		2056.5; 60MB, 28 °C, pH 7–7.5, NaCl 3%[8]
36.642.6BH, 25 °C, pH 7–7.5,
NaCl 3%,
Tetradecane 2%		[9]
Phenotypic characterization (Idiomarina sp. 185, [18]; Idiomarina sp. A19, this study)
Utilization as
carbon source		Strain 185 Strain A19 Biochemical testsStrain 185Strain A19
Arabinose-- Citrate utilization+-
Glucose-- Ornithine Decarboxylase--
Inositol, Saccharose-- H2S production--
Maltose-- Tryptophan Deaminase+-
Mannitol, Sorbitol-- Indole production--
Mannose-- Amygdaline--
Mellibiose, Rhamnose-- Reduction of nitrates ++
N-Acetyl-Glucosamine-- Glucose fermentation+-
Potassium gluconate--
Biochemical test Adipic acid-+
Urease+- Malate-+
β-galactosidase-- Trisodium Citrate--
Gelatinase+- Phenylacetic acid --
Arginine Dihydrolase+- Capric acid--
Lysine
Decarboxylase 		--
* MB, Marine Broth; BH, Bushnell Haas Broth; CO, crude oil; BP, biphenyl; ** ST, surface tension.
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Rizzo, C.; Papale, M.; Lo Giudice, A. Idiomarina sp. Isolates from Cold and Temperate Environments as Biosurfactant Producers. J. Mar. Sci. Eng. 2022, 10, 1135. https://doi.org/10.3390/jmse10081135

AMA Style

Rizzo C, Papale M, Lo Giudice A. Idiomarina sp. Isolates from Cold and Temperate Environments as Biosurfactant Producers. Journal of Marine Science and Engineering. 2022; 10(8):1135. https://doi.org/10.3390/jmse10081135

Chicago/Turabian Style

Rizzo, Carmen, Maria Papale, and Angelina Lo Giudice. 2022. "Idiomarina sp. Isolates from Cold and Temperate Environments as Biosurfactant Producers" Journal of Marine Science and Engineering 10, no. 8: 1135. https://doi.org/10.3390/jmse10081135

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