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Evaluation of Conventional and Response Habib et al. Microb Cell Fact (2018) 17:44 https://doi.org/10.1186/s12934-018-0889-8 Microbial Cell Factories RESEARCH Open Access Evaluation of conventional and response surface level optimisation of n‑dodecane (n‑C12) mineralisation by psychrotolerant strains isolated from pristine soil at Southern Victoria Island, Antarctica Syahir Habib1, Siti Aqlima Ahmad1, Wan Lutf Wan Johari2, Mohd Yunus Abd Shukor1, Siti Aisyah Alias3, Khalilah Abdul Khalil4 and Nur Adeela Yasid1* Abstract Background: Biodegradation of hydrocarbons in Antarctic soil has been reported to be achieved through the utilisa- tion of indigenous cold-adapted microorganisms. Although numerous bacteria isolated from hydrocarbon-contami- nated sites in Antarctica were able to demonstrate promising outcomes in utilising hydrocarbon components as their energy source, reports on the utilisation of hydrocarbons by strains isolated from pristine Antarctic soil are scarce. In the present work, two psychrotolerant strains isolated from Antarctic pristine soil with the competency to utilise die- sel fuel as the sole carbon source were identifed and optimised through conventional and response surface method. Results: Two potent hydrocarbon-degraders (ADL15 and ADL36) were identifed via partial 16S rRNA gene sequence analysis, and revealed to be closely related to the genus Pseudomonas and Rhodococcus sp., respectively. Factors afecting diesel degradation such as temperature, hydrocarbon concentration, pH and salt tolerance were studied. Although strain ADL36 was able to withstand a higher concentration of diesel than strain ADL15, both strains showed similar optimal condition for the cell’s growth at pH 7.0 and 1.0% (w/v) NaCl at the conventional ‘one-factor-at-a-time’ level. Both strains were observed to be psychrotrophs with optimal temperatures of 20 °C. Qualitative and quantita- tive analysis were performed with a gas chromatograph equipped with a fame ionisation detector to measure the reduction of n-alkane components in diesel. In the pre-screening medium, strain ADL36 showed 83.75% of n-dode- cane mineralisation while the reduction of n-dodecane by strain ADL15 was merely at 22.39%. The optimised condi- tion for n-dodecane mineralisation predicted through response surface methodology enhanced the reduction of n-dodecane to 99.89 and 38.32% for strain ADL36 and strain ADL15, respectively. Conclusions: Strain ADL36 proves to be a better candidate for bioaugmentation operations on sites contaminated with aliphatic hydrocarbons especially in the Antarctic and other cold regions. The results obtained throughout strongly supports the use of RSM for medium optimisation. Keywords: Biodegradation, Diesel fuel, Psychrotroph, Response surface methodology, GC-FID analysis *Correspondence: [email protected] 1 Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Habib et al. Microb Cell Fact (2018) 17:44 Page 2 of 21 Background degradation. In the conventional approach, individ- Antarctica, once a pristine ice-covered landmass is being ual factors are optimised by changing one factor at a contaminated with pollutants, largely hydrocarbons. time while keeping other variables at a constant value. Petroleum hydrocarbons are widely used in Antarctic as Although the ‘one-variable-at-a-time’ method can be human demands for heating, transportation and power implemented easily in helping the selection of optimum generation [1]. One of the common hydrocarbon fuels analysed parameters, it is meticulous, time-restraining used in the Antarctic region is diesel. Diesel oil, a com- and overlooks interactions among factors afecting a plex combination of aliphatic and aromatic hydrocarbons particular response. Contrariwise, statistical analysis is obtained from the middle-distillate, gas-oil fraction such as response surface methodology (RSM) measures during fractional distillation of petroleum [2]. Tere is and interprets the responses obtained from a batch run also the presence of branched-chain hydrocarbons and while studying the interactions between the individual aromatic compounds, although in lesser amounts [3]. factors assessed. RSM is also more benefcial in conduct- However, due to their immense production and use as ing experiment as it reduced experimental time and runs fuels for transportation, contamination in the polar pris- [19]. Comparative analysis between the conventional and tine soil are likely bound to occur [4, 5]. Oil spills and response surface method has been studied previously leakage from the underground storage tanks and pipe- with the latter approach proved to be more successful in lines are also likely to pollute the unfrozen soil active determining optimal point in studied parameters [20, 21]. layer due to their low viscosity and mobile properties Te aim of this study was to isolate and identify the [6]. Aislabie et al. [7] reported that oil spills frequently diesel-degrading bacteria from pristine soils collected occur near to the research and fuel stations where fuel from Scott Coast and Ross Island, Antarctica. Respective storage and refueling of aircrafts and vehicles are heav- strains identifed by 16S rRNA sequence analysis were ily operated. Te contamination can also cause risks for then characterised by their biodegradative abilities and humans and other living organisms if diesel fuel reaches growth at optimised parameters. Degradation of n-dode- groundwater reservoirs and water bodies [8]. Besides, cane was also estimated at two diferent level of optimi- the freeze–thaw cycle and the lower temperatures in the sation approaches—conventional ‘one-factor-at-a-time’ Antarctic means that spilt fuel may persist longer, which and statistical method to ascertain the efciency of both may indicate that recovery of the Antarctic ecosystem, is methods in optimising the mineralisation of dodecane. slower than in temperate climates [9, 10]. Tese negative efects toward organism are of great concern, and tech- Methods niques to remove the pollutants are strongly demanded. Samples and media Due to the remoteness from proper infrastructure for Soil samples collected from pristine Antarctic areas were remediation of the Antarctic environment, bioremedia- labelled as 1–43. Te collected samples were kept in tion is acknowledged as the adequate approach to recu- appropriate coolers (3–5 °C) during transportation from perate contaminated soils as it is simple, economical and the Antarctic mainland to Malaysia. Diesel fuel used causes lesser environmental impact [1, 11, 12]. Since throughout the experiment was purchased from the local the Antarctic Treaty outlaws the introduction of foreign gas station (PETRONAS) in Selangor, Malaysia. Alkane organisms, microbes that are indigenous to the Antarc- analytical standard C8–C20 was purchased from Sigma- tic soil are required for the application of bioremedia- Aldrich, USA. For the isolation of hydrocarbon-degrad- tion [13]. Besides, indigenous microbes that are able to ing bacteria and growth optimisation, a standardised degrade hydrocarbon contaminants are already adapted Bushnell-Haas (BH) broth was used [22]. Te BH media to the pollutant presence for growth and survival towards is composed of (g/l): MgSO4 (0.2); CaCl2 (0.02); K2HPO4 the extreme conditions exist in soils which are typically (1.0); KH2PO4 (1.0); NH4NO3 (1.0) and FeCl3 (0.05); cold, dry, alkaline and low in nutrients during the sum- with the addition of 20 g bacteriological agar for solid mer seasons [14]. Several hydrocarbon-degrading bacte- media. Te pH of the media was adjusted to 7.0 ± 0.2 ria that commonly isolated from polluted Antarctic soils at 25 °C before sterilisation. Diesel was the sole carbon are typically allocated to the genera Acinetobacter, Pseu- source used in all experiments. Uninoculated sterile BH domonas, Rhodococcus or Sphingomonas [1, 5]. Tese agar and broth were used as controls for confrmation of bacterial species are known to degrade both alkanes [15, strain ability to utilise diesel as a sole carbon source. 16] and/or aromatic hydrocarbons [13] aerobically. Previously isolated bacteria able to utilise hydrocar- Strain isolation and screening bons are reported to depend strongly on the physical and One gram of each soil sample was weighed and sus- medium factors [17, 18]. Terefore, an optimisation pro- pended in 10 ml of sterile saline solution (0.9% w/v cess must be done to achieve maximum hydrocarbons NaCl), incubated at 10 °C on a shaking incubator at Habib et al. Microb Cell Fact (2018) 17:44 Page 3 of 21 160 rpm for 1 h, before serially diluted up to 107. Aliquots agarose gel electrophoresis. Te PCR products were puri- (0.1 ml) of each dilution were spread onto the BH agar fed using GeneJET Gel Extraction and DNA Cleanup Kit (BHA) supplemented with 0.5% (v/v) diesel fuel sterilised
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