bs_bs_banner Dynamics of bacterial populations during bench-scale bioremediation of oily seawater and desert soil bioaugmented with coastal microbial mats Nidaa Ali, Narjes Dashti, Samar Salamah, Naser possible oil concentration as one lot in the beginning Sorkhoh, Husain Al-Awadhi and Samir Radwan* of bioremediation, addition of vitamins, and slowing Microbiology Program, Department of Biological down the seawater flow rate. Sciences, Faculty of Science, Kuwait University, PO Box 5969, Safat, 13060, Kuwait. Introduction Summary Remediation of sites contaminated with xenobiotic com- pounds is achieved by physical and chemical methods, This study describes a bench-scale attempt to e.g. land filling and incineration (Kuiper et al., 2004). bioremediate Kuwaiti, oily water and soil samples However, the physical removal of pollutants from all con- through bioaugmentation with coastal microbial mats taminated sites on earth is obviously very costly rich in hydrocarbonoclastic bacterioflora. Seawater (Rosenberg, 1993). In addition, incineration is associated and desert soil samples were artificially polluted with with air pollution, and land filling frequently leads to 1% weathered oil, and bioaugmented with microbial leachates in the form of gases and liquids which can mat suspensions. Oil removal and microbial commu- pollute the ground water (Kuiper et al., 2004). The much nity dynamics were monitored. In batch cultures, oil more cost-effective and more environmentally friendly removal was more effective in soil than in seawater. technology of bioremediation implies the use of microbial Hydrocarbonoclastic bacteria associated with mat activities in pollutant biodegradation (Atlas and samples colonized soil more readily than seawater. Pramer, 1990). It comprises two major practices. The predominant oil degrading bacterium in seawater ‘Bioaugmentation’ (inoculation or seeding), which implies batches was the autochthonous seawater species the introduction of suitable oil-degrading microorganisms Marinobacter hydrocarbonoclasticus. The main oil into the contaminated site. The second practice is degraders in the inoculated soil samples, on the other ‘biostimulation’, whose objective is to enhance the activ- hand, were a mixture of the autochthonous mat ities of indigenous (autochthonous) pollutant-degrading and desert soil bacteria; Xanthobacter tagetidis, microorganisms via environmental management, e.g. the Pseudomonas geniculata, Olivibacter ginsengisoli addition of nutrients and other growth-limiting factors, and others. More bacterial diversity prevailed in sea- especially nitrogen and phosphorus (Atlas and Bartha, water during continuous than batch bioremediation. 1998; Radwan, 2009). Bioremediation commonly is rec- Out of seven hydrocarbonoclastic bacterial species ommended as an alternative technology to the use of isolated from those cultures, only one, Mycobacte- chemicals and other toxic materials for removing hydro- rium chlorophenolicum, was of mat origin. This result carbon contaminants (Piskonen and Itävaara, 2004). too confirms that most of the autochthonous mat bac- As already mentioned, bioaugmentation implies teria failed to colonize seawater. Also culture- the inoculation of the contaminated sites with independent analysis of seawater from continuous laboratory grown, hydrocarbon-degrading microorgan- cultures revealed high-bacterial diversity. Many of isms (Al-Awadhi et al., 1996; Van Limbergen et al., 1998; the bacteria belonged to the Alphaproteobacteria, Kuiper et al., 2004). This leads to the introduction of addi- Flavobacteria and Gammaproteobacteria, and were tional gene pools complementary to the already existing hydrocarbonoclastic. Optimal biostimulation prac- ones, with the purpose of enhancing degradation of con- tices for continuous culture bioremediation of seawa- taminants (Domde et al., 2007). In a study on the effect of ter via mat bioaugmentation were adding the highest bioaugmentation with a consortium of bacteria on the Received 2 April, 2015; revised 9 September, 2015; accepted 9 remediation of hydrocarbon contaminated waste water, September, 2015. *For correspondence. E-mail: samir.radwan@ ku.edu.kw; Tel. +96524987145; Fax +96524847054. the water chemical oxygen demand, which reflects the Microbial Biotechnology (2016) 9(2), 157–171 organic substance content, dramatically decreased doi:10.1111/1751-7915.12326 Funding Information This work has been supported by Kuwait Uni- (Domde et al., 2007). Obviously, the proper consortia of versity, Research Grant RS02/12. microorganisms should be used in order to complete the ª 2016 The Author. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 158 N. Ali et al. degradation process (Kapley and Purohit, 2001; ganism (DiGregorio et al., 2015), the two practices Moharikar et al., 2003; Domde et al., 2007). In literature (autochthounous bioaugmentation and biostimulation) reports, exogenous pure cultures as well as unidentified could be regarded as two faces of one coin. Autochtho- mixtures of microorganisms have been used for nous microorganisms of a habitat are the natural inhabit- bioaugmentation (Atlas and Bartha, 1998). Based on their ants, contributing to biochemical activities therein. Their ability to degrade a wide range of organic compounds, counterparts, the allochthonous microorganisms are species of Pseudomonas have been frequently selected foreign survivals which do not contribute significantly to (Atlas and Bartha, 1998). Evidently, the bioaugmented activities in the habitat. organisms should be adapted to physicochemical param- The following snap shots summarize the history of the eters of the contaminated site. Imported Arthrobacter ‘autochthonous bioaugmentation (ABA)’ concept, and strains, in contrast to locally isolated ones, failed to colo- contribute to highlighting the objectives of this study. nize local oil-polluted soils due to their inability to compete About two and half decades back, one of our group with the already existing strains (Radwan et al., 1997). (Radwan, 1991) warned from using imported microbial Although proper microorganisms may be inoculated, they cocktails, instead of depending on indigenous microor- may fail to remove the pollutant (El Fantroussi and ganisms for combating the greatest man-made oil spill in Agathos, 2005). Reportedly, this could be due to the the history of mankind (the spill associated with the 1990– absence of a single bacterium that possesses the entire 1991 occupation of Kuwait by the Iraqi forces). Experi- set of enzymes needed to biodegrade the pollutant. mental studies supported the validity of this concept Another five reasons have been suggested (Goldstein (Vecchioli et al., 1990; Weber and Corseuil, 1994). et al., 1985): the contaminant concentration is too low to However, it was Ueno and colleagues (2007) who coined support bacterial growth, presence of inhibitors that sup- the term ‘autochthonous bioaugmentation (ABA)’, which press microbial growth and/or activity, reduction of bacte- necessitates the use of natural microbial inhabitants of an rial numbers due to protozoan grazing, presence of better environment for its bioremediation. With this background utilizable sources of carbon and inability of the microbial in mind, the major objective of this paper was to study, in cells to spread and reach the pollutant. bench-scale experiments, the feasibility of using local Biostimulation, the second bioremediation, practice microbial mats from Kuwaiti coasts, instead of laboratory- implies, among others, the addition of nutrients, usually grown microbial cocktails, as bioaugmentation materials nitrogen, phosphorous and trace elements (Korda et al., for bioremediation of local oil-contaminated seawater and 1997). Enhancing effects of biostimulation on hydrocar- desert soil samples. We selected microbial mats on the bon biodegradation have been documented (Bossert and basis of our earlier report (Sorkhoh et al., 1992) that they Bartha, 1984; Leahy and Colwell, 1990; Atlas, 1991; were the primary colonizers of coastal oil sediments, and Margesin and Schinner, 1998, Namkoong et al., 2002, consequently the first sign of self-cleaning of the dead Jimenez et al., 2007; For review see Nikolopoulou and coasts that had been heavily polluted during the Iraqi Kalogerakis, 2009). On the other hand, a few investiga- occupation of Kuwait. Reportedly, such coastal mats were tors found that the rate of hydrocarbon degradation was rich in hydrocarbonoclastic bacteria, well adapted to the not affected following the addition of nutrients (Seklemova Kuwaiti conditions. There are still only a very few studies et al., 2001). It has been reported that the percentage of worldwide on the ABA strategy (Hosakawa et al., 2009), oil degraded was inversely proportional to the concentra- and almost none on the contaminated Kuwaiti habitats. tion of the contaminating oil (Rahman et al., 2002). These facts highlight the need for the current study. Bioremediation in the field is unpredictable because of the lack of knowledge of the persisting microorganisms in the site (Head, 1998). Results Ideally, biostimulation should be coupled with Oil removal in batch culture bioaugmentation (Odokuma and Dickson, 2003; Coppotelli et al., 2008; Nikolopoulou et al., 2013a,b). The Kuwait map in Fig. 1