Biostimulation of indigenous microorganisms for bioremediation of oily hypersaline microcosms from the Arabian Gulf Kuwaiti coasts

Highlights

• Hypersaline microcosms from Kuwait coast harbor hydrocarbonoclastic bacteria and archaea.

• Those microorganisms could be biostimulated by the addition of cations and vitamins.

• Such amendments were effective in oil-removal in the halostressed microcosms.

• Cations are known to serve as osmoregulators in halophilic bacteria and archaea.

• Results of this study pave the way for field bioremediation for halostressed sites.

Abstract

Hypersaline soil and water samples were collected in summer and winter from the “sabkha” area at the Kuwaiti shore of the Arabian Gulf. Physicochemical parameters were analyzed, and found suitable for microbial oil-removal. Summer- and winter-microcosms were treated with individual cation (K⁺, Ca²⁺, Mg²⁺, Fe³⁺) salts, and with animal blood and commercial yeast, as cost-effective vitamin sources. Those microcosms were exposed to the open environment for six winter and six summer months, and analyzed for their hydrocarbonoclastic microorganisms at time zero and in two month intervals. The hydrocarbonoclastic microbial communities in the microcosms consisted of halophilic bacteria and haloarchaea. The constituent bacterial species varied according to the season. Three species, Dietzia kunjamensis, Marinobacter lacisalsi and Halomonas oxialensis consistently occurred both in summer- and winter-samples, but the remaining species were different. On the other hand, the haloarchaeal communities in summer and winter were quite similar, and consisted mainly of Haloferax spp and Halobacterium spp. Treating the microcosms with cations and with vitamin-containing natural products enhanced microbial numbers and oil-removal. The effectiveness of the cations in oil-removal was in the order; Fe³⁺ (94%) > Ca²⁺ (89%) > Mg²⁺ (85%) > K⁺ (82%). Thus, oily microcosms amended with trivalent and divalent cations lost most of the oil, and those amended with commercial yeast and with animal blood, as vitamin sources, lost 78% and 72% oil, respectively.

Introduction

Unlike studies on oil-bioremediation for nonextreme environments (Radwan, 2009), those for extreme environments are still rather few (Margesin and Schinner, 2001, Le Borgne et al., 2008, Martins and Peixoto, 2012, Jurelevicius et al., 2013; Fathepure, 2014, Ali et al., 2016a, Ali et al., 2016b, Xiao et al., 2016, Edbeib et al., 2016, Corsellis et al., 2016). Hypersaline areas, namely those with salinities above that of seawater (Grant et al., 1998), are spread globally, and represent extreme environments, that become occasionally polluted with waste hydrocarbons (Lefebvre and Moletta, 2006, Dastgheib et al., 2012). Such areas may be bioremediated via the activities of halophilic/halotolerant hydrocarbonoclastic microorganisms (Oren, 2002). During the past few decades, interesting studies have been published on halophilic microorganisms with hydrocarbonoclastic potential from hypersaline environments (Oren et al., 1992, Emerson et al., 1994, Margesin and Schinner, 2001, Garcia et al., 2004, Nicholson and Fathepure, 2005, Zhao et al., 2009, Al-Mailem et al., 2010, Bonfa et al., 2011). Those microorganisms comprised, bacteria (e.g. Marinobacter sedimentalis, Halomonas salina, Pseudomonas sp.), archaea (e.g. Halobacterium salinarum, Haloferax larsenii) and fungi, which biodegraded oil and pure aliphatic and aromatic hydrocarbons at high salinities. Therefore, they may be useful in oil bioremediation for saline environments (Oren, 2002). Two bioremediation strategies are known (Bartha, 1986). The first involves “bioaugmentation” (i.e. inoculation) of the oily environment with exogenous, hydrocarbonoclastic microorganisms, and the second involves the “biostimulation” of hydrocarbonoclastic microorganisms already inhabiting the oily site. The latter approach can be achieved via fertilization with nutrients, e.g. N and P, addition of surfactants and by optimizing physical conditions, e.g. pH, aeration and others. Biostimulation, which depends on indigenous microorganisms well adapted to the sites' environmental conditions, is preferred by many authors over bioaugmentation (Radwan, 1991, Radwan et al., 1995, Nikolopoulou et al., 2013). More information about researchers' experiences on biostimulation and bioaugmentation strategies is available in our earlier publications (e.g. Ali et al., 2016a, Ali et al., 2016b).

Hypersaline environments in Kuwait are coastal areas locally called the supertidal “sabkhas”. In earlier studies, our group recorded in those sabkhas, extremely halophilic bacteria and archaea, capable of hydrocarbon utilization at high salinities (Al-Mailem et al., 2010, Al-Mailem et al., 2012, Al-Mailem et al., 2014a). Growth and hydrocarbon consumption by such microorganisms were enhanced by specific amendments such is K- and Mg- salts as osmoregulators (Al-Mailem et al., 2013), and by pure vitamins (Al-Mailem et al., 2014b).

The major objective of this work was to elaborate on our earlier laboratory studies. Therefore, we considered the amendment-based (biostimulation) bioremediation of oily, hypersaline microcosms that were exposed to the open environment during the bioremediation process. We confirmed our earlier laboratory results and in addition, report on new cations and vitamin-rich, natural products (instead of the costly, pure vitamins) with more pronounced biostimulatory effects. Achieving microcosm biostimulation under open conditions is obviously an important transitional step between the in vitro study and the targeted field-bioremediation. In this context, Kuwait, is a small oil-producing country, that belongs to the semiarid region with harsh weather. In the dry summer, the maximum temperature frequently exceeds 50 °C and in the mild, rainy winter, the night temperature frequently drops to zero °C. Due to legal (e.g. oil production and transport) and illegal (e.g. deliberate disposal of oily wastes by vehicles) activities, the Arabian Gulf water body and shores are frequently polluted with hydrocarbons.