microorganisms Review Microbial Eukaryotes in Oil Sands Environments: Heterotrophs in the Spotlight Elisabeth Richardson 1,* and Joel B. Dacks 2,* 1 Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada 2 Division of Infectious Disease, Department of Medicine, University of Alberta, Edmonton, AB T6G 2G3, Canada * Correspondence: [email protected] (E.R.); [email protected] (J.B.D.); Tel.: +1-780-248-1493 (J.B.D.) Received: 6 May 2019; Accepted: 14 June 2019; Published: 19 June 2019 Abstract: Hydrocarbon extraction and exploitation is a global, trillion-dollar industry. However, for decades it has also been known that fossil fuel usage is environmentally detrimental; the burning of hydrocarbons results in climate change, and environmental damage during extraction and transport can also occur. Substantial global efforts into mitigating this environmental disruption are underway. The global petroleum industry is moving more and more into exploiting unconventional oil reserves, such as oil sands and shale oil. The Albertan oil sands are one example of unconventional oil reserves; this mixture of sand and heavy bitumen lying under the boreal forest of Northern Alberta represent one of the world’s largest hydrocarbon reserves, but extraction also requires the disturbance of a delicate northern ecosystem. Considerable effort is being made by various stakeholders to mitigate environmental impact and reclaim anthropogenically disturbed environments associated with oil sand extraction. In this review, we discuss the eukaryotic microbial communities associated with the boreal ecosystem and how this is affected by hydrocarbon extraction, with a particular emphasis on the reclamation of tailings ponds, where oil sands extraction waste is stored. Microbial eukaryotes, or protists, are an essential part of every global ecosystem, but our understanding of how they affect reclamation is limited due to our fledgling understanding of these organisms in anthropogenically hydrocarbon-associated environments and the difficulties of studying them. We advocate for an environmental DNA sequencing-based approach to determine the microbial communities of oil sands associated environments, and the importance of studying the heterotrophic components of these environments to gain a full understanding of how these environments operate and thus how they can be integrated with the natural watersheds of the region. Keywords: Oilsands; microbial eukaryote; 18s; microbial ecology; HNF; reclamation; molecular ecology; remediation; meta-genomics 1. Introduction The term “Eukaryote” is often considered synonymous with multicellular organisms such as animals, plants, and fungi. However, multicellular diversity only captures a minor fraction of overall eukaryotic diversity (Figure1). There is tremendous insight to be gained from studying the diversity of microbial eukaryotes in nature, both from an ecological and a cell biological perspective. In this review, we consider the impact of high-throughput sequencing on our understanding of microbial eukaryotic species and communities in the context of hydrocarbon-associated environments, and in particular the oil sands of Northern Alberta. Microorganisms 2019, 7, 178; doi:10.3390/microorganisms7060178 www.mdpi.com/journal/microorganisms Microorganisms 2019, 7, 178 2 of 14 Microorganisms 2019, 7, x FOR PEER REVIEW 2 of 14 FigureFigure 1. 1.Diversity Diversity ofof eukaryotes,eukaryotes, basedbased uponupon Adl et al. [1]. [1]. Groups Groups with with noted noted resistance resistance to to hydrocarbons,hydrocarbons, or or ability ability to degradeto degrade hydrocarbons, hydrocarbons, are are indicated. indicated. 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Protist Diversity Diversity and and Environmental Environmental Abundance:Abundance: New New Technology Technology Uncover Uncover New New Realities Realities WithWith the the advent advent of of environmental environmental DNADNA (eDNA)(eDNA) and high-throughput sequ sequencingencing analyses analyses of of microbialmicrobial community community composition, composition, thethe opportunityopportunity to understand the the diversity diversity and and abundance abundance of of eukaryoteseukaryotes has has exploded exploded [1]. Though[1]. Though the development the development of this newof this technique new technique has brought has technological brought challenges,technological reviewed challenges, in Thomsen reviewed inter in aliaThomsen [2], important inter alia information [2], important has information been obtained has frombeen all obtained from all manner of global environments that has forced microbiologists to re-examine manner of global environments that has forced microbiologists to re-examine some widely held some widely held preconceptions about microbial eukaryotic diversity and abundance. In this preconceptions about microbial eukaryotic diversity and abundance. In this review, we consider review, we consider all microbial eukaryotes, also known as protists, including those from groups all microbial eukaryotes, also known as protists, including those from groups which also include which also include multicellular organisms such as Opisthokonta and Chloroplastida [1]. multicellular organisms such as Opisthokonta and Chloroplastida [1]. 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Amplicon-driventaxonomically informative studies, using genes taxonomically obtained via eukaryotic-specific informative genes primers obtained most via commonly eukaryotic-specific the 18S primersribosomal most subunit commonly [7], theare 18Snow ribosomal effectively subunit mapping [7], protist are now diversity, effectively abundance, mapping and protist microbial diversity, abundance,ecology on and a global microbial scale. ecology Researchers on a globalin this scale. field Researchersare also in the in process this field of are developing also in the unified process ofprotocols developing for unifiedprotist taxonomic protocols forclas protistsification taxonomic via eDNA, classification most recently via the eDNA, EukRef most initiative recently [8,9]. the EukRefThis vast initiative and expanding [8,9]. This field vast is andfar beyond expanding the scope field of is this far beyondreview, thebut see scope Massana of this et review, al. [10] butfor a see review of protistan diversity in eDNA experiments. Nonetheless, we include here a few vignettes Massana et al. [10] for a review of protistan diversity in eDNA experiments. Nonetheless, we include illustrating the types of insights possible through this approach. here a few vignettes illustrating the types of insights possible through this approach. Firstly, it is not accurate to assume that the environmental factors driving bacteria and protists Firstly, it is not accurate to assume that the environmental factors driving bacteria