Extremophilic Microfactories: Applications in Metal and Radionuclide Bioremediation

Metals and radionuclides (M&Rs) are a worldwide concern claiming for resilient, efficient, and sustainable clean-up measures aligned with environmental protection goals and global change constraints. The unique defense mechanisms of extremophilic bacteria and archaea have been proving usefulness towards M&Rs bioremediation. Hence, extremophiles can be viewed as microfactories capable of providing specific and controlled services (i.e., genetic/metabolic mechanisms) and/or products (e.g., biomolecules) for that purpose. However, the natural physiological plasticity of such extremophilic microfactories can be further explored to nourish different hallmarks of M&R bioremediation, which are scantly approached in the literature and were never integrated. Therefore, this review not only briefly describes major valuable extremophilic pathways for M&R bioremediation, as it highlights the advances, challenges and gaps from the interplay of ‘omics’ and biological engineering to improve extremophilic microfactories performance for M&R clean-up. Microfactories’ potentialities are also envisaged to close the M&R bioremediation processes and shift the classical idea of never ‘getting rid’ of M&Rs into making them ‘the belle of the ball’ through bio-recycling and bio-recovering techniques.


INTRODUCTION
Metals and radionuclides (M&Rs) are problematic pollutants sourced from different industrial sectors, nuclear power plants, electronic waste and, especially, from mining activities. Undoubtedly, mining industry nurtures a plethora of human needs (Carvalho, 2017), but past and ongoing mining activities represent a huge footprint of environmental (Marques et al., 2014) and health injuries Venkateswarlu et al., 2016). Still, future societal demands will force industrial growth, thus making M&R pollution a never-ending threat, mainly due to their persistence and non-biodegradability (Gupta and Diwan, 2017).
Bioremediation, though being often viewed as an 'old fashion' and slow-acting technology, it has been thriving for M&R reclamation. Its sustainability, low-cost and efficiency totally rival conventional remediation techniques (Li and Zhang, 2012). In particular, microbially-based bioremediation relies on microbes abilities, biogenic products and/or components to scavange, transform or immobilize M&Rs (Lloyd and Lovley, 2001;Voica et al., 2016). Despite the interesting nature-based self-healing character of this approach, the tolerance of microbes to high M&R levels, extreme physical (e.g., radiation), chemical (e.g., acidic pH) and climate changing conditions may constrain (in situ) bioremediation efficiencies (Ramos et al., 2011). Extremophilic microbes, though, have attractive skills as bioremediation tiny factories (microfactories), which performance can even be improved and customized for M&R clean-up. This review will hence focus: (1) the peculiar mechanisms of extremophilic bacteria and archaea exploitable as 'microfactories' for M&R bioremediation, (2) the groundbreaking opportunities leveraged from nature and science ('omics' plus biological engineering) interplay to enhance extremophilic-microfactories-based bioremediation, (3) M&R recovery and recycling conducted by extremophilic microfactories as a sustainable and valueadded management of M&R-bioremediation-resulting wastes. Overall, this is an innovative overview on the potentialities of extremophilic bacteria/archaea in three M&R bioremediation hallmarks: application, optimization, end-waste management (Figure 1).

M&R Sensing
Sensing M&Rs presence is the alarm button to trigger the activation/repression of specific regulatory proteins and downstream cascades that control the expression of resistance genes/operons encoding enzymes and biomolecules for M&R detoxification (Das et al., 2016). Along this, the uptake/efflux of M&Rs across extremophiles cell wall and membranes may occur through energy-dependent P-type ATPases (copA1 Af , copA2 Af , and copB Af ), resistance nodulation cell division carriers (cusA Af , cusB Af , and cusC Af ) and/or chaperones (cusF Af and copC Af ) as in the copper-resistant Acidithiobacillus ferrooxidans (Navarro et al., 2009). A comprehensive description of the genetic characteristics and functioning of regulatory and M&R uptake/efflux systems was performed namely for thermoacidophilic, acidophilic, and metalophilic bacteria and/or archaea (Nies, 2000;Orell et al., 2010Orell et al., , 2013Navarro et al., 2013;Wheaton et al., 2015).
Despite the great biotechnological potential of extremophilic microfactories, remediation efficiency, biomass productivity, and economic profitability may be challenging, especially for in situ applications due to extreme optima, interspecies competition and interaction/communication inhibiting bioremediation, and limited proliferation (Ramos et al., 2011;Johnson et al., 2015). Consequently, extremophiles-based M&R bioremediation may become less attractive considering mining industry demands, hence requiring function-directed improvements.

UPGRADING EXTREMOPHILIC MICROFACTORIES FOR ENHANCED M&R BIOREMEDIATION
'Omics' and biological engineering are leading the achievement of more proficient extremophilic microfactories for M&R bioremediation to keep pace with current economical, technological, social, and climate change defiance.
Despite the relevance of this knowledge, the study of M&R-constrained gene expression and regulation through transcriptomics assumes a pivotal role for bioremediation purposes. DNA microarrays applied to the As-resistant Thiomonas spp. evidenced genomic rearrangements and horizontal gene transfer (Arsène-Ploetze et al., 2010), which can be exploited to transfer M&R resistance. Other studies observed overexpression of metallochaperones and enzymes mediating metal-phosphate granules formation in acidophilic bacteria (A. ferooxidans) (Navarro et al., 2009) and (thermo)acidophilic archaea (Sulfolobus metallicus, Metallosphaera sedula) challenged with M&Rs (Orell et al., 2013;Wheaton et al., 2016).
Nevertheless, many transcripts keep with unassigned functions or activities. Proteomics, through 2-DE gel coupled with mass spectroscopy (e.g., LC-MS, MALDI-ToF, and ICPL) tools and protein libraries, is thereby elucidating the type and abundance of proteins synthesized under M&R stress (Desai et al., 2010). Among them, those associated with EPS and biofilm production, oxidative stress responses, regulation of RND-type Cus system and metal uptake/efflux pumps (Zivanovic et al., 2009;Almárcegui et al., 2014;Martínez-Bussenius et al., 2016) are hugely relevant.
Additionally, metabolomics enlightens the metabolic fingerprinting of M&R-induced responses on extremophiles (Mosier et al., 2013). A diverse toolbox of chromatographic (GC and HPLC) and spectroscopic (FTIR and NMR) techniques have hence been employed to harness the genetic, transcriptional and translational processes sustaining the cellular biomolecules/metabolite fluxes (i.e., fluxomics) (Hold et al., 2009;Desai et al., 2010) leading to M&R sequestration. In silico reconstruction of unknown metabolic networks relevant for M&R bioremediation based on sequenced genomes (Sun et al., 2009), robust bioinformatics and libraries, is becoming a powerful fine-tuning approach, though requiring a cautious analysis of metal-microbe interactions to prevent misinterpretations (Johnson et al., 2015). In this context, community-level meta-omics (metagenomics, metatranscriptomics, and metaproteomics) offer great avenues by avoiding extremophiles culturing hurdles, while uncovering pools of novel biomolecules/enzymes for M&R bioremediation without previous sequence data ( Mohamed et al., 2013), and providing ecologically-relevant analysis of community shifts and functions (Cowan et al., 2015;Garris et al., 2016;Mirete et al., 2016) along the bioremediation processes. Multi-'omics' integration, either at individual or community levels, constitutes another robust approach to unravel new M&R-bioremediation agents or monitoring bio-devices. Indeed, Wilkins et al. (2011) used a proteogenomic analysis to develop a biomarker of G. sulfurreducens activity during uranium bioremediation.

Engineering Extremophic Microfactories for M&R Bioremediation
The advent of DNA recombinant techniques for tailoring existent abilities or to create synthetic ones, in natural or newly designed extremophiles, is a doorway to boost M&R bioremediation.

Genetic/Metabolic Engineering of Whole-Microbes and Communities
The peculiar mechanisms of extremophiles can be customized through the insertion of genes or gene clusters, as to enhance their skills and robustness for M&R attenuation (Cárdenas et al., 2010). Metal-resistant (Cupriavidus metallidurans; Rojas et al., 2011), radiophilic (D. radiodurans;Brim et al., 2000) and thermoacidophilic (D. geothermalis; Brim et al., 2003) bacteria were transformed with genes/clusters encoding enzymes involved in individual-or multi-M&Rs reduction/oxidation. This is biotechnologically worthy given the mixture of M&Rs in mine/nuclear wastes. In order to enhance M&R bioprecipitation, D. radiodurans was indeed manipulated to express a Cd(II)binding synthetic phytochelatin (EC20) and metallothionein (smtA) (Chaturvedi and Archana, 2014), and a periplasmic acid phosphatase (PhoN), which hydrolysed organic into inorganic phosphates to precipitate uranium (Appukuttan et al., 2006(Appukuttan et al., , 2011. However, some extremophiles are more resistant to transformation due to their robust protective mechanisms (Chen et al., 2011;Liu et al., 2011), hence forcing the construction of adapted vectors (Meng et al., 2013).
Recently, the innovative project NANOBINDERS (PTDC/AAG-REC/3004/2014) is enrolled in the creation of biogenic nanopolymers functionalized to bind M&Rs (the NANOBINDERS) from uranium mine effluents. These M&R-binding nanopolymers are self-assembled inside a host transformed with new constructs harboring nanopolymer-and metal-binding-peptides-encoding genes, originally obtained from microbes isolated in a uranium mine. Such functional nanobeads are a revolutionary option to engineered microbes, which in situ release still raises regulatory concerns, besides genetic instability and limited efficiency under real scenarios (de Lorenzo, 2009). Alternatively, manipulated extremophiles may house suicidal genes (Paul et al., 2005) activated upon undetectable M&R levels, for future in situ application.
Native microbial communities have been used to rehabilitate M&R-contaminated areas by natural attenuation (Shukla et al., 2017), which can be engineered through biostimulation (i.e., supplementation of compounds/substrates enhancing microbial proliferation and activities) and/or bioaugmentation measures (i.e., addition of microbes endowed with particular functional mechanisms). In mesocosms, Ñancucheo and Johnson (2011) reduced acid mine drainage production when consortia of metabolically-cooperating microalgae, acidophilic and/or acid-tolerant bacteria were added to promote iron-and/or sulfate-reduction of tailings. The biostimulation of Geobacter sp. activity by acetate enrichment had enhanced uranium reduction, thereby proving successful in situ bioremediation (Anderson et al., 2003). However, metabolic fluxes modeling (Hold et al., 2009) should be applied in the future to design novel extremophilic consortia with tailored co-activities for M&R clean-up.

Synthetic Biology
Synthetic biology (SynBio) moves beyond the simple transformation of genes or operons to the insertion of newly designed and constructed synthetic biological parts and circuits to create robust, profitable, programmable and customized microfactories (Martínez-Garcia and de Lorenzo, 2016). The genetic instability and limited performance of engineered microbes for M&R bioremediation, can be overcome by DNA de novo synthesis and genome editing tools [e.g., Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs)-and protein Cas; Mougiakos et al., 2016Mougiakos et al., , 2017. Hence, extremophiles can be engineered as novel platforms (or chassis) housing stable and multiple designed functions together with the transcriptional and translational machinery to be active from laboratory-to field-scale extreme scenarios (Adams, 2016). Likewise, Gerber et al. (2015) took advantage of Deinococcus genetic plasticity and robustness to construct a chassis towards different applications. Deinococcus spp. was also successfully engineered with metal-resistance genes, demonstrating their genetic flexibility for heterologous and co-expression of different metal-resistance determinants under extreme temperature and radiation (Brim et al., 2000(Brim et al., , 2003. Thus, constructing extremophilic-microfactories chassis is an appealing new wave for enhanced M&R bioremediation, especially if universal genetic toolkits could be created (Adams, 2016).
The joint endeavor of SynBio and metabolic engineering of communities is indeed enabling the design and building of synthetic consortia (Shong et al., 2012) for M&R remediation. Developing synthetic consortia demands the engineering of cell-cell communication, i.e., quorum-sensing (QS) systems (Brenner et al., 2008). QS modulates behavioral, metabolic and structural dynamics in microbial communities through signaling molecules [e.g., acyl homoserine lactones family (AHL)] (Shong et al., 2012). QS is pivotal namely in EPS synthesis and biofilm formation (Farah et al., 2005;McDougald et al., 2012), as well as AHLs can mediate copper resistance in A. ferrooxidans (Wenbin et al., 2011). Thus, putting effort on the modulation or creation of new QS systems and signaling molecules biosynthesis can fine-tune the pool of relevant functions in extremophilic consortia for M&R removal (e.g., biomass production and EPS synthesis) (Brune and Bayer, 2012;Shong et al., 2012).

GENERATING RICHNESS FROM BIOREMEDIATED M&Rs
A discouraging issue in M&R bioremediation is the disposal of final wastes. Nevertheless, extremophilic microfactories can be successfully explored for M&Rs recycling and/or recovery after being remediated, thereby sustainably closing M&R bioremediation processes.

Microbial Fuel Cells (MFCs)
Microbial fuel cells (MFCs) can be a valuable recovery method since it uses the catalytic activity of microbes to generate electrical energy from the oxidation of organic substrates (Mathuriya and Yakhmi, 2014). They are composed by an anode that captures electrons from substrate oxidation, a cathode and an intermediate cation-specific membrane. Since M&Rs have a high redox potential, they can be reduced and precipitated by receiving electrons from the cathode. Geobacter sulfurreducens biofilms possessing electronconductive nanowires (pili) have been exploited for increased electricity generation into MFCs (Reguera et al., 2006). The generated electrons can be further used to reduce U(VI) into removable U(IV), hence synergizing Geobacter sp. skills for bioremediation-derived-waste recycling. An anoxic sludge containing Geobacter spp. recovered the noble metal silver, while generating electrical energy in a MFC (Ho et al., 2017). Other extremophiles have indeed been used in MFCs (Shrestha et al., 2018), especially towards metal recovery (Dopson et al., 2015).

Recycling Through Nanoparticles (NPs) Production
Extremophiles biosynthesize economically-lucrative inorganic NPs through intracellular or extracellular transformation/precipitation of M&Rs (Ulloa et al., 2016), hence providing another profitable M&R recycling strategy. The metaltolerant Cupriavidus metallidurans CH34 mineralized Au and precipitated it as nanoparticulate Au 0 in the periplasm (Reith et al., 2009), whilst the alkalotolerant Rhodococcus sp. accumulated Au-NPs in the cytoplasm and cell wall (Ahmad et al., 2003). Spherical Ag-NPs were also extracellularly synthesized at 80 • C by the thermophilic Ureibacillus thermosphaericus (Juibari et al., 2011). CdS-NPs with enhanced stability under acidic conditions were produced by Acidithiobacillus spp., being a gainful option for Cd turnover (Ulloa et al., 2016). Uraninite, which is a worthy biogenic NP for uranium in situ remediation (Bargar et al., 2008), was synthesized by Geobacter metallireducens upon extracellular U(VI) reduction (Gorby and Lovley, 1992). The precious metal Pd(II) used in many industrial sectors was precipitated in G. sulfurreducens biofilms as Pd(0)-NPs and easily extracted by centrifugation (Yates et al., 2013).

CONCLUDING REMARKS
Extremophiles enclose a pool of genetic and metabolic opportunities that can be harnessed as microfactories for multiple M&R-bioremediation hallmarks. Nevertheless, the complexity of extremophiles' cellular mechanisms, interspecies relationships and M&R interplay under real scenarios of co-occurring extreme conditions may rise some challenges. Multi-'omics' applied side-by-side with genetic engineering techniques are covering knowledge gaps, allowing gene expression and metabolic pathways customization for improved bioremediation. Synthetic biology, however, through an iterative design-build-test-analyze framework is further revolutionizing M&R bioremediation and opening new perspectives on the creation of robust extremophilic recombinants or chassis with de novo-designed biologically-, energetically-, and economically-viable traits for specific M&R removal processes. Future trends will hence target adaptation or construction of multi-skilled extremophilic microfactories to sustainably empower M&R bioremediation and end-waste recycling, while keeping up with global changes, natural resources availability, and cost-efficiency requirements.

AUTHOR CONTRIBUTIONS
CM was responsible for doing all the tasks concerning the development of the work: conceptualized the idea and goals of the mini-review, performed literature search and revision, and wrote the manuscript.

FUNDING
This work was funded by the Portuguese Foundation for Science and Technology (FCT) and by the European Regional Development Fund (ERDF) through the Portugal 2020 Partnership Agreement between Portugal and the European Union, the Competitiveness and Internationalization Operational Program (COMPETE 2020) and the Regional Operational Program Lisboa (POR-Lisboa), under the project NANOBINDERS (PTDC/AAG-REC/3004/2014). CM was granted as an Invited Scientist within the project NANOBINDERS (ref. BCC/UI88/5626/2016). Thanks are due for the financial support to CESAM (UID/AMB/50017 -POCI-01-0145-FEDER-007638), to FCT/MCTES through national funds (PIDDAC), and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020.