Towards a sustainable biobased industry – Highlighting the impact of extremophiles

Accepted Manuscript Title: Towards a sustainable biobased industry – Highlighting the impact of extremophiles Authors: Anna Kruger, ¨ Christian Sch¨afers, Carola Schr¨oder, Garabed Antranikian PII: DOI: Reference:

S1871-6784(16)32667-X http://dx.doi.org/doi:10.1016/j.nbt.2017.05.002 NBT 970

To appear in: Received date: Revised date: Accepted date:

5-1-2017 28-2-2017 3-5-2017

Please cite this article as: Kruger, ¨ Anna, Sch¨afers, Christian, Schr¨oder, Carola, Antranikian, Garabed, Towards a sustainable biobased industry – Highlighting the impact of extremophiles.New Biotechnology http://dx.doi.org/10.1016/j.nbt.2017.05.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Towards a sustainable biobased industry – Highlighting the impact of extremophiles Anna Krüger, Christian Schäfers, Carola Schröder and Garabed Antranikian Institute of Technical Microbiology, Hamburg University of Technology (TUHH), Kasernenstr. 12, D-21073 Hamburg, Germany Anna Krüger ([email protected]) Christian Schäfers ([email protected]) Carola Schröder ([email protected]) Garabed Antranikian ([email protected])

Corresponding author Anna Krüger ([email protected]), Tel.: +49(0)40-42878-3638, Fax: +49(0)40-428782582

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Highlights 

Transition of the oil-based economy to a bioeconomy is expected to solve current global challenges



Biotechnology plays a key role in this transformation towards a sustainable biobased industry



Benefits of extremozymes have been recognized for several industrial applications



Increasing numbers of robust biocatalysts are identified by sophisticated “omics” analyses

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Abstract The transition of the oil-based economy towards a sustainable economy completely relying on biomass as renewable feedstock requires the concerted action of academia, industry, politics and civil society. An interdisciplinary approach of various fields such as microbiology, molecular biology, chemistry, genetics, chemical engineering and agriculture in addition to cross-sectional technologies such as economy, logistics and digitalization is necessary to meet the future global challenges. The genomic era has contributed significantly to the exploitation of nature´s biodiversity also from extreme habitats. By applying modern technologies it is now feasible to deliver robust enzymes (extremozymes) and robust microbial systems that are active at temperatures up to 120 °C, at pH 0 and 12 and at 1000 bar. In the post-genomic era, different sophisticated “omics” analyses will allow the identification of countless novel enzymes regardless of the lack of cultivability of most microorganisms. Furthermore, elaborate protein-engineering methods are clearing the way towards tailor-made robust biocatalysts. Applying environmentally friendly and efficient biological processes, terrestrial and marine biomass can be converted to high value products e.g. chemicals, building blocks, biomaterials, pharmaceuticals, food, feed and biofuels. Thus, further application of extremophiles has the potential to improve sustainability of existing biotechnological processes towards a greener biobased industry.

Keywords:

Bioeconomy;

biotechnology;

extremophile;

biocatalyst;

digitalization;

bioinformatics; metagenomics

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Introduction In the face of global challenges, a rethinking process was initialized and strategies towards a sustainable biobased industry were developed in several countries across the globe [1,2]. As defined by the European Commission, these challenges are the growing global population, rapid depletion of many resources, increasing environmental pressures and climate change [3]. Further challenges addressed in current literature comprise energy security, food and water security and soil destruction [4]. Therefore, long-term objectives for the transition to a global bioeconomy will be to ensure food and health security, make energy provision more sustainable, make more efficient use of resources and produce new biobased materials [5]. Thus, the ecological acceptability of the whole value chain is one of the key aspects that need to be considered in future designs of bioeconomy. The intended transition from an oilbased to a biobased economy can only be achieved if research centers, universities, politics, industry and civil society are willing to work together on national and international level. An interdisciplinary approach is crucial to develop sustainable and integrative bioeconomy policy strategies [1]. Initial concepts towards greening of industrial processes included the biorefineries, which took traditional oil-based refineries as a model for the development of refineries that use renewable resources as feedstock [6]. In the beginning, starch-containing biomass was utilized for the production of biofuels. First-generation biorefineries thus entailed the discussion whether or not potentially edible biological material should be applied for the production of energy [4]. However, this problem could be solved by switching to lignocellulosic biomass, such as agricultural residues, energy crops and woody materials, as sustainable substrates. Lignocellulosic substrates are the most abundant organic materials on the planet [7]. Besides terrestrial biomass, marine biomass, including micro- and macroalgae, are proposed as renewable resource for a regenerative bioeconomy.

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Among the biggest drawbacks in current application of lignocellulosic feedstock as substrate are the recalcitrance of the material and the resulting necessary pretreatment, respectively. Lignocellulose consists of three main constituents: cellulose, hemicellulose and lignin. The fractionation of these components is essential for an economic processing of the biomass [7]. Although the required fractionation can be achieved by various pretreatment strategies, recalcitrance of lignocellulosic materials remains a problem, since state-of-the-art pretreatment steps are too expensive. The development of efficient, environmentally friendly and low-priced processes for the pretreatment of lignocellulosic materials thus forms a major task on the way towards a broader application of sustainable biorefineries in a future bioeconomy [7]. In this context, the low lignin content of macroalgae and its resulting reduced recalcitrance presents another benefit of this so-far underexploited marine biomass. Other important sectors delivering feedstock for a biobased economy are the food and waste industries. Especially in the food industry enormous amounts of waste streams are generated in various processes. Here, several problems could be simultaneously overcome: generating cheap feedstock for biorefineries, reducing energy consumption and the amount of waste that needs to be deposited. This is in accordance with the principle of the bioeconomy to enable economic growth decoupled from increasing greenhouse gas emissions as major reason for the anthropogenic climate change [4]. Enhanced by a growing public awareness of the climate change and related global problems caused by exploiting the Earth’s limited fossil resources, policy makers have now started to change course and face the challenge to overcome the oil-based industry towards a completely new and sustainable biobased industry as part of a global bioeconomy. Therefore, innovative technologies have to be developed in order to meet the future challenges and contribute to the 17 Sustainable Development Goals (SDGs) of the United Nations [8]. The use of robust biological systems and processes that are provided by extremophilic microorganisms will open new paths in industrial research and application. The exploitation of nature’s biodiversity from exotic habitats will lead to products that are needed for diverse industrial sectors and human health.

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Towards a sustainable biobased industry A sustainable biobased industry (also termed industrial bioeconomy) is thought to play a crucial role during the next decades on the way towards a global bioeconomy (also termed biobased economy, knowledge-based bioeconomy or low-carbon bioeconomy) [2,6,9]. The aspired transition of the industry will be achieved by application of technologies developed in natural and engineering sciences with impact on various sectors [6]. According to the Organization for Economic Co-operation and Development (OECD), bioeconomy is defined as “the application of biotechnology to primary production, health and industry”. Essential for this ongoing development is the understanding of cellular processes down to the molecular level and the application of biomass as renewable feedstock [10]. The potential of the bioeconomy concept is immense. If designed properly, an economy completely relying on renewable resources for the production of materials, chemicals and energy is thought to be one possible solution to the global challenges. Moreover, it is expected to be the answer to the general sustainability problem with regard to environmental and socio-economic needs on a regional, national and international level [11].

The replacement of petrochemical processes by sustainable biotechnological processes was successfully started as biorefineries, e.g. for the production of biofuels and bioplastics. Widening the application of industrial biotechnology will smooth the way towards a competitive bioeconomy [12]. Starting from already existing integrated biorefineries, a stepby-step transition of the oil-based economy will have to be carried out over the next decades. Integrated biorefineries were previously defined as “industrial sites that transform biomass in a sustainable way into food and feed, biomaterials, biofuels and high-value chemical products” [6]. By using the complete biomass for either food or non-food products, a circular principle without waste generation is created that is able to produce a wider range of products than classical, fossil refineries [6]. This principle was applied to produce firstgeneration biofuels and further developed to overcome the resulting food or fuel discussion. In order to produce a wide range of various value-added chemical building blocks and

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materials, second-generation biorefineries will also have to rely on green chemistry processes [13]. However, during the evolution of the biorefinery concepts, synergism remains a central point of the biorefinery within a bioeconomic society [1]. Another principle that needs to be fulfilled on the way to a sustainable circular bioeconomy is to avoid waste. In biorefineries, waste can be utilized as potential renewable feedstock by applying cascades of biotechnological processes, thereby closing the loop from cradle to cradle [14]. Management of biogenic wastes, which is generated from various sectors, such as chemical, paper and textile industries or food and feed production, is not only important with regard to the sustainability paradigm, but also when it comes to securing biological resources for the emerging bioeconomy [15]. Finally, in order to overcome current limitations of the integrated biorefinery approach, a global bioeconomy requires in addition cross-sectional technologies, such as digitalization, logistics, economy and entrepreneurship. Other factors such as knowledge transfer, communication, training, social competences and sustainability are crucial for the implementation of bioeconomy of the future (Figure 1).

Despite the fact that to date more than 30 countries worldwide have developed ideas, strategies or policy agendas towards a bioeconomy, it has to be stated that the bioeconomy is still in its infancy. There are not only different definitions of the bioeconomy, but also varying research focuses [16]. Research in natural and engineering sciences, however, is essential to enable the implementation of a sustainable biobased industry on the way towards a global bioeconomy [17]. Of course, this development needs to be monitored, leading to new challenges related to assessing the bioeconomy efficiency. On the one hand, this is due to the definition problem, which activities and the corresponding data belong to the bioeconomy or the non-bioeconomy [18]. As depicted in Figure 1, various sectors of bioeconomy include agriculture, forestry, fisheries, food, feed, paper and pulp production, chemical, biotechnological and energy industries. And although the bioeconomy is said to be at its beginning, it has already taken on an impact that is hard to neglect: In 2012, more than

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22 million people were employed in the European bioeconomy sector and created a turnover of ca. 2 € trillion [19].

Figure 1: Contributions of extremophiles and their enzymes to bioeconomy

Terrestrial and marine biomass as a major resource shaping bioeconomy As the biobased industry gains momentum, new sectors, such as biomaterials and green chemistry, are currently developing [20]. Besides the impact of biotechnology on agricultural production and processing industries as one of the major drivers of the bioeconomy, the new sectors are now becoming further drivers shaping the bioeconomy [21]. Among others, this leads to an increased demand for renewable biomass as sustainable substrate [20]. Therefore, new challenges arise from an intensified biomass production, e.g. mass growth of fast-growing crops with the concomitant problems of monocultures. One proposed solution is biomass production in semi-natural plant communities by applying a multifunctional agriculture [22]. Equally important are soils for the development of a sustainable bioeconomy, since a bioeconomic society depends on fertile soils that are not readily exploited, but subjected to a sustainable long-term utilization [23]. Again, different drivers of the bioeconomy, such as food security and environmental conservation, are likely to end up competing for biomass as resource. It is important to analyze these trends and to develop technical strategies and integrated solutions in order to secure a sustainable biomass supply within the growing value chain nets of an emerging bioeconomy [16]. In this context, the development of standardized life cycle assessment methods for the analysis of bioeconomy value chains appears to be inevitable [19]. This is also true for developed regions, which already exhibit a high land footprint, such as the European Union (EU). Without a functional monitoring system, a growing bioeconomy in the EU is likely to increase land-use pressures abroad [24].

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The land footprint of the EU bioeconomy is one example for the narrow path towards a sustainable biobased industry, which needs to carefully balance environmental and economic needs. However, there are several other aspects concerning the transition to a bioeconomy that are currently subject to controversial discussions. General criticism was mentioned with regards to recent conceptualizations of the bioeconomy, which were found to contain ambiguities, difficult assumptions and contradictions from an political-economic point of view [25]. Another criticism is that several competing visions, e.g. the agri-biotechnology, are trying to establish their technology-driven program towards an agricultural industry rather than enabling alternative so-called agro-ecological approaches [26-28]. In Europe, agribiotechnology has been widely refused by the public with genetically modified (GM) food being a symbol for fears related to threats like economic globalization and genetic pollution [29]. In addition to terrestrial biomass, another important resource for renewable carbon for the development of biobased economy consists of macro- and microalgae. Macroalgal seaweeds such as brown, green and red algae contain valuable polymers such as alginate, laminarin, fucoidan, xyloglucan, carrageen as well other products such as proteins, enzymes, nitrogen and phosphorous. Microalgae are a valuable resource for the synthesis of triglycerides, phospho- and glycolipids, carbohydrates and proteins as well as high-value products, e.g. carotenoids, phytosterols and antibiotics [30].

On the way towards a sustainable biobased industry, food and non-food productions chains will depend on biotechnological processes, which apply genetically modified organisms (GMOs) in order to be sustainable. Thus, bioeconomy might receive a similar negative public attention as agri-biotechnology [31]. These societal concerns have to be taken seriously, because they are likely to become a major drawback for bioeconomic products entering the market. The end consumer’s lacking trust in novel bioproducts can only be counteracted by providing the public with the required knowledge to understand the benefits of the bioproducts. This means that transparency and participation from the beginning will ensure

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an improved communication between bioeconomy stakeholders and the public and guarantee the end user’s acceptance of novel biobased products [2].

Extremophiles as a toolkit for bioeconomy Organisms, which are able to thrive under conditions that diverge from our human conditions, are defined as extremophiles. The name is derived from the observation that they literally seem to “love the extreme”, i.e. their preferred conditions for growth and metabolic activities are “extreme” from a human perspective. Vivid examples for extremophiles are organisms growing at elevated temperatures, so-called thermophiles. The latter can be further distinguished according to their preferred temperatures: Generally, only thermophilic prokaryotes are found at temperatures exceeding 62 °C. Moreover, hyperthermophiles, whose preferred growth temperatures lie above 80 °C, consist mostly of Archaea. However, Bacteria are also found that grow at temperatures up to 100 °C. The most extreme thermophile currently known is Methanopyrus kandleri. It was found to grow at temperatures up to 122 °C [32]. Another example for organisms loving extreme conditions is growth at pH values significantly differing from a neutral range. At an acidic pH around 3, acidophiles were described from all three domains of life. Alkaliphiles on the other hand show growth at pH values up to 12. Other extremes include preferred growth at temperatures below 0 °C, at high salt concentrations or high pressure as well as resistance to high radiation doses or tolerance towards high solvent concentrations. Often, extremophiles are identified, which thrive under more than one extreme condition. For instance, the combination of acidic and high-temperature conditions is found in so-called thermoacidophiles, such as the Archaeon Picrophilus torridus, which is able to grow at pH 0 and 65 °C [33]. Thermoalkaliphiles such as Anaerobranca gottschalkii grow optimally at 60 °C and pH 9. All of these biological systems are ideal candidates as source of robust cell factories for countless industrial applications.

In order to enable growth under these harsh conditions, the cells have come up with several adaptations, which have been only partly understood to date. Besides adaptations of

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membrane components and the production of protective molecules, such as compatible solutes, especially extracellular enzymes of extremophiles have to be stable and active under the inhospitable conditions of their exotic habitats. In general, no single mechanism has been identified so far, which could explain the extreme properties of enzymes from extremophiles, so-called extremozymes. It has been proposed, however, that a charged outer surface and stable ion bonds could account for thermostability of extremozymes. Furthermore, stability towards elevated temperatures might be supported by an increased amount of acidic and basic amino acids leading to a tightly packed enzyme core [34]. The resulting superior properties of extremozymes allow for a wide range of reactions beyond the borders of conventional biocatalysis, such as biocatalytic processes in non-aqueous solutions, in the presence of high solvent concentrations, at high pressures, extremes of pH or temperatures up to 140 °C [35]. Since extremozymes already exhibit many of the features, which are usually aimed at by various random or directed protein-engineering approaches, they have a great potential for application in industrial biotechnology. Moreover, they are an excellent starting point for further improvements of desired enzyme properties including stability, stereo- and regioselectivity, tolerance towards solvents or improved substrate affinity. A novel approach within the field of synthetic biology is the incorporation of noncanonical amino acids into enzymes, resulting in so-called congeners. Due to their fraction of synthetic amino acids, these congeners have been found to exhibit superior activity and stability spectra than the respective wild-type enzymes [36,37]. Although these experiments are currently performed with suitable auxotrophic E. coli strains, we assume that similar stains could be designed based on Thermus and Sulfolobus species. Concluding, the application of synthetic biology approaches like directed evolution, DNA shuffling, gene fusions, rational protein design or the incorporation of non-canonical amino acids into extremozymes as robust starting points offers great potential for the design of tailor-made biocatalysts for application in environmentally friendly processes in various industrial sectors [38-40].

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Extremozymes and biomass bioconversion A major field of application for extremozymes is biomass conversion in integrated biorefineries. In order to avoid the food versus fuel debate, non-edible lignocellulosic biomass, such as rye straw, was also applied as resource for the production of high-value products and biofuels [41]. However, as previously discussed, the recalcitrance of lignocellulosic materials necessitates various pretreatment procedures to fractionate and utilize its three components cellulose, hemicellulose and lignin [42,43]. Further enzymatic processing then depends on the selected fraction: Cellulose consists of D-glucose monomers linked by β-1,4-glycosidic bonds, whereas hemicellulose is a heterogeneous polysaccharide made up of β-1,4-glycosidic linked D-xylose units and other sugars including D-glucose, D-arabinose, D-mannose and D-galactose. These monomers are valuable starting materials for polymer production, building blocks for pharmaceutical precursors or substrates for fermentation processes. Therefore, stable cellulases and hemicellulases, respectively, can be applied for polysaccharide degradation to oligomers and monomers [44]. The third lignocellulosic component, lignin, has a very complex structure, which contributes most to the recalcitrance of this biomass. It can, however, be degraded to valuable aromatic compounds by the concerted action of oxidative enzymes, such as laccases, peroxidases and oxidases [45]. Efficient pretreatment approaches are now under development, which aim at combining hydrothermal procedures at elevated temperature and pressure with the benefits of enzymatic degradation processes [46]. This process combination has several benefits including energy savings by omitting cooling steps and improved processing possibilities at high temperatures, e.g. enhanced substrate accessibility at simultaneously reduced viscosities. However, such an advanced pretreatment procedure can only be feasible when applying thermostable extremozymes, since commercially available enzymes from mesophilic organisms are not suitable for a targeted continuous processing of lignocellulosic biomass in an integrated biorefinery at high temperatures [47]. The latter also offers

great

potential

for

the

development

of

metabolically

engineered

robust

microorganisms. At the envisioned high-temperature processes, the contamination risk with

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potentially pathogenic microorganisms can be significantly reduced. Moreover, with enzymes still being the most efficient, but also most expensive means in second-generation biorefinery concepts, there is a huge potential for process improvement using extremophiles and their enzymes.

Ideal extremozymes for application in a sustainable biorefinery with lignocellulosic waste material as feedstock should exhibit high specific activities at high temperatures combined with superior thermostability. Several enzymes with these desired properties have already been described and a selection of extremozymes with high potential for application in industrial processes is assembled in Table 1. Examples for thermoactive and thermostable cellulases (endoglucanases, cellobiohydrolases and β-glucosidases) comprise extremely stable endoglucanases from Dictyoglomus thermophilum and from an archaeal enrichment culture [48,49]. Hemicellulases (endoxylanases and β-xylosidases) with similar extreme properties were recently identified in different Thermotoga spp. and Acidothermus cellulolyticus [50,51]. Thus, biomass utilization for the integrated production of chemicals, polymers, energy carriers and biofuels based on lignocellulosic resources will be taken to the next level by development and implementation of tailor-made extremophiles and their robust biocatalysts [52].

Although the competition with the food and feed industry has led to neglect of the starchbased biorefinery for the production of first-generation biofuels, starch degradation for food technologies is another important sector for the application of extremophiles and extremozymes. In contrast to lignocellulose, starch consists of α-1,4-linked glucose molecules, which either form linear amylose or the branched amylopectin polymer, the latter of which also containing α-1,6-linked glucose moieties. Similar to second-generation biorefineries, starch-containing biomass needs to be treated with synergistically acting glycoside hydrolases. The required α- and β-amylases, glucoamylases, α-glucosidases and pullulanases have also been identified from extremophilic microorganisms [40,53]. However,

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traditional starch processing to glucose applying liquefaction and saccharification currently requires an intermediate cooling step due to usage of amylolytic enzymes derived from mesophilic organisms [54,55]. This process could be significantly improved by utilizing thermostable starch-hydrolyzing enzymes without cofactor requirement [56,57]. As indicated in Table 1, several amylolytic extremozymes have already been described, including extremely heat-active α-amylases from Pyrococcus furiosus and Methanococcus jannaschii as well as other industrially relevant α-amylases of extremophilic origin [58-61]. Together with a recently described thermoactive pullulanase from Thermococcus kodakarensis KOD1, many options seem to be available for creating more sustainable starch processes based on extremozymes [62]. Additionally to the aforementioned applications in biorefineries of the first and second generation, there are several other possible areas of application for robust biocatalysts. The most prominent one, Thermus aquaticus DNA polymerase, has brought molecular biology to a new level. However, with today’s need for biomass diversification, extremozymes will gain increasing importance in the modification of various waste streams as so-far underexploited biomass resource, such as chitin or pectin.

Chitin is a structural polysaccharide found in fungi, insects and crustaceans. It consists of Nacetylglucosamin molecules linked by β-1,4-glycosidic bonds. Recently reported chitinolytic extremozymes were proposed to be applicable as biofungicides and bioinsecticides [63,64]. Further possible fields comprise enzymatic chitin-based fertilizer production, e.g. from marine waste [65,66]. Pectin is another structural polysaccharide and the respective degrading enzymes have promising applications in different industrial sectors ranging from beverage production to wastewater treatment [67].

Further waste streams, especially from the food industry, contain proteins and lipids. Proteases are widely applied in the leather, textile, detergent and food industry as well as for pharmaceutical applications, and many proteolytic enzymes have been subjected to

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extensive protein engineering [68]. Here, cold-active enzyme candidates are essential for energy savings during textile cleaning at reduced temperatures [69]. On the other hand, there is also a need for thermoactive and heat-stable proteases, which are able to withstand harsh process conditions [70]. Lipids as waste can be bioconverted by lipolytic enzymes, i.e. esterases hydrolyzing short-chain acyl esters or lipases catalyzing the hydrolysis of longchain acyl esters. Lipolytic enzymes are used at low temperatures for the synthesis of pharmaceutical components and other temperature-sensitive building blocks, thus exploiting their enantio- and regioselectivity for the production of high-value chemicals [71,72]. Thermostable lipases have also been tested as model extremozymes for innovative improvements by incorporation of non-canonical amino acids resulting in increased enzyme activity and a broadened substrate spectrum [37].

All in all, there are versatile fields of industrial applications for a wide range of extremozymes. Future challenges will be to meet the demand for new tailor-made stable enzymes by identifying novel biocatalysts from the vast potential of uncultivable microorganisms through utilization of “omics” technologies. Other tasks will comprise the development of suitable production systems for these extremozymes and, furthermore, sophisticated proteinengineering methods for further adjusting the robust biocatalysts to the desired industrial applications on the way towards a sustainable biobased industry.

Table 1: Selected extremozymes with high potential for industrial applications (adapted from [73])

Genomics of extremophiles as megatrend for a sustainable bioeconomy In addition to the unique biological systems available on our planet, further cross-sectional technologies are needed to develop a sustainable bioeconomy. The trend towards a greener or biobased industry depends heavily on the digitalization, representing another important transformation that took place within industry and economy. The term digitalization is

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ubiquitous and includes key aspects like the common usage of digital content and automatic workflows as well as the usage of global acting networks traversing modern societies and affecting science, culture, media and security. Developments reflecting the enormous significance of digitalization in science are the so-called “omics”, including (meta)-genomics, (meta)-transcriptomics,

(meta)-proteomics

or

metabolomics

[74-78].

These

“omics”

technologies depend on the storage, processing and analysis of large datasets enabled by the advent of digitalization in sciences. Thus, none of these technologies could have been realized without the digital revolution including computer systems with high performance processors and high capacity memory storages as well as the inventions of next-generationsequencing (NGS) technologies. In this case, both the scientific as well as the technological improvements went side by side and were mutually supportive. The increased impact of digitalization resulted in a decrease of sequencing costs and an increased amount of produced data (big data) [79]. The challenge to deal with these enormous amounts of datasets has led to the evolution of novel research fields, such as bioinformatics or research data management, that have been steadily growing over the last decade. The sequencing of whole genomes of various extremophilic microorganisms in the last two decades contributed dramatically to the understanding of the strategies how extremophiles survive in extreme environments such as hot springs, solfataric fields and saline lakes or at high pressure in the deep ocean. Furthermore, insight could be obtained on their metabolic pathways, bioconversion of various substrates, transport mechanisms, DNA processing, enzymology and energetics.

Over 120 genomes of hyperthermophiles have been completely sequenced and are publicly available. Interesting strains belonging to extremophilic Archaea and Bacteria, that grow at elevated temperatures have been studied in detail such as the genera Pyrococcus, Thermotoga and Thermus. Genomes from other thermophiles that are also adapted to life at high pH (Anaerobranca gottschalkii) or low pH (Sulfolobus sp., Thermoplasma sp. and Picrophilus sp.) deliver enzymes that are robust under various extreme conditions and are

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suitable for industrial application. Another example of an attractive candidate for application is Bacillus halodurans, which produces enzymes that are active at extreme alkaline conditions (pH>11). Furthermore, these technologies will lead to the identification of further novel metabolic pathways, which can be used for the generation of new synthetic metabolic pathways with optimal performances on biodegradable substrates representing a driving force towards a sustainable white biotechnology.

Genomics for extremozyme discovery In addition to the knowledge gained on the survival strategies of extremophiles, genome sequencing provides us with a huge number of genes that encode extremozymes, which are of interest for basic as well as applied research. Research in this field shall yield better insight into the mechanism of action of robust enzymes and deliver information on the secrets behind the 3D structure of extremozymes. The combination of computational and structure-based analysis with evolutionary driven approaches, such as directed evolution or synthetic biology, has been significantly accelerated by the identification of novel extremozymes with high potential for industrial applications in the last years [36,80-82]. In order to achieve this development various kinds of screening approaches, including sequence-based or activity-based (function-based) studies, have been established. In activity- or function-based screening approaches the identification of novel enzymes is performed by high-throughput methodologies using dye-coupled substrates, protein chip analysis, single cell fluorescent assays or complementation studies of process-specific mutants [83-87]. Especially with entering the post-genomic era and the availability of lots of sequence

information

in

public

databases,

sequence-based

approaches

using

metagenomics, metatranscriptomics or metaproteomics will dramatically accelerate the discovery of novel biological systems [61,74,82,88-92].

For the heterologous production of extremozymes in larger amounts, various mesophilic or thermophilic hosts including Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris,

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Thermus thermophilus or Sulfolobus solfataricus have been successfully used [93-99]. The production in bulk quantities is crucial to implement a sustainable bioeconomy strategy. The potential application of extremophiles in biofuel synthesis has been explored and an overview of the different biofuels is given by Barnard et al. [100].

Metagenomics and analysis of microbial communities Metagenomics is defined as a genomic analysis of all microorganisms that are present in an environmental sample under study [76]. Therefore, DNA from extreme habitats can be cloned directly into various kinds of cloning plasmids, i.e. phagemids, fosmids or bacterial artificial plasmids (BACs). Since more than 95 % of microorganisms are unculturable the advances in the field of genomics had significant impact on understanding microbial physiology in extreme habitats. Applying the metagenomic approach it is now possible to analyze the microbial communities in these habitats and study the influence of environmental factors such as changes in temperature, pH, salt concentration on the diversity of extremophilic Archaea and Bacteria. These studies have led to new insights into microbial metabolism and evolutionary relationships in a microbial community. Beginning in the early 2000s, metagenomic analyses have evolved from the pioneering works of Venter, Torsvik and colleagues to a well-established and commonly used methodology to describe microbial ecosystems, including extreme environments, such as volcanic areas, salterns or antarctic soils [101-106].

In addition to the question which microorganisms are present in a metagenome sample, further industrially relevant topics, e.g. what kind of changes or shifts microbial communities undergo during fermentation processes or what kind of starting cultures provide an optimal distribution and adaption to the corresponding industrial process, were investigated by metagenomics. Such approaches were reported for various industrial applications including biogas production or food fermentation processes [75,107,108]. A deeper understanding of the influence of environmental conditions on microbial communities, the microbial adaptation

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and the underlying regulation processes can be gained by the combination of metagenomics with expression studies like metatranscriptomics or metaproteomics. The latter ones enable studies of transcribed genes and translated proteins, respectively, at a specific time point and in response to a given environment. Here again, the development of NGS technologies has brought these studies to the next level with regard to the direct sequencing of RNA, lacking the previously needed cDNA synthesis or cloning steps and resulting in an increasing amount of datasets [109,110]. Hence, the combination of these methodologies ensures a deeper understanding of functional and regulating networks in the cells of each individual microorganism within the microbial consortium of interest [78,104,111]. Moreover, the provided information on gene, transcript or protein level can be used for the identification of novel targets or for metabolic engineering of industrial production strains [91,112]. Therefore, pre-existing pathways can be improved by protein engineering or novel ones can be built from scratch, which are most suitable for the corresponding industrial process. Furthermore, high-throughput screening approaches for the identification of mutated variants or improved production strains have been successfully established [82,113-116].

Metagenomics for discovery of novel extremozymes Screening of metagenomic libraries from extreme ecological niches enables the identification of robust enzymes possessed by non-cultivable microorganisms that represent the majority of all existing microorganisms [117,118]. Activity- or function-based approaches allow the identification of promising active enzymes without knowing their coding sequences. Disadvantages linked to function-based approaches are the dependency on cultivable microorganisms and resulting low expression levels in the heterologous hosts. In contrast to this, shotgun metagenomics can be performed, during which the environmental DNA is sequenced directly from the sample, skipping any cloning steps and resulting in whole sequences of protein encoding genes. This allows a functional description of large parts or complete genomes of each microorganism that is present in the studied consortium. These new metagenomic methods depend on the possibility to store, process and analyze vast

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amounts of data, which could only be achieved by digitalization in sciences. Therefore, metagenomics is a great example of how the digitalization has accelerated scientific progress in the field of extremophiles. On the other hand, it is interesting to note that extremozymes, e.g. DNA polymerases from the thermophiles Thermus aquaticus and Pyrococcus furiosus, have revolutionized life sciences during the last three decades. Hence, various industrially relevant extremozymes like DNA-modifying enzymes, lipases, esterases, proteases, amylases, cellulases have been discovered by using metagenomics, thus showing its importance in the search for novel biocatalysts and, consequently, for a sustainable industry [74,119-125]. Moreover, since 2010 several hundred so-called “commercial useful enzymes” (CUEs), have been made publicly accessible by the “MetaBioMe” database, all of which were identified by metagenomics techniques. Classification of these CUEs was performed into nine application categories, specified as agriculture, energy, food and nutrition, biotechnology, biosensor, environment and health [126]. Crucial requirements for shotgun metagenomics are bioinformatics tools to assemble the generated short reads to continuous sequences (contigs or scaffolds) after sequencing, thereby enabling gene prediction and functional assignments of the metagenomic sequences. Especially the reconstruction of each individual genome sequence within a metagenome requires the complex bioinformatics workflows successfully developed in the last years [77,126-129]. Altogether, the adoption of the reported trends into industrial applications offers a promising way to switch from conventional processes to more biobased processes leading towards an industrial revolution and into a sustainable technology landscape.

Conclusions and future perspectives Global challenges including the growing population, depletion of fossil resources and climate change demand concerted actions from various players from politics, academia, industry and civil society. The general goal is to apply an integrated approach in the transition of the oilbased economy towards a sustainable economy completely relying on biomass derived from

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terrestrial and marine sources as renewable feedstock. In order to fulfill the demand for industrial sustainability, for instance by considering social and environmental aspects besides economic facets in industrial processes, extremophiles and their enzymes have been identified as suitable means for a greener chemistry. In the post-genomic era, different sophisticated “omics” analyses allow the identification of novel enzymes regardless of the lack of cultivability of most microorganisms. Furthermore, elaborate protein-engineering methods are clearing the way towards tailor-made robust biocatalysts. Thus, further application of extremozymes has the potential to develop novel biotechnological processes and products for a sustainable biobased industry. In order to overcome current limitations of the integrated biorefinery approach, however, a global bioeconomy requires in addition cross-sectional technologies, such as digitalization, robotics, logistics and economy. Other factors such as knowledge transfer, entrepreneurship, communication, training, social competences and sustainability are crucial for the implementation of bioeconomy of the future. However, a smooth transition can only be accomplished when paying attention to lessons learned from the past, such as avoiding future conflicts between food and fuel and avoid onesided technology-driven approaches. Again, lessons learned from lacking public acceptance of agri-biotechnology and its products should be kept in mind when promoting a biobased industry [11]. As the success of the bioeconomy will depend on the public acceptance of its products, it has been suggested to support the market introduction of biobased products by establishing certifications, quality labels and education campaigns [2]. All in all, besides the technology-based innovations at the center of the ongoing transformation process, it appears to be essential to combine sustainable management practices with a circular economy approach to meet environmental, economic and social needs [6].

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Figure Caption Figure 1: Contributions of extremophiles and their enzymes to bioeconomy. Cross-sectional technologies and other factors influencing a new emerging bioeconomy.

28

Table Table 1: Selected extremozymes with high potential for industrial applications (adapted from [73]) Enzyme (source/organism)

Topt (°C)

pHopt

Specific activity (U/mg) or unique property

Reference

α-Amylase (Halorubrum xinjiangense)

70

8.5

487

[130]

α-Amylase (pilot-plant biogas reactor)

80

7.0

1,000

[61]

α-Amylase (Pyrococcus woesei)

100

5.5

Inactivation by autoclaving for 8 h at 120 °C

[131]

Pullulanase (Thermococcus kodakarensis KOD1)

100

5.5-6.0

Application in maltose syrup production

[62]

Pullulanase (Thermotoga neapolitana)

80

5.0-7.0

25

[132]

Pullulanase/Amylase (Thermococcus kodakarensis KOD1)

100

5.6-6.0

118

[62]

Glucoamylase (Picrophilus oshimae)

90

2.0

Thermoacidophilicity

[133]

α-Glucosidase (Geobacillus toebii E134)

70

6.8

5

[134]

β-Amylase (Salimicrobium halophilum LY20)

70

10.0

573

[135]

CGTase (Anaerobranca gottschalkii)

70

8.0

Application in βcyclodextrin production

[136]

Cellulase Cel12E (archaeal metagenome)

90-95

5.5

692

[137]

Endoglucanase (Alicyclobacillus vulcanalis)

80

3.5-4.5

Application in secondgeneration biorefinery

[138]

Endoglucanase (Archaeal enrichment)

109

6.8

4

[49]

Endoglucanase (Dictyoglomus thermophilum)

60-85

5.0

5

[48]

29

β-Glucosidase (Thermotoga thermarum DSM 5069T)

90

4.8

142

[139]

Endoxylanase (Acidothermus cellulolyticus 11B)

90

6.0

350

[50]

Endoxylanase (Thermotoga thermarum)

95

7.0

146

[51]

Endoxylanase (Thermotoga petrophila)

95

6.0

2,600

[140]

Chitinase (Sulfolobus tokodaii)

70

2.5

0.08

[63]

Chitinase (Bacillus thuringiensis subsp. kurstaki)

110

9.0

4.7

[64]

Chitinase (Thermococcus chitinophagus)

70

7.0

Heat-resistance

[141,142]

Pectinase (Thermotoga maritima)

80

6.4

Application in industrial food processing

[143]

Lipase (Thermoanaerobacter thermohydrosulfuricus)

75

8.0

12

[144]

Lipase (Metagenomic enrichment culture)

70

8.0

12

[119]

Protease (Coprothermobacter proteolyticus)

85

9.5

4

[70]

30