Algal Research 56 (2021) 102288

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Algal Research

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Review article Algal cellulose, production and potential use in plastics: Challenges and opportunities

Enio Zanchetta a, Eya Damergi a,b, Bhavish Patel b,*, Tobias Borgmeyer a, Horst Pick a, Adrian Pulgarin a, Christian Ludwig a,b a Ecole Polytechnique F´ed´erale de Lausanne (EPFL), School of Architecture, Civil and Environmental Engineering (ENAC), Environmental Engineering Institute (IIE), Ludwig Group (GR-LUD), Station 6, CH-1015 Lausanne, Switzerland b Paul Scherrer Institute (PSI), Energy and Environment Division (ENE), Bioenergy and Catalysis Laboratory (LBK), Chemical Processes and Materials Group (CPM), CH-5232 Villigen PSI, Switzerland

ARTICLE INFO ABSTRACT

Keywords: The development of new biodegradable plastic materials from renewable sources is a major challenge for plastic Bioplastic industries to provide sustainable alternatives to petroleum plastic. Bio-based plastics are generally obtained from Algae photosynthetic biomass such as higher plants, crops and more recently algae. This review aims to summarise the Nanocellulose current state of bioplastic production in general with particular emphasis on algal cellulose and its derivatives Cellulose (nanocellulose) for bioplastic applications. Despite the potential of algae as a feedstock for nanocellulose, little Biorefinery information is available on strain selection regarding cellulose content and downstream processing. This study lists possible optimization opportunities to increase the cellulose yield of algal biomass and the current status of its conversion to nanocellulose. Moreover, the findingsof this review provide insight into existing knowledge and future direction in the algal cellulosic bioplastic domain based on algal bioplastic life cycle assessment studies. Finally, the review gives an overview of the main standards used for biodegradability certifications in view to limit the access to the biodegradable label when the required quality is not reached.

1. Introduction the environment directly or indirectly, e.g., oceans, non-controlled landfills [2]. Due to the disparity and lack of efficient plastic waste It is incontestable that plastics play an important role in our modern management infrastructures, and the so-called “throw away mentality”, society. Unfortunately, the social and economic benefitsof plastics come 8 million tons (Mt) of plastic waste end up in the ocean annually [3]. In with severe environmental detriment [1]. Since its commercial devel­ addition, the greenhouse gas (GHG) emissions from the plastic lifecycle opment in the 1950s, fossil-based plastics have caused serious envi­ contribute up to 15% towards the total global carbon emission [2]. ronmental problems leading to the onset of an imbalance on the Under the effect of abiotic factors, littered plastics tend to decompose equilibrium of our ecosystem. In 2018, the plastic industry produced slowly into plastic debris. Subsequently, the formed microplastic parti­ 359 million metric tons (Mt) of plastic, nearly half of which was utilised cles (sizes from 1 mm to 1 μm) extensively pollute our marine envi­ for single-use and packaging material. Moreover, only 25% of plastic ronment at all levels e.g., smothering marine organisms that make up waste generated was recycled, 22% being disposed to landfills and/or the base of our food chain [4]. If the current strong growth of plastic incineration while the remaining 42% is assumed to have been lost into usage continues as expected, by 2025, the ocean will accumulate 1 ton of

Abbreviations: ABA, abscisic acid; BR, brassinosteroids; CesA, cellulose synthase; CF-PBR, Circular flow photobioreactor; CK, cytokinins; CNC, cellulose nano­ crystals; CNF, Cellulose nanofibrils; ETH, ethylene; GA, gibberellins; GHG, greenhouse gas; GOBASE, Organelle Genome Database; IAA, indole acetic acid; IBA, indolebutyric; IM, indomethacin; LCA, life cycle assessment; MFC, microfibrillated cellulose; Mt, metric tons; NAA, napthylacetic acid; NC, nanocellulose; NCBI, National Center for Biotechnology Information; NFC, nanofibrillatedcellulose; NGOs, non-governmental organizations; OPR, open pond reactor; PBAT, polybutylene adipate terephthalate; PBR, photobioreactor; PBS, polybutylene succinate; PCL, polycaprolactone; PHAs, poly-hydroxy-alkanoates; PLA, poly-lactic acid; PVA, polyvinyl alcohol; SA, salicylic acid; SDGs, sustainable development goals; SL, strigolactones; TAG, triacylglycerol; TCs, terminal complexes; UN, United Nation; WVP, water vapour permeability. * Corresponding author. E-mail address: [email protected] (B. Patel). https://doi.org/10.1016/j.algal.2021.102288 Received 31 July 2020; Received in revised form 26 March 2021; Accepted 27 March 2021 Available online 22 April 2021 2211-9264/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). E. Zanchetta et al. Algal Research 56 (2021) 102288 plastic for every 3 tons of fish, and by 2050, the mass of plastics in the 2018 [7]. ocean will be greater than that of fish [5]. These are some serious In order to progress from non-renewable and non-recyclable plastics environmental repercussions that will without doubt cause irreversible to cost-efficient bio-based materials with reduced environmental foot­ and unmanageable damage to marine life, and consequently human life prints, it is envisioned that new bioplastic materials should be aligned too. with the United Nation’s (UN) sustainable development goals (SDGs) Under pressure from non-governmental organizations (NGOs) and [8]. awareness of consumers, many companies that utilise plastics in their By definition, bioplastics are plastics made from organic renewable products are now looking at their plastic policy in terms of recyclability sources such as terrestrial crop biomass, agricultural waste and fast- and pushing towards a new generation of easily recyclable products with growing microorganisms. The term bioplastics can be misleading as it view of “Design to recycle”. However, plastic recycling is not such a can refer to plastic material that is either biodegradable or bio-based or straight-forward process as it requires complex infrastructure and both as shown in Fig. 1. It should be noted that biodegradable plastic diverse waste-streams to separate different types of plastics that cannot may not necessarily be bio-based and vice versa. For instance, poly­ be melted together. Without separating the plastic according to their butylene succinate (PBS), polybutylene adipate terephthalate (PBAT) chemical composition, the resulting material will exhibit structural and polycaprolactone (PCL) are biodegradable plastics obtained from weaknesses rendering the recycled product with limited application. fossil sources. Although biodegradable fossil-based polymers look Furthermore, recycling can become counterproductive when biode­ interesting as an alternative, their production from petrochemicals dis­ gradable and non-biodegradable plastics are not differentiated during qualifiesthem from carrying the label of CO2 neutral. Other plastics are the separation and recycling process. Plastic polymers are generally referred as oxo-degradable due to the addition of some chemicals that blended with other functional additives such as antistatic agents, dyes, accelerate their degradation (prooxidant additive) [9]. Nevertheless, stabilizers, flame retardants, etc., to improve their performance (flexi­ oxo-degradable plastics are far from being a sustainable solution for the bility, resistance, etc.). These chemicals create additional recycling environment. Incorporated additives are generally insufficient to pro­ problems as some of them are known to be toxic and not compatible for vide complete mineralization of the plastic, thus the non-decomposed all applications. Moreover, some plastic additives are known to exert fraction will contribute to microplastic pollution [10], and still persist endocrine-disruptive activities that cause reproductive disorders in in the environment. Unfortunately, biodegradable bioplastics generally human and animals and can be responsible for an increased risk of possess poor mechanical properties such as tensile strength, oxygen and cancer development [6]. A list of the most commonly used additives in water permeability which are relevant parameters to determine the plastic materials and their functionalities is outlined in Hahladakis et al. suitability for different applications [11]. In order to meet the minimum

Renewable raw materials

Bio-based Bio-based + Biodegradable Biopolymers Biopolymers Bio Polyethylene (Bio-PE) Polylacc acid (PLA) Bio-Polyethylene terephthalate Polyhydroxyalkanoates (PHA) (Bio-PET) Starch Bio-Polyvinyl chloride (Bio-PVC) Cellulose Nanocellulose (NC)

Non-Biodegradable Biodegradable

Convenonal polymers Biopolymers Polyethylene (PE) Polybutylene adipate terephthalate Polypropylene (PP) (PBAT) Polyethylene terephthalate (PET) Polybutylene succinate (PBS) Fossil-based Polycaprolactone (PCL) + Biodegradable Non-Biodegradable

Fossil-based raw materials

Fig. 1. Example of bioplastics currently utilised, segmented by feedstock type and biodegradability. Adapted from [18].

2 E. Zanchetta et al. Algal Research 56 (2021) 102288 application requirements without compromising the biodegradability of biomass and this includes cellulose and NC. It is envisioned that the bioplastics, renewable additives should partially be accounted cellulose-based bioplastic derived from algal biomass could substan­ within the novel material in order to meet the current expected per­ tially minimise the impact on the environment and food supply formance standard of bioplastics. compared to older generation feedstock, particularly ones associated Bio-based and biodegradable biopolymer such as cellulose, starch, with lignocellulosic material. In some cases, highly pure cellulose can be alginate, chitin, polylactic acid (PLA), chitosan, and their derivatives, extracted from algal biomass. This could reduce the cost allocated for revealed to be promising candidates with regards to their abundant the chemical treatments needed for the removal of lignin and hemi­ availability from numerous resources and their biodegradability [12]. celluloses as in the case of wood-derived cellulose. Among them, cellulose is by far the most abundant biopolymer obtained It is understood that the route forward for a truly renewable, bio- from nature and present in almost all photosynthetic organisms such as based bioplastic will come in the form of utilising a 3rd generation plants, algae, tunicates, as well as some bacteria. Moreover, techno­ feedstock. Ultimately, CNF/NFC/MFC based material production is logical advancement can now produce advanced cellulosic biomaterials sufficientlyadvanced to be produced at the technical level, but the lack by manipulation of native cellulose fibreat the nano scale. In particular, of a novel feedstock has led to a bottleneck. Microalgal biomass has clear cellulose fibrehas outstanding strength properties with high stiffness (an advantages as highlighted above to fulfil this gap but, there is a severe elastic modulus up to 220 GPa) and tensile strength (around 3 GPa), lack of understanding in the fieldof utilising it for bioplastic production. light weight and low density (around 1.6 g⋅cm 3) that can be used to Attempts to produce bioplastic from algal nanocellulose (CNC or CNF) improve the mechanical properties of bioplastics without affecting the are very rare. Only one recent study reported the use of algal CNC (red biodegradability process [13]. Depending on the extraction and con­ algae waste from agar production) as reinforcing filler in polyvinyl version procedure applied on the cellulosic biomass, two types of alcohol (PVA) or chitosan-based bio-composite films[ 19]. In the case of nanocellulose (NC) can be extracted from cellulose: cellulose nano­ PVA, the addition of algal CNC (8 wt%) improved the tensile strength crystals (CNC), and cellulose nanofibrils(CNF). The latter is also called and elastic modulus of the composite by a factor of three while only nanofibrillated cellulose (NFC) or microfibrillated cellulose (MFC) minutely affecting the transparency of the film.Other studies suggested depending on the field of application. that CNC produced from Posidonia sp. [20,21] or CNF from Nanno­ These nanocellulosic materials exhibit interesting mechanical prop­ chloropsis sp. [22] could also have interesting properties to be used as erties, which are different than their cellulosic precursors present in the reinforcing filler for fibres-reinforced composites. However, the use of source biomass material, and thus provides an opportunity to explore algal CNC or CNF should not be limited solely as reinforcing additives in their properties as novel biomaterials for potentially wider bioplastic other polymer matrices. CNC and CNF can be used directly to produce applications. Several studies have shown that NC can enhance the me­ self-standing films. Usually, CNF-based films show similar barrier chanical properties of bioplastic when coated or incorporated with other properties (to oxygen or water vapour) than conventional petro-based biopolymers. For instance, when NC was added in the formulation of plastics (e.g., cellophane), and improved barrier properties (e.g., to starch-based edible films bioplastic for food packaging application, the oxygen) compared to CNC-based films, thus better suited for applica­ result showed an improvement in water vapour permeability (WVP) and tions such as packaging [23]. Algal CNF seems to have a substantial, and the mechanical strength of the film [14]. Despite the useful features of unexplored potential for use in the production of high-performance NC, its production is energy-intensive, consisting of different mechani­ bioplastics. Given the plethora of algal species available, the different cal and chemical steps to remove lignin and defibrillatecellulose fibres. cultivation options/conditions, extraction/conversion technologies as As a consequence, this high energy consumption remains a barrier to­ well as end of life choices, the task associated with understanding the wards commercialisation of NCs [15]. gaps will be based on further research. As such, the following sections Based on the original source of the biopolymer, we can distinguish will provide a holistic overview of the important advancements made in three generation of bioplastics: 1st generation bioplastics derived from the field associated with the aforementioned unit operations, ranging agricultural crop-based feedstocks (corn, soy, sugar cane) and ligno­ from the fundamentals of cellulose production and purification to the cellulosic biomass (trees). Typical biopolymers extracted from 1st gen­ end of life scenarios, as well as provide direction where a positive eration feedstocks are mainly cellulose, starch, protein, and further change is lacking. bioprocessed polymers such as PLA, poly-hydroxy-alkanoates (PHAs), and NC. Bioplastics of 1st generation require the use of arable land, 2. Cellulose feedstocks nutrients and freshwater, thus competing with food production and consequently not fully aligning with the UN SDGs. The switch to bio­ 2.1. Cellulose in plants, animals and bacteria plastics using these sources has the potential to affect the world’s food supply while sourcing materials from forests will impact the biodiversity Cellulose is a water-insoluble polymer composed of glucose mole­ and potentially result in more deforestation. cules in which individual glucose units are connected via acetal linkages In order to overcome the detrimental properties associated with 1st generation bioplastics, 2nd generation bioplastics incorporated agri­ cultural waste as well as discarded biomass as a feedstock. All of the above listed biopolymers from 1st generation can also be produced from 2nd generation feedstock. However, these resources are limited and insufficient in term of volume and quality to replace global plastic de­ mand with bioplastics [16]. The disruption presented in the supply Cellulose chain associated with such feedstock also prevents wide scale com­ mercial opportunities. Lastly, 3rd generation bioplastics produced from fast-growing microorganism such as bacteria, and specifically microalgae can theo­ retically address the growing problems related to biomass production as they can be located on non-arable land. Microalgae is of particular in­ terest since it has high growth rates compared to terrestrial plants and can use CO2 rich off-gases, saline and/or wastewater, allowing efficient Cellulose chain nutrient recycling (e.g., nitrogen and phosphorous) [17]. A wide range of biopolymers can theoretically be provided from algal Fig. 2. Cellulose and cellobiose chemical structures.

3 E. Zanchetta et al. Algal Research 56 (2021) 102288 between the C1 and C4 carbons of the glucopyranose ring (Fig. 2). It can In this context, the composition of the cell wall, and the content of cell reach a high degree of polymerization with up to 15,000 glucose units wall compounds (i.e., its purity related to cellulose), is relevant for the [24]. Cellulose also has a distinct hydrophilic and hydrophobic char­ quality of extracted nanocellulosic products. Cellulose is the major load- acter due to the presence of both equatorial hydroxyl groups and axial bearing component of the cell walls of plants and several algal taxa [33]. hydrogen atoms that confers its stability via the coplanar orientation of Many algal taxa were reported to contain cellulose and hemicelluloses the individual glucopyranose rings [25]. [33–36] (e.g., xyloglucans, xylans, mannans, (1 → 3),(1 → 4)-β-glucan) Cellulose chains in plants (terrestrial and algae) and some bacteria in their extracellular covering (Table 1), such as the green algae tend to self-assemble into microfibrils, which undergo further aggrega­ Chlorophyta (e.g., Trebouxiophyceae, Ulvophyceae, Chlorophyceae) tion into larger assemblies, leading to cellulose fibres characterized by and Charophyta (e.g., Charophyceae), or the red algae Rhodophyta. In the presence of crystallites and disordered amorphous regions. To date, the phylum Ochrophyta, the Phaeophyceae class (brown algae) were four different polymorphs of cellulose exist: cellulose (I), cellulose (II), generally reported to contain cellulose and hemicelluloses (e.g., sulfated cellulose (III) and cellulose (IV) illustrated in Fig. 3. These polymorphs xylofucoglucan), while a more limited number of species from Xantho­ differ in polarity of the constituting chains and hydrogen bond ar­ phyceae (yellow green algae) and Chrysophyceae class (golden algae) rangements. Cellulose (I) is the most widespread crystalline form pre­ were found to use cellulose as structural polysaccharide for their cell sent in nature where cellulose (II), (III) and (IV) are mainly obtained wall. In some Dinophyta (thecate dinoflagellates), a complex extracel­ synthetically via chemical and thermal treatments. Cellulose allomorphs lular covering called amphiesma can also include an armour of cellulosic differ from one to the other with respect to chain alignment and inter­ plates (theca). Other matrix polysaccharides may also be found in the chain hydrogen bonding, which creates different geometries. cell wall of green, red or brown algae such as pectins, ulvans, agars, As shown in Fig. 3, two sub-allomorphs designated as Iα (triclinic) carrageenans or alginates [33–35] and can be relevant co-products for and Iβ (monoclinic) are found in cellulose (I) which differ in terms of increasing the economic viability of algal cellulose production. their microfibrils arrangement (parallel and antiparallel) [18]. Gener­ However, despite its potential relevance to find adapted extraction ally, cellulose Iβ is commonly found in higher plants while cellulose Iα is methods for cellulose, little information is available on the cell wall the predominant form of cellulose in algae and some bacteria. Different ultrastructure of many species and a high structural diversity can be chemical and thermal treatments allow the conversion of cellulose (I) to observed, sometimes within the same taxon [37,38]. It is usually known the other listed form of polymorphs. For instance, cellulose (I) can be that brown algae contain a thick cell wall composed mainly of alginates converted to cellulose (II) using different concentration of an alkaline and fucoidans, the cellulose fraction (cellulose microfibrils)is generally solution (mercerization). In this step, the orientations of microfibrilsare smaller but plays a crucial role by associating with alginates for struc­ completely disrupted and the original parallel chain (hydrogen bonding tural purposes [35]. The cell wall of red algae generally contains pattern is O6-H-O3) in cellulose (I) is switched to anti-parallel chain sulphated galactans (agars, carrageenans) which are not always associ­ (hydrogen bonding is O6-H-O2) in cellulose (II). The conversion of ated with cellulose. For instance, the cell wall of Chondrus crispus was cellulose (II) to cellulose (III) is generally performed using diamine or shown to contain a layer of cellulose microfibrilsembedded in a matrix NH3 (liquid) treatment and results in two sub-allomorph Cellulose (IIII) of carrageenans [39], while Gracilaria verrucosa is bi-layered with an and cellulose (IIIII). Finally, the last polymorph of cellulose can be ob­ inner layer of agar and an outer layer mainly composed of cellulose with tained via hydrothermal conversion of cellulose (III) in the presence of a small fraction of xylose and mannose [40]. Among green algae, glycerol [27]. Chlorella (Trebouxiophyceae) is known to have a main fibrillar layer of polysaccharides (cellulose, hemicellulose and pectin blend) which can 2.2. Cellulose in algal biomass be protected in some cases by a highly resistant algaenan outer layer [34,41,42]. The diversity of the cell wall structure between and within 2.2.1. Cellulose biosynthesis pathway in microalgae Chlorella species was found to be even higher. For instance, Yamada Information on the sites of cellulose synthesis, its diversity and et al. [43] differentiated three main types of cell wall structures from evolution of cellulose-synthesizing enzyme complexes (terminal com­ different strains of Chlorella vulgaris. A large range of cell wall structures plexes) in microorganisms is a key factor for the industrialization pro­ was also observed within the Chlorophyceae family. For example, the cess. According to Tsekos et al. [28], cellulose synthesis is conducted by cell wall of Scenedesmus quadricauda was shown to be trilaminar where membrane-bound cellulose synthase terminal complexes (TCs), con­ pectic and cellulosic layer can be clearly differentiated and are protected taining cellulose synthases (CesA). The arrangement of cellulose mi­ by an algaenan layer [44,45], while Oedogonium bharuchae showed a crofibrils(size, shape, crystallinity, and intra microfibrillarassociations) two-layered cell wall composed of a mix of cellulose, pectins and gly­ is directly related to the geometry of the terminal complexes (TCs) as coproteins followed by a cellulose-free layer containing extensin and shown in Fig. 4. and by Tsekos et al. [28]. Typical arrangement of TCs in arabinogalactan proteins [46]. A pectic layer was also found in some higher plants follows a hexagonal structure commonly known as rosettes Charophyta (e.g., Penium margaritaceum, Chara corallina) where it linked 2+ structure. However, a more diverse organization of TCs can be found an inner layer of cellulosic microfibrils to an outer “Ca -pectin” layer within the algae domain, this includes rosette, single and multiple rows [35]. In contrast, pectins were not found in the cell wall of some Ulvo­ arrangement [29,30]. Rosette structures are found in Charophyceae, phyceae, but rather ulvans serving as matrix polysaccharides [35]. while linear TCs divides into three categories: (i) single rows include Nannochloropsis (Ochrophyta/Eustigmatophyceae) is also known to brown algae (Phaeophyceae) and some red algae (Rodophyta), (ii) have a very special cell wall containing a porous inner layer composed of multiple rows are found in Ulvophyceae, Chlorophyceae, some Rodo­ struts connecting the cell membrane to a porous cellulose-based layer phyta, and Dinophyta, and (iii) diagonally arranged multiple rows can coated by an algaenan outer layer [37]. be found in Xanthophyceae [31]. Based on current knowledge in the field, Nannochloropsis sp. is the only microalgae studied for its cellulose production potential from 2.2.2. Cellulose potential in algal biomass which cellulose has been extracted [22,47]. However, there is not much information about other species, thus, more research is needed to a. Cellulose bearing families identify other interesting species for cellulose production. Some of these might have a high potential of cellulose concentration, this could Several studies have focused on the content of specific algal prod­ include microalgae from the Trebouxiophyceae class such as Chlorella ucts, such as pigments, vitamins, lipids, carbohydrates, and proteins to sp. and Oocystis sp. [36,41,48], Chlorophyceae class such as Scenedesmus produce a wide range of products. However, only few showed interests sp., Coelastrella sp., Chlorococcum sp., Selenastrum sp. [41,49–51], in algae as a source of cellulose or nanocellulose (see Table 4 for details). Charophyta phylum such as Chaetosphaeridium sp. and Staurastrum sp.,

4 E. Zanchetta et al. Algal Research 56 (2021) 102288 I I II II Cellulose II Cellulose IV Triclinic Cellulose III Cellulose III Cellulose IV One monoclinic One monoclinic Two monoclinic Cellulose I Cellulose I Two orthorhombic Two orthorhombic One monoclinic rao /I 60/40 25/75 I I II II ) ]. ) 26 Cellulose II Cellulose [ Cellulose III III Cellulose Cellulose IV IV Cellulose Cellulose III Cellulose IV IV Cellulose Cellulose I Cellulose Cellulose I Cellulose (Parallel chains) (Parallel chains) (Parallel chains) (One parallel chain) parallel (One An-parallel chains) (Two parallel chains) parallel (Two (An-parallel chains) ( Valonia Castanea sava (Two an-parallel chains) geometry Plant ( Algae ( Source of cellulose I I Cellulose sub-allomorphs Cellulose sub-allomorphs crystal their and pathways ) production to Cellulose I to Cellulose I Chemical structure Chemical allomorphs to Cellulose II ) II Cellulose 3. 3 Fig. Nave Synthec 1 5 Cellulose I 2 Cellulose II Cellulose III Cellulose IV (Parallel chains) (Parallel (An-parallel chains) (An-parallel Cellulose allomorphs 4

(L) liquiddiamine ortreatment (g) evaopraon /(g) evaopraon Cellulose III (fromtreatment NaOH (g) evaopraon / Hydrothermal treatment (from Cellulose III

87 88 58 138 3 3 3 lscmdls(GPa) modulus Elasc

4. NH 1.(mercerizaon) treatment NaOH 2. NH 5. 260°C treatment Thermal in glycerol 3. NH Decreasing sffness Decreasing

5 E. Zanchetta et al. Algal Research 56 (2021) 102288 Absence - Single rowsof TCs Low content/ TCs arrangement + Six-fold symmetry TCs Parallelogram with with Parallelogram diagonally Medium content/ Medium ]. ++ 32 [

2 nm

25 nm from 2 nm 2 synthase cellulose complexes terminal TCs: 20 nm structure Microfibril

3.5 nm

High content/

10 nm 10

3.5 nm 3.5 1.5-2 nm 1.5-2 adapted +++ organisms, different in (TCs) complexes Cellulose Cellulose microfibril microfibril terminal synthesizing cellulose Lignin Hemicelluloses Other* Lignin Hemicelluloses Cell level of Cellulose Cellulose macrofibril macrofibril +++ +++ ++ -/++ -/++ -/+++ morphology and Cell wall Cell wall Cell wall Organization 4. Fig. Cellulose feedstock Trees Microalgae Macroalgae

6 E. Zanchetta et al. Algal Research 56 (2021) 102288

Table 1 Overview of the major polymer found in the extracellular covering of different algal taxa, adapted from [33–35].

Algal taxa Crystalline Hemicelluloses Matrix polysaccharidesa polysaccharides

Chlorophyta (green algae) Cellulose Xyloglucans, xylans, mannans, glucuronan, (1 → 3)-β-glucan, (1 → 3),(1 → 4)- Ulvans, pectins β-glucan Charophyceae (green Cellulose Xyloglucans, xylans, mannans, (1 → 3)-β-glucan, (1 → 3),(1 → 4)-β-glucan Pectins algae) Rhodophyta (red algae) Cellulose, Xylans, mannans, glucomannans, sulfated (1 → 3),(1 → 4)-β-glucan, (1 → 3),(1 → 4)- Agars, carrageenans, (1 → 4)-β-mannan, β-xylan porphyran (1 → 4)-β-xylan, (1 → 3)-β-xylan Phaeophyceae (brown Cellulose Sulfated xylofucoglucan, sulfated xylofucoglucuronan, (1 → 3)-β-glucan Alginates, fucoidans algae) Dinophyta Cellulose – –

a Matrix polysaccharides may form composite layers including cellulose and/or hemicelluloses, or separate layers. The precise ultrastructure can be species dependant and should be looked on a case by case basis.

Chrysophyceae class such as Dinobryon sp. or Dinophyta phylum Tables 2 and 3 show the cellulose content quantified by various [35,36,52]. Evidence of cellulose is not clear for Chlamydomonas sp. studies for both micro- and macro- algae species, respectively in total (Chlorophyceae) as contradictory statements were reported in the dry weight of the algae and in the cell wall dry weight. Only a limited literature [53,54]. More exotic species from Coccolithophyceae class number of studies have quantified cellulose from microalgae, which (Haptophyta phylum) such as Pleurochrysis sp. were also reported to mostly focused on Nannochloropsis sp. and Chlorella sp. Nannochloropsis produce cellulosic scales instead of the usual calcite layer [36]. gaditana and occulata were found to have a high cellulose content in Among macroalgae, most of the studies for cellulose production their cell wall (respectively 75 and 80 wt% on dry weight basis [37,59]) focused on Ulvophyceae class with species such as Ulva sp. (Ulvales while Chlorella pyrenoidosa contained poor cellulose amount in its order), or Cladophora sp., Valonia sp., Boergesenia sp. (Cladophorales covering (15.4 wt% [53]). On the other hand, when looking at the total order) (e.g., [55–58]). Many other species from the Cladophorales order dry weight of the algae, Nannochloropsis gaditana was found to reach 25 are known to contain a well-organized and highly crystalline cellulose wt% cellulose [47], while Chlorella vulgaris ranged between 10 and 47.5 such as Siphonocladus sp., Apjohnia sp., Dictyosphaeria sp., Chaetomorpha wt% [48]. This notable difference in content could be explained by the sp., Rhizoclonium sp. [36], and might be worthy of more extensive large influenceof the culture conditions on the constitutive elements of studies to identify potential for cellulose production. Several other re­ the algae such as cellulose, and highlights the importance of good searchers found good cellulose potential in Posidonia sp. (Tracheophyta culturing design needed for maximizing cellulose production. For phylum, Monocots class) from which cellulose has been extracted instance, it was shown in the study of Aguirre and Bassi [48] that the [20,21]. Nitella sp. (Charophyceae class) was studied mainly for its cell nitrate concentration played an important role on the cellulose content wall properties [36], excluding aspects regarding cellulose extraction. of Chlorella vulgaris (ranging from 10 to 47.5 wt%). Another factor that Cellulose seems to be present in most red algae (Rodophyta phylum) and could potentially nuance the lower cellulose content found in total dry all brown algae (Phaeophyceae class), with interesting potential re­ weight basis for Nannochloropsis sp. is the cell division mode of this ported in red algae Ptilota sp., Rhodymenia sp., and brown algae Fucus algae. Microalgal cell division generally occurs either by desmoschisis, sp., Laminaria sp. A limited amount of species from Xanthophyceae class, where the daughter cell inherit an equal proportion of the mother cell such as Tribonema sp. (filamentous microalgae) or Vaucheria sp. (mac­ wall to form their coverings, or eleuteroschisis, where each daughter roalgae) were known to contain cellulose [36]. cells produce a new cell wall after complete dissolution of the mother cell wall [60]. As Nannochloropsis sp. undergo eleuteroschisis [37], the b. Cellulose, hemicellulose and lignin content in algal biomass measured cellulose content could vary depending if the shed cell walls

Table 2 Cellulose, hemicellulose and lignin content in total dry weight basis of algal feedstock. n.d.: not determined, det.: detected (either directly or indirectly).

Phylum/class Strain Cellulose Hemicellulose Lignin [%] References [%] [%]

Microalgae – Mix of microalgae & cyanobacteria from waste water 7.1 16.3 1.5 [61] treatment plant Chlorophyta/ Chlorella vulgaris 10–47.5 n.d. n.d. [48] Trebouxiophyceae Ochrophyta/ Nannochloropsis gaditana 25 det. n.d. [47] Eustigmatophyceae Macroalgae Chlorophyta/Ulvophyceae Cladophora glomerata 21.6 det. n.d. [55] Chlorophyta/Ulvophyceae Ulva lactuca 12.4 n.d. n.d. [56] 6.0 12.2 9.8 [66] 16.6 32.5 1.5 [67] Chlorophyta/Ulvophyceae Ulva prolifera 19.4 14.4 9.4 [68] Chlorophyta/Ulvophyceae Ulva pertusa 6.7 16.8 n.d. [69] Chlorophyta/Ulvophyceae Ulva sp. 40.7 7.1 7.9 [70] Ochrophyta/Phaeophyceae Cystosphaera jacquinottii 4.6 6.1 19 [71] Ochrophyta/Phaeophyceae Fucus vesiculosus, Laminaria digitata 8 n.d. n.d. [72] Rodophyta/Florideophyceae Gelidium elegans 17.2 29.5 4.5 [73] Tracheophyta/Monocots Posidonia oceanica 32.5 23.3 28.2 [20] 31.4–40.0 21.8–25.7 29.3–29.8 [21] 40.0 20.8 29.8 [74] Tracheophyta/Monocots Posidonia australis 20.2 11.7 14.5 [75]

7 E. Zanchetta et al. Algal Research 56 (2021) 102288

Table 3 Cellulose, hemicellulose and lignin content in the cell wall of algal feedstock. n.d.: not determined, det.: detected (either directly or indirectly), abs.: absent.

Phylum/class Strain Cellulose [%] Hemicellulose [%] Lignin [%] References

Microalgae Chlorophyta/Trebouxiophyceae Chlorella pyrenoidosa 15.4 31 n.d. [53] Ochrophyta/Eustigmatophyceae Nannochloropsis gaditana 75 det. n.d. [37] Charophyta/Zygnematophyceae Staurastrum sp. 72 4.0 1.2–5.6 [52] Macroalgae Chlorophyta/Ulvophyceae Valonia ventricosa 75 det. abs. [65] Chlorophyta/Ulvophyceae Cladophora rupestris 28.5 abs. n.d. [76] Chlorophyta/Ulvophyceae Ulva lactuca 19 det. n.d. [76] Chlorophyta/Ulvophyceae Chaetomorpha melagonium 41 det. n.d. [76] Chlorophyta/Ulvophyceae Enteromorpha sp. 21 det. n.d. [76] Ochrophyta/Phaeophyceae Fucus serratus 13.5 det. n.d. [76] Ochrophyta/Phaeophyceae Laminaria digitata 20 det. n.d. [76] Ochrophyta/Phaeophyceae Laminaria saccharina 18 det. n.d. [76] Ochrophyta/Phaeophyceae Halidrys siliquosa 14 det. n.d. [76] Ochrophyta/Phaeophyceae Himanthalia lorea 8 det. n.d. [76] Rodophyta/Florideophyceae Ptilota plumosa 24 det. n.d. [76] Rodophyta/Florideophyceae Rhodymenia palmata 7 det. n.d. [76] were retained or not during the harvesting process. Nanocellulose pro­ are more prevalent during autumn and winter [62,63], thus there is duction might also be considered in a biorefineryapproach as proposed some affect from seasonality in such studies. by Lee et al. [22], where the cellulose content of Nannochloropsis oce­ Compared to microalgae, a larger number of studies is available for anica leftover (after lipid and protein extraction) was 34 wt% and rep­ the compositional analysis of macroalgae, especially for cellulose, resented 47 wt% of the total carbohydrate content. hemicellulose and lignin. Most of the studies assessed macroalgae from Regarding cellulose purity, it was found that Chlorella pyrenoidosa the Ulvophyceae class such as Ulva sp., Cladophora sp., Valonia sp. and can contain two-fold hemicellulose mass, compared to cellulose in its Phaeophyceae class such as Fucus sp. and Laminaria sp. (Tables 2 and 3). cell wall [53] (Table 3). Again, this does not necessarily mean that Some red algae (Rhodophyta) and Tracheophyta such as Posidonia sp. Chlorella sp. has a poor cellulose purity, as also suggested by the high were also quantified.The cellulose content is variable depending on the cellulose content from the study of Aguirre and Bassi [48]. But this is strain, ranging from 4 to 40 wt% of the total dry weight and up to 75 wt most likely due to poorly optimized culture conditions. Gunnison and % of the dried cell wall for Valonia sp. It appears that certain macroalgae Alexander [52] also showed that some microalgae species such as such as Posidonia oceanica has a significantamount of lignin (around 30 Staurastrum sp. (Charophyta/Zygnematophyceae) can contain lignin in wt%) coupled with a high cellulose content (30–40 wt%). Ulva sp. has the range of 1.2–5.6 wt% of its cell wall, high cellulose content (70 wt% the advantage of containing less lignin (up to 10 wt%) with a variable of cell wall mass) and a low hemicellulose fraction (4 wt%). However, no but important potential for cellulose (6–40 wt%). Other strains such as other studies known so far reported any quantification of the hemicel­ Cladophora sp. seem to have a great potential for cellulose (20–45 wt%), lulose or lignin content in microalgae species. To build on this, Ververis which is highly crystalline [64]. However, there was no clear evidence et al. [61] provided further insights into this question as they compared of the purity of the cell wall, and contradictory statements regarding the the composition of several organic materials, including a freshwater possible existence of lignin was also deduced. Finally, strains such as microalgae slime (Chlorella sp., Scenedesmus sp., and other microalgae Valonia sp. seem to have a very pure and highly crystalline cellulosic cell and cyanobacteria) collected from a waste water treatment plant, for use wall [64,65]. This suggests that different methods should be used to as paper pulp supplement. It was found that the slime contained 7.1 wt% purify cellulose from other compounds depending on the composition of cellulose, 16.3 wt% hemicellulose and a low fraction of lignin (1.5 wt%). the algae, and will be developed in a later part of this review. It was suggested that the cellulose and hemicellulose content could come from Scenedesmus sp., Ulothrix sp., Stigeoclonium sp. or Chlorella sp. It was noted that Scenedesmus sp. usually dominates in spring, while the others

Algae strain

Strategy 3: Strategy 1: Strategy 2: Genec engineering Opmizaon of biomass Opmizaon of cellulose producvity content in biomass (stress) Strategy 4: Phytohormones Opmizaon of algal biomass

Conversion to nanocellulose

Conversion to bioplasc

Fig. 5. Optimization strategies of algal biomass for nanocellulose production.

8 E. Zanchetta et al. Algal Research 56 (2021) 102288

3. Optimization of algal biomass for cellulose and nanocellulose effective biomass production and extracellular polysaccharide accu­ production mulation [103,104]. To improve the nutrient uptake of microalgae in mixotrophic cultivation, low-strength ultrasound has been used to in­ Herein, we outline different strategies that could potentially increase crease the transport of nutrients into algal cells that consequently pro­ the overall yield of cellulose produced by microalgae (Fig. 5). One moted biomass yields [105]. Ideally, the carbon present in wastewater approach for achieving this goal is the optimization of cell culture should be utilised by algae, negating the need of an exogenous source. conditions to improve the growth rate and cell density [77–80]. Typically, municipal waste water contains <10% sugars, with the ma­ Obtaining higher biomass yields in shorter times is essential for the use jority of carbon sources linked to protein or/and fibre[ 106]. In order to of microalgae at a commercial production scale. Other strategies aim at liberate C for microalgae uptake, a co-culture of bacteria is needed to potentially influencing cellulose synthesis by applying specific stress ensure release what is the correct growth phase [107], presumably conditions like low nitrogen, sulphur, or phosphorus, or high salinity under heterotrophic conditions. It should be noted that not all micro­ [81–83]. It is also possible to influencemetabolic pathways directly on a algae species are suitable for use in wastewater treatment and a general genetic level to drive photosynthesis and CO2 fixationto higher growth consensus is not available. Studies conducted thus far have not been rates and biomass yields, or accumulation of specific products. Newly conclusive for direct comparison due to variability in experimental developing genetic engineering approaches and genome editing tech­ conditions [107] and as such the cultivation of different microalgae nologies are currently paving the way for the construction of industrial species in wastewater should not be treated equally without taking the microalgal strains with desired and optimized properties [84–90]. environmental factors into account. Finally, the use of phytohormones either exogenously added or geneti­ cally engineered for endogenous expression show great potential of 3.2. Specific stress conditions boosting high biomass production, and accumulation of desired prod­ ucts, especially under abiotic stress conditions [91,92]. Eukaryotic microalgae possess remarkable adaptability to most abiotic stresses. Evidence for stress-induced increase in the cellulose 3.1. Optimization of culture conditions (phototrophic, heterotrophic, content of microalgae is rare [81] and may deserve more attention. mixotrophic) Here, we focus on specific stress conditions, which can be purposely applied to stimulate the overall carbohydrate production, and could Achieving high productivity represents a major challenge in com­ potentially be linked to increased cellulose synthesis. Future optimiza­ mercial applications for microalgae, and consequently also for the pro­ tion of algal biomass for cellulose production would require specifically duction of cellulose at an industrial scale. Microalgae often exhibit targeting the cellulosic compound for quantification. Common stress remarkable individual flexibility in their mode of cultivation. Whether strategies for carbohydrate increase include nutrient limitation/deple­ phototrophically, mixotrophically or heterotrophically grown, each tion (e.g., nitrogen, phosphorus, sulphur), or high salinity (osmotic mode represents technical challenges of its own. Mixotrophic culture is stress) [82,83]. A decrease of temperature below optimal conditions was gaining increasing attention because the metabolic capability of some also reported to improve the carbohydrate content of Chlorella vulgaris microalgae to use both photosynthesis and organic carbon leads to [108], or excessive light intensity (photoinhibition) slightly increased higher productivity than either phototrophic or heterotrophic processes the glucose content of Scenedesmus obliquus [109] at the cost of alone [77,79]. Mixotrophic cultivation modes, which combine waste­ decreased growth rates. However, the efficiency and energy consump­ water treatment with the production of high-value biomass, have future tion of such techniques would make them difficult to implement for potential to allow large-scale manufacturing of raw materials from large-scale carbohydrate production. microalgae in a cost-effective, and sustainable approach. In general, there are two possible pathways of carbon fixation in 3.2.1. Medium induced stress by deprivation or excess of specific microalgae: (i) autotrophic, which relies on photosynthetic growth and compounds fixation of inorganic carbon (i.e., carbon dioxide) through the Calvin- One of the main interest for microalgae cultivation under nutrient Benson cycle, and (ii) heterotrophic, based on the assimilation of limitation/depletion was the promotion of lipids and other added value organic carbon in the absence of light [93]. Some microalgae species products, including carbohydrates [110–112]. Microalgae grown under show metabolic versatility but more species are obligate photoauto­ nitrogen limitation generally tend to redirect their carbon flow from trophs rather than heterotrophs [78]. Heterotrophic culturing of protein synthesis to lipid or carbohydrate synthesis pathways [113]. microalgae has been reported to enhance the biomass concentration by This effect was observed for Nannochloropsis oceanica, where the pro­ as much as 25-fold compared to phototrophic conditions and ultra-high portion of triacylglycerol (TAG) and glucose increased in response to N- biomass yields of more than 200 g⋅L 1 of culture have been recently depletion [114]. Several studies admitted that glucose accumulated in reported [79,80]. Nannochloropsis sp. mainly in the form of β-1,3-glucan [114,115], thus Nevertheless, a number of commercially relevant microalgae (e.g., targeting specific genes involved in their biosynthesis (e.g., UDPG Chlorella vulgaris, Arthrospira platensis (former: Spirulina platensis), pyrophosphorylase and β-1,3-glucan synthase). Very recently, Jeong Chlamydomonas reinhardtii, and Dunaliella salina), were shown to exhibit et al. [81] targeted the cell wall evolution and genes involved in cellu­ a more flexible growth mode. For instance, they can also grow hetero­ lose synthesis of Nannochloropsis salina under nitrogen starvation, trophically and even showed improved biomass yields under mixo­ showing that glucose could also accumulate in the cellulosic wall of the trophic conditions as compared to a photoautotrophic or heterotrophic cell. The microalga exhibited substantial thickening of its cell wall growth mode alone [94–97]. Mixotrophic cultivation of many micro­ cellulosic layer and increased cellulose content when grown under ni­ algae strains show additive or synergistic effects of photoautotrophic trogen deficiency for 2 days (compared conditions: 427.5 mg⋅L 1 1 and heterotrophic metabolic activities, leading to an overall increases in NaNO3; without nitrogen 0 mg⋅L NaNO3). This effect was reasonably productivity [98–100]. Several studies, which analysed the microalgal explained by an enhanced transcript level of genes involved in cellulose energy metabolism, showed that mixotrophic cultures resulted in higher biosynthesis (e.g., UDPG pyrophosphorylase and cellulose synthases), energetic efficiency because the amount of energy dissipated was min­ which was detectable during nitrogen starvation conditions. Nitrogen imal which presumably increases their productivity [101,102]. limitation induced a 1.5-fold increase of the cellulose content compared The most prominent advantage of mixotrophic or heterotrophic to non-stressed conditions, respectively, 9% and 6% of the total biomass growth, compared to phototrophic cultivation is the possibility to grow dry weight, while increasing cell size (1.18-fold increase, from 2.73 μm microalgae in wastewater to utilise organic carbon, thereby offering an to 3.24 μm) and cell wall thickness (1.54-fold increase, from 52 nm to excess of carbon flowin addition to atmospheric inorganic CO2 for cost- 80 nm). Calcofluor white, a common dye for cellulose detection,

9 E. Zanchetta et al. Algal Research 56 (2021) 102288 revealed that at least some of the cell wall thickening was attributed to cost-effective. Furthermore, stress conditions often have pleiotropic ef­ an increase of the cellulosic layer. On the other hand, the specificgrowth fects in microalgae by influencingnot only the carbohydrate production rate decreased significantlyunder stressed conditions (control: 0.53 d 1, but also lipid, pigments or antioxidant contents and should be envi­ nitrogen deficiency: 0.09 d 1). sioned in a biorefinery concept to increase overall economic viability. Similarly, it would be worthwhile to try other common nutrient starvation strategies used for carbohydrate increase such as low phos­ 3.3. Genetic engineering for improving microalgal biomass and phorus or sulphur [82,83] to investigate the extent to which they could productivity promote cellulose synthesis or cytoplasmic sugar accumulation in Nan­ nochloropsis sp. The effect of nitrogen, phosphorus or sulphur depletion Despite their potential to boost the production of bioproducts, ge­ on potential cellulose accumulation in other microalgae species still netic engineering techniques in microalgae have been lagging behind remains undemonstrated. A clearer picture of the glucose accumulation those available for higher plants, fungi and bacteria [84]. However, pathways either in the form of starch or cellulose seems necessary. recent advances in genome sequencing [126] and genome editing A number of studies reported the overall increase of carbohydrates technologies [85] promise enhanced biotechnological exploitation of under salinity stress (high salinity) in freshwater species such as Chlor­ microalgae by genetic engineering of strains with optimized physio­ ella sp. or Scenedesmus sp. [116–118]. Several reviews pointed that logical properties (metabolic engineering), making them more suitable microalgae may produce low molecular weight organic compounds for commercial utilisation [84]. (osmolytes). For instance, carbohydrates, to reduce the osmotic stress The availability of genomic sequence data is critically required for generated by an increase in salinity [82,113,119]. However, adapting to genome editing approaches. First microalgal nuclear genome sequences such changes of turgor pressure could also involve other mechanisms were reported from Thalassiosira pseudonana [127], Chlamydomonas such as variation in cell size or increase in cell wall thickness, respec­ [128] and Phaeodactylum [129]. The number of publicly accessible ge­ tively, decreasing the tension applied to the cell wall or increasing its nomes is currently estimated to range between 40 and 60 and is pre­ tensile strength [120]. Cellulose is known to be the major contributor to dicted to significantly increase in the near future [84,130]. Genetic cell wall strength in land plants [121], similarly cellulose bearing engineering of microalgae may not only involve manipulations of nu­ microalgae could increase their cellulosic wall thickness to increase the clear DNA but also modifications on the level of chloroplast and mito­ mechanical strength of their cell wall for osmotic acclimation. This topic chondrial genomes for which quite a number of sequences are already may deserve more attention in the vision of optimizing cellulose pro­ available at the NCBI organelle database and the organelle genome duction in microalgae. database GOBASE [131]. In addition to the detailed genome sequence information and the determination of gene functions, also molecular 3.2.2. Growth limitation under stress and possible strategies of mitigation techniques and synthetic toolbox DNA elements as for instance gene Stress-induced stimulation of specificcompounds is often associated delivery methods, plasmids, selectable markers, promoters, ribosome with decreased growth rates caused by oxidative stress [110]. This binding sites, transcriptional regulators (enhancer/silencer) and tran­ limitation was addressed by many reviews [82,83,110–112,122]. A scription terminators are prerequisites for the genetic engineering of trade-off is generally found in terms of stress intensity to maximize the economically viable microalgal strains with desired properties overall productivity of the targeted product. Ideal stress intensities are [84,132,133]. species dependant. For instance, salinity values ranging from 0.06 Metabolic pathways can be modified by overexpression or silencing 1 1 mol⋅L to 0.4 mol⋅L of NaCl increased the carbohydrate fraction of of certain genes to achieve a higher yield of desired products [86]. In this freshwater species such as Scenedesmus obliquus (from 28 wt% to 52 wt% context, significanteffort has been undertaken to increase lipid contents of the total dry weight) or Chlorella vulgaris (26 wt% to 47.5 wt% of dry and change the fatty acid composition for biofuel production [87]. weight) [116]. However, the maximum carbohydrate productivity was However, the enhancement of photosynthesis and biomass productivity 1 1 1 reached at 0.3 mol⋅L for Scenedesmus (22.2 mg⋅L .d ) and 0.08 of microalgae has also been addressed by genome editing. These factors 1 1 1 mol⋅L for Chlorella (12.6 mg⋅L ⋅d ) due to a differing decline in have primarily shown to be determined by light utilisation efficiency growth rates. and CO2 fixationrate [88]. First encouraging results on the engineering Different cultivation strategies have been proposed to mitigate the of microalgal strains for higher photosynthetic efficiencywere obtained conflict between biomass growth and content of targeted compounds using the CRISPR-Cas9 method [89]. In another study, the CO2 fixation [112,119,122]. These strategies usually involve multi-stage cultivation capacity of Chlorella vulgaris could be enhanced by a genetic modifica­ where ideal growth conditions maximize biomass production, while tion, to achieve an enhanced expression of the Calvin cycle enzyme stress conditions increase the content of specificproducts. One reported aldolase [90]. strategy used intermittent nitrogen supply (3 to 10 cycles of starvation) The emergence of genomic sequence databases and the determina­ during fed-batch culture [123,124]. The most widely used strategy tion of gene-specificfunctions represent an indispensable basis to allow consists of two-stage cultures where high cell densities are produced genetic engineering of microalgae towards increased metabolic output. prior to application of the stress [112,119,122]. Mixotrophic or het­ Moreover, significant recent advances provide molecular tools and erotrophic cultivation could be envisioned to increase biomass produc­ synthetic toolbox DNA elements in combination with genome editing tivity during the firststage [112]. The application of a salinity-gradient, technologies, which now allow directed gene manipulation with high in case of stress from high salinity, was also shown to reduce growth precision in shorter duration. All these developments have the potential inhibition during the second stage by improving the adaptation of cells to shift algal biotechnology towards profitable bioengineered produc­ to the stress conditions [125]. Conversely, two-stage strategies require tion strains, which can economically compete with other existing sys­ higher investment and operating costs since more photobioreactors/ tems for the production of bioproducts. ponds and a transfer of the biomass between them would be needed Genetically engineered microalgae using classical recombinant DNA [119]. Additionally, a longer cultivation time is needed in case the two techniques or gene-editing procedures currently remain in the EU under stages is envisioned in the same culturing container, which also avoids the GMO (Genetically modified organism) legislation. This means that the possibility of continuous or semi-continuous cultures. Comparative its products must receive approval prior to any commercialisation assessments (e.g., techno-economic) would be required between one- [134]. Risk assessments related to genetically engineered microalgal and two-stage culturing strategies to evaluate the most cost-effective strains aim to predict their potential damage on the environment as well solution for cellulose production. Culturing design involving interme­ as on human and animal health [135]. This includes also the analysis of diate harvesting (e.g., nutrient depletion) should be avoided to decrease the risk of their deliberate escape into the environment, which may costs [119]. In that sense, strategies such as high salinity could be more potentially cause the spreading of recombinant DNA by horizontal gene

10 E. Zanchetta et al. Algal Research 56 (2021) 102288 transfer to other organisms or to their wild-type counterparts by sexual carbohydrate production in microalgae. However, the oxidative damage reproduction. The use and design of transgenes should therefore be caused by nitrogen depletion dramatically reduces cell growth rates. In a considered not to confer a selective advantage in nature. Horizontal study by Yu et al. [152], a combination of auxin-related phytohormones gene transfer mediated by viruses for instance is regarded as a rare event (indolebutyric acid, IBA; napthylacetic acid, NAA) together with a ni­ but has nevertheless been documented in algae [135,136]. To improve trogen depletion condition significantly increased cell productivity, biosafety issues related to genetically modified microalgae, legislative increased cell sizes and maintained normal growth rates in Scenedesmus rules should be harmonized, because they are unfortunately not equal and Chlorella species. Increased resistance of Chlorella to N-stress was around the world [137]. Furthermore, analytical procedures should be also observed for various other phytohormones such as another auxin adapted to better trace the potential transfer of transgenes and their (Indole-3-acetic acid, IAA), cytokinin (kinetin), and gibberellin (gib­ impact on the natural environment [137]. berellic acid, GA), resulting in increased biomass productivity in each case [153]. On Nannochloropsis species, abscisic acid showed improved 3.4. Phytohormones for improving biomass yield and stress resistance growth under nitrogen depletion [154]. This phytohormone also improved the resistance to an excess of salinity applied on Chlamydo­ Phytohormones comprise diverse classes of low molecular weight monas (freshwater species) resulting in a higher growth [155]. Even if chemical messengers in plants that play essential roles as regulators of this alga probably does not produce cellulose, the finding that phyto­ growth and development. Nine categories of phytohormones have been hormones could improve the resistance to several stress conditions could identifiedto date comprising: auxins, cytokinins (CK), gibberellins (GA), be relevant to find optimum stress strategies for cellulose production. abscisic acid (ABA), ethylene (ETH), brassinosteroids (BR), salicylates This area of research deserves more attention. (SA), jasmonates (JA) and strigolactones (SL) [138]. These natural compounds occur and act at very low concentrations (fmol to pmol⋅g 1 4. Conversion of algal biomass to cellulose and nanocellulose of fresh weight) and represent promising targets for improving plant productivity and tolerance to environmental stress factors [139,140]. Only a limited number of studies have reported the use of algae for Phytohormones have been identifiedin multiple microalgal lineages. cellulose or nanocellulose (CNF, CNC) production (see Table 4). Most of Often their functional role remains unclear but increasing evidence the studies focused on macroalgae species, ranging from Ulvophyceae suggests that their physiological effects might be similar to those found class such as Valonia ventricosa, Cladophora glomerata, Boergesenia for­ in higher plants [91]. The research on microalgal phytohormones is still besii, and Ulva lactuca. Posidonia oceanica, some brown algae (Phaeo­ in its infancy but has the potential to reveal attractive perspectives for phyceae) such as Cystosphaera jacquinottii, Fucus vesiculosus, Laminaria achieving higher biomass yield and increased productivity of metabo­ digitata, as well as red algae (Rodophyta) such as Gelidium elegans were lites such as carbohydrates, lipids and proteins. The exogenous appli­ also studied for cellulose production. Only Lee et al. and Baba Hamed cation of phytohormones alone or in conjunction with specificabiotic or et al. [22,47] reported the use of microalgae (Nannochloropsis salina, biotic stress conditions (high salinity, nitrogen or phosphorus depletion, oceanica and gaditana) for cellulose and CNF production. etc.) as well as the genetic engineering of endogenous phytohormone The production of CNF from algal biomass involves multiple steps: (i) systems are presumably important additional building blocks, which a purificationstep consists of breaking down the biomass and separating may help to establish microalgal productions at an industrial scale. the cellulose pulp from other impurities; (ii) pre-treatment of the cel­ lulose pulp (mechanical, biological, or chemical) can be used to facili­ 3.4.1. Phytohormones to improve the growth rate and carbohydrate tate the fibre delamination prior to CNF formation and reduce the accumulation of microalgae energy demand linked to CNF production [156,157]; (iii) the last step In some microalgae, different phytohormones were shown to induce involves the mechanical disintegration of the fibres where cellulose higher growth rates and increased biomass yields [92], sometimes bundles are completely delaminated to individual fibrils.Alternatively, increased monosaccharides content [141]. For instance, some brassi­ cellulose nanocrystals (CNC, also called whiskers) can be produced by nosteroids (BRs), which are polyhydroxylated steroidal hormones, strong acid hydrolysis (typically around 60% H2SO4 [130]) from puri­ stimulated increases of two-to-three fold in the growth, and up to three- fiedcellulose pulp. Usually, CNCs are characterized by their smaller size fold in the monosaccharide content, of Chlorella vulgaris when used at (2–15 nm wide, 100–500 nm long) compared to CNF (20–50 nm wide, concentrations of between 10 12 and 10 8 mol⋅L 1 [142]. Also, the 500–1500 nm long) [158], as well as higher crystallinity [159]. growth of Chlorella vulgaris in presence of salicylic acid (between 10 6 Large-scale production of cellulose (e.g., pulp and paper industry) and 10 4 mol⋅L 1) increased the cell number and monosaccharide usually involves lignocellulosic biomass that requires strong delignifi­ content by up to 40% [143]. The synthetic auxins indomethacin (IM, cation processes to break the lignin shell surrounding holocellulose 10 7 mol⋅L 1) and naphthyl-3-acetic acid (NAA, 5 ppm) substantially bundles (cellulose and hemicellulose blend) for access. The most com­ increased the biomass productivity of two Chlorella species (Chlorella mon processes entail a combination of aqueous chemical treatment at vulgaris, Chlorella sorokiniana) by about 70–80% on average compared elevated temperature and pressure (“cooking”) to extract a cellulose with the control, along with an increased monosaccharide content for IM pulp, and subsequent bleaching to achieve higher purity (e.g., removal [144,145]. Additionally, all other types of phytohormones (cytokinins, of residual lignin). The chemicals used in these processes usually gibberellins, abscisic acid, ethylene, jasmonates and strigolactones) generate a large number of air and water pollutants, including volatile improved the growth rate of Chlorella species [146–151], along with emissions of chlorine, chloroform, carbon tetrachloride or sulphur, and increased monosaccharide/glucose content for cytokinins, jasmonates, emissions of chlorinated organic compounds (e.g., dioxins), or chlor­ and abscisic acid [146,148,150]. In summary, all nine categories of ophenols in aqueous waste streams [160]. Using algal biomass as an phytohormones have shown improved growth on microalgae species alternative feedstock would ease the purification process since most of (literature shown for Chlorella, but other species showed similar im­ the algae species were reported as lignin-free [161], thus avoiding the provements, e.g., [147]) followed in a number of cases by an increase of need for strong “cooking” processes and possibly limiting the use of monosaccharide or glucose production. More research would be needed bleach. The purification step for algae serves mainly for removal of to identify if cellulose content is also specifically increased by phyto­ hemicelluloses, other polysaccharides such as pectins or lipidic frac­ hormones supplementation. tions. Some algae species were reported to contain a certain proportion of lignin (e.g., Posidonia sp., Ulva sp.). In that case, softer cooking and/or 3.4.2. Synergistic effects between abiotic stress and phytohormones on the bleaching might be required. In general, non-wood species are easier to productivity of microalgae cook than wood chips, therefore conventional Kraft pulping can be Nitrogen starvation is a useful approach to enhance lipid and replaced by soda cooking (sodium hydroxide only), thus reducing the

11 E. Zanchetta et al. Algal Research 56 (2021) 102288

Table 4 Processes used for cellulose purificationand conversion to CNF and CNC from algal biomass. The cellulose is firstpurified from the other components of the biomass (in some cases CNF or CNC was also produced). The conversion to CNF includes a series of three potential steps: (i) Mechanical pre-treatment (not reported so far for algae), (ii) Biological or chemical pre-treatment, (iii) and mechanical treatment; while for CNC, a unique chemical treatment is required. For each step (purification, CNF, or CNC), the processes used are displayed in chronological order. CNF: Cellulose nanofibrils,CNC: Cellulose nanocrystals, TEMPO: 2,2,6,6-tetramethylpiperidine- 1-oxyl.

Algae strains Cellulose purification Conversion to CNF: Conversion to CNF: Conversion to References pre-treatment main treatment CNC: chemical (biological or (mechanical) treatment chemical)

Nannochloropsis salina, Ethanol hexane, TEMPO oxidation – – [22] Nannochloropsis oceanica Sodium hydroxide, Sodium chlorite Nannochloropsis gaditana Ethanol toluene, – – – [47] Sodium hydroxide, Sodium chlorite Posidonia oceanica Ethanol toluene, – – Hydrochloric acid [20] Hot water, Sodium chlorite, Potassium hydroxide Soda-anthraquinone, – – Sulfuric acid, [21] Sodium hypochlorite, Ultra-sonication Sodium hydroxide Ulva lactuca Ethanol, – – – [56] Ammonium oxalate, Sodium chlorite, Sodium hydroxide, Hydrochloric acid Cladophora sp., Sodium hydroxide, – – Hydrochloric acid [57] Valonia ventricosa, Hydrochloric acid or Boergesenia forbesii or Sulfuric acid Potassium hydroxide, Sodium chlorite Cladophora glomerata Sodium chlorite, Enzymatic hydrolysis Blending, – [55] Sodium hydroxide, Micro-fluidization Hydrochloric acid Sodium chlorite, – – – [165] Sodium hydroxide, Hydrochloric acid Cladophora sp. Sodium hydroxide, – – – [166] Hydrochloric acid Valonia ventricosa Sodium hydroxide, Sulfuric acid Ultrasonication – [58] Hydrochloric acid Sodium hydroxide, – – – [167] Hydrochloric acid, Sodium chlorite, Potassium hydroxide Cystosphaera jacquinottii Hot water, – – – [71] Ethanol toluene, Sodium hydroxide, Sodium chlorite Fucus vesiculosus, Supercritical fluid, – – – [72] Laminaria digitata Hydrochloric acid, Sodium carbonate, Hot water Gelidium elegans Ethanol toluene, – – Sulfuric acid [73] Sodium hydroxide, Hydrogen peroxide Red algae waste Hot water, – – Sulfuric acid, [19] (from agar production) Sodium hydroxide, Ultra-sonication Sodium chlorite quantity of pollutants produced [160]. candidates, which could be a very promising feedstock for CNF pro­ Very little is known about the influence of the algal cellulose struc­ duction if they exhibit similar “easy to delaminate” cellulose substruc­ ture on subsequent CNF formation. As mentioned previously, the ge­ ture. Coupled with its fast growth, decent cellulose content (at least for ometry of the TCs has an influenceon the cellulose microfibrilstructure, Nannochloropsis sp.), and absence of lignin, microalgae could be a which changes depending on the algae species. Many algal species differ serious competitor to wood-based CNF. from the classical rosette structure found in lignocellulosic biomass. Some specificmicroalgae such as Nannochloropsis sp. were found to have a porous structure of thin cellulose microfibrils [37], which makes the 4.1. Cellulose purification delamination process much easier compared to lignocellulosic biomass [22]. In that case, CNF can be produced without any mechanical treat­ Most of the cellulose purification treatment reported for algae con­ ment, thus significantlyreducing the energy consumption linked to CNF sisted of a combination of bleach (mainly chlorite), alkaline hydrolysis production. It would be worthwhile to investigate other microalgae (mainly sodium hydroxide), and dilute acid hydrolysis (hydrochloric acid), in some cases with additional hot water extraction (Table 4).

12 E. Zanchetta et al. Algal Research 56 (2021) 102288

Solvent-based (ethanol, ethanol-hexane, ethanol-toluene) or supercriti­ Cladophora, Boergesenia and Posidonia. Stronger hydrolysis conditions cal fluidextraction was also occasionally reported as techniques for lipid were reported by Bettaieb et al. and Kassab et al. [19,21] on pulp from ◦ extraction. Posidonia oceanica and red algae waste using 64% H2SO4 at 55 C for 20 The cellulose purificationof Nannochloropsis species was reported as to 40 min. The CNC was further sonicated for 5 min for reduction of a three-step process: lipid extraction, sodium hydroxide treatment and aggregate size. The crystallinity increased after the acid treatment and bleaching. Ethanol/hexane (1:1, v/v) or ethanol/toluene (32:68, v/v) is the aspect ratio of the CNC was unsurprisingly low with an average used for the defatting step, the lipid-free biomass is further treated by diameter of the nanocrystals of ≈10 nm and length of ≈300 nm. ◦ 2% or 4% NaOH at 80 C for 2 h to remove the hemicellulose, followed ◦ by bleaching at 70 C in acetate buffer (pH 4.8–4.9) using 1.7% NaClO2 4.3. Production of cellulose nanofibrils for 4 h or 6% to 10% NaClO2 for 2 h [22,47]. As no lignin is expected in Nannochloropsis sp., the bleaching step could be used to remove the Algal CNF can also be produced from previously purified cellulose pigments. However, a high concentration of bleach could have a nega­ pulp. Xiang et al. [55] achieved successful CNF production from Cla­ tive effect on the crystalline structure of cellulose. For instance, a 10% dophora glomerata using a combination of enzymatic hydrolysis (cellu­ NaClO2 concentration was reported to create more amorphous sites on lase enzyme blend containing cellulases, ß-glucosidases, and the semi-crystalline cellulose structure [47]. hemicellulases) and micro-fluidization.While cellulases helps to liberate For macroalgae species, a broader range of processes were proposed individual fibrils by hydrolysing the amorphous region of cellulose for cellulose purification. High lignin content species such as Posidonia (endoglucanases) or attacking the reducing and non-reducing ends of oceanica (about 30% lignin) needed strong delignification process to cellulose chains to produce tetra- and di-saccharides (cellobiohy­ obtain purifiedcellulose pulp. Bettaieb et al. [21] proposed a three-step drolases), the β-glucosidases further hydrolyses the tetra- and di- procedure: (i) biomass delignification using soda-anthraquinone (Soda- saccharides to glucose [168]. It was also shown that other enzymes AQ) treatment (20% NaOH and 0.1% anthraquinone) for 2 h at a tem­ such as hemicellulases (xylanase), laccases, and lytic polysaccharide ◦ ◦ perature ranging between 150 C and 170 C, (ii) bleaching with 12% monooxygenases (LPMO) increases cellulose accessibility to cellulase NaClO for 15 min at pH 12, (iii) alkaline treatment to remove hemi­ enzymes and improves fibre delamination [169–171]. In the case of ◦ cellulose using 4% NaOH at 80 C for 2 h. Usually, alkaline hydrolysis Xiang et al. [55], a 2% (w/v) cellulose suspension was mixed in a sodium treatment can remove lignin and a significant part of hemicellulose acetate buffer at pH 4.8 with an enzyme charge of 3 FPU⋅g 1 glucan, the ◦ [162], here anthraquinone was added to NaOH to catalyse delignifica­ solution was incubated at 50 C and shaken at 150 rpm between 1 h to tion [163]. Similarly, Tarchoun et al. [20] reported a softer treatment on 48 h. Cellulose samples with and without enzymatic treatment were milled dried Posidonia: (i) dewaxing using ethanol/toluene (1:2, v/v) soaked in water overnight before disintegration in a blender for 30 s at treatment, (ii) hot water extraction for 5 h to remove sugars, colouring 30000 rpm. The suspension diluted to 0.2 wt% was then passed 10 to 20- ◦ matter, and starch, (iii) bleaching at 70 C using 1.7% NaClO2 in acetic times in a 200 μm microfluidizer chamber and an additional 10 to 20- acid solution (pH 3–4) for 1 h to remove lignin (the process was repeated times in an 87 μm chamber. This produced fibrils of 10–40 nm diam­ ◦ 7-times), (iv) 5% KOH treatment at room temperature for 24 h and 90 C eter and several μm in length with a crystallinity ranging between 80% for 2 h to remove hemicelluloses and residual pectin. It should be noted and 85% which showed that the pre-treatment decreased the crystal­ that at concentration beyond 8 to 9% of NaOH, cellulose mercerization linity, but had little change to the diameter distribution. However, the occurs which leads to irreversible conversion of the cellulose structure use of endoglucanase, exoglucanase and ß-glucosidases blend is ques­ from native cellulose I to cellulose II [164]. However, this can be tionable since it was shown that endoglucanase alone helped reduce beneficial for further pre-treatment as the hydrogen-bond becomes depolymerization compared to multi-enzyme blend while producing a disordered (swelling), resulting in increasing the accessibility of chem­ better separation of the nanofibrils [172]. icals or enzymes between the fibres. Hanley et al. [58] proposed to isolate cellulose microfibrils from Lower lignin content macroalgae such as Ulva lactuca (about 10% previously purified cell wall of Valonia ventricosa by a combination of lignin reported [66]) could also be purified under similar bleaching strong acid hydrolysis (67% H2SO4 at room temperature for 30 min and ◦ conditions than the soft treatment for Posidonia oceanica. After lipids and 70 C for another 30 min) and ultra-sonication (2 min on a 1 wt% sus­ extractives removal (ethanol treatment), and ulvan extraction using pension). This may be surprising since strong acid hydrolysis would tend ◦ ammonium oxalate, the biomass was bleached at 60 C using 2% NaClO2 to produce nanocrystals rather than nanofibrils.However, this treatment in 5% acetic acid solution until biomass discoloured and turned white. was found to isolate fibrilsof several μm in length and 7–12 nm in width, An alternative strategy for hemicellulose extraction was reported here which is closer to CNF than CNC. This result could be explained by the ◦ using 0.2% NaOH at 60 C treated overnight, followed by boiling at 5% extraordinary resistance of Valonia cell wall to acidic treatments as it ◦ HCl, then at 30 C [56]. appeared that in 72% sulfuric acid, the cell wall does not swell and the High hemicellulose content in macroalgae such as that found in red microfibrilsare hardly affected [65]. In that case, the residence time was algae Gelidium elegans (about 30% hemicellulose [66]) was also purified much less (1 h) than CNC production from Valonia sp. reported above (3 under similar alkali conditions as reported above. A 2% NaOH treatment days), showing the decrease of treatment severity. ◦ at 80 C for 2 h was able to decrease hemicellulose from 29.5 to 8.2% Lee et al. [22] showed that it was possible to produce CNF from while decreasing the lignin content slightly (from 4.5 to 3.4%). An microalgae (Nannochloropsis sp.) without any mechanical treatment to alternative bleach treatment using hydrogen peroxide (30% H2O2 at drastically reduce the energy consumption usually needed for the me­ ◦ 80 C for 1.5 h, twice) was used afterwards to remove residual hemi­ chanical shearing of the cellulose pulp. The previously purifiedcellulose cellulose and lignin, (decreasing to 2.0% and 0.7% respectively) slurry was subjected to TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) showing the efficiencyof the combination between alkali treatment and oxidation to enhance delamination to individual fibril. TEMPO oxida­ bleach for purification. tion is one among many chemical pre-treatments that could be used to introduce repulsive ionic charges between elementary cellulose fibres, 4.2. Production of cellulose nanocrystals and help their delamination by weakening the cohesion of hydrogen bonds between the fibres [158,168]. Chemical pre-treatments can tune After cellulose purification, cellulose nanocrystals were sometimes cellulose with new properties by functionalization of its surface and produced using concentrated hydrochloric or sulfuric acid. Imai et al. produce a unique grade of CNF [168]. TEMPO/NaBr/NaClO was mixed and Tarchoun et al. [20,57] reported the production of algal CNC using (gently) to the cellulose pulp for 2 h, while the pH was maintained at 10 9% HCl solution boiled from 30 min to 12 h or using 40% H2SO4 solution using a 2% NaOH solution [22]. The isolated fibrils were 1–9 μm and ◦ mixed at 70 C for 3 days on cellulose pulp originating from Valonia, about 10 nm wide with an exceptionally high crystallinity (91.7%). It

13 E. Zanchetta et al. Algal Research 56 (2021) 102288 was suggested that the efficientfibrillation to CNF (without mechanical 4.4.1. Cellulose purification treatment) could be due to two factors: (i) the material was never dried The literature on algal cellulose production revealed that a combi­ during the process which helps to avoid tight agglomeration of cellulose nation between alkaline hydrolysis (NaOH, sometimes KOH) and bleach fibres through hydrogen bonding and allows reagents to find space be­ (NaClO2, sometimes NaClO), with potential additional solvent-based tween the fibrils, (ii) the cellulose layer of Nannochloropsis sp. has a lipid extractions, was efficient in most cases to extract a purified cellu­ delicate fibrous substructure (fluffy and porous) composed of thinner lose pulp from raw biomass. However, comparative studies of the best fibrils compared to the one of red algae and land plants arranged purificationprocedure for specificalgae species is still lacking. It might through linear rows or rosettes, no such structure was found in Nanno­ be worth trying other processes to see if the use of a bleaching agent can chloropsis sp. [22,37]. Further research is needed to understand if other be avoided or limited. For instance, dilute acid hydrolysis or hot water pre-treatments would lead to the same efficiency of delamination. For extraction was not often reported. Dilute acid hydrolysis (typically <4 TEMPO oxidation, one improvement could be to use TEMPO/NaClO/ wt%) using various mineral or organic acids at elevated temperature NaClO2 instead of TEMPO/NaBr/NaClO as the former has been reported was shown to efficiently hydrolyse hemicellulose [175], while more to overcome cellulose depolymerization [173,174]. However, the use of severe conditions (higher temperature or concentration) tend to bleaching agents such as NaClO or NaClO2 should be avoided if possible hydrolyse cellulose to individual glucose units [176]. Liquid hot water due to their toxic potential. Additionally, the chemicals generally used treatment was also shown to cause significant hemicellulose solubili­ for TEMPO oxidation are difficultto recover and expensive [158]. Other sation and partial lignin degradation, while possessing the advantage of cellulose functionalization (chemical pre-treatment) or pre-treatment being chemical-free [162]. without functionalization (e.g., enzymatic hydrolysis) should be tested Other selective solvent-based purification processes could also be to findmore cost-effective and environmentally benign solutions of CNF used for algal biomass fractionation. They include for instance organo- production from Nannochloropsis sp. solvation and ionic liquids. Organo-solvation can involve a variety of organic or aqueous-organic solvent mixture (with or without acid catalyst) to efficiently remove hemicellulose and lignin (if necessary) 4.4. Future opportunities for algal nanocellulose production [175], while ionic liquids are able to specifically remove certain com­ ponents (e.g., lignin) depending on the solvent used [162]. The current literature on the production of cellulose or nanocellulose (CNF or CNC) from algae is very scarce. Herein, we propose a bigger 4.4.2. Production of cellulose nanofibrils picture of the processes that can be used to produce CNF or CNC from The production of algal CNF reported only the classical processes algal biomass to identify future opportunities (Fig. 6). It should be noted that are generally used for nanofibrillation, biological (enzymatic) pre- that by changing the combination of cellulose purification processes, treatment, classical chemical pre-treatment (TEMPO oxidation) [158], pre-treatment and mechanical treatment, various grades of CNF with and mechanical treatment (high-pressure homogenization, in that case unique properties (fibril morphology, crystallinity, degree of polymeri­ with a microfluidizer) [168]. In addition to the fact that it would be zation, etc.) can be obtained from the same biomass [168]. Post- interesting to test other algae species using these conventional pro­ treatment could also be added afterwards to functionalize CNF with cesses, it also seems relevant to test other pre-treatment or mechanical specific properties (modification by adsorption, molecular or polymer treatment processes to produce CNF with unique properties. grafting) [158,168] or fractionate the nanofibrils (centrifugation, The available chemical pre-treatments can be separated in two main filtration) [168].

Producon of CNC

Chemical treatment

Cellulose - Acid hydrolysis Algal biomass Producon of CNF purificaon

Mechanical Biological/chemical Mechanical Post-treatment - Alkaline hydrolysis pre-treatment pre-treatment treatment - Bleaching - Dilute acid hydrolysis - Liquid hot water - Blending Biological modificaon Convenonal: -Chemical -Organo-solvaon -Refining - Enzymac hydrolysis - Homogenizaon modificaon - Ionic liquids - Grinding - Grinding - Fraconaon Chemical modificaon -Refining of hydroxyls groups: -Tempo oxidaon Emerging: -Carboxymethylaon -Extrusion - Caonizaon - Blending -Sulfoethylaon - Ultrasonicaon - Phosphorylaon - Cryo-crushing Chemical modificaon -Steam explosion by ring opening: - Ball milling - Periodate oxidaon - Aqueous counter -Ozonaon collision

Fig. 6. Possible conversion processes from algal biomass to nanocellulose materials. A first purification step is required to separate cellulose from the other com­ ponents of the biomass. A combination of several purificationtreatments can be considered depending on the biomass composition. In a second step, the cellulose can be converted to cellulose nanocrystals (CNC) or cellulose nanofibrils (CNF). A large variety of processes is available for CNF conversion. Usually the mechanical treatment can be sufficient to achieve cellulose fibrillation and CNF formation, however a pre-treatment step (biological, chemical, mechanical) can facilitate the fibrillation or in some specific cases directly achieve total fibrillation to CNF.

14 E. Zanchetta et al. Algal Research 56 (2021) 102288 categories: (i) modificationof cellulose hydroxyls groups, (ii) opening of policymakers, requested by conscientious customers, developed by re­ the cellulose ring and introducing the aldehyde groups (dialdehyde searchers, commercialised by industry and praised by most as a “greener cellulose) which are then modified to other functional groups [158]. and better” solution. However, there remains the problem of whether Hydroxyl groups can be modified to carboxyl (TEMPO oxidation) these new products offer the complete consumer experience (and ex­ [174,177,178], carboxymethyl (carboxymethylation) [179,180], sulfo­ pectations) as advertised by industry, in particular pertaining to nate (sulfoethylation) [181], and phosphoryl (phosphorylation) groups biodegradability and compostability promised by producers as a supe­ [182,183], or by introduction of cationic ammonium sites (cationiza­ rior alternative to current non-renewable material [209–211]. Tradi­ tion) [184–186]. For the ring-opening methods, periodate oxidation can tionally, industry had focused predominantly on the development of a be used to cut the C—C bond between C2 and C3 positions on the cel­ stable, long-lasting product capable of sealing perishable goods and lulose ring to form aldehyde groups, which can be turned to alcohol increasing their edibility duration [212]. Recently, the focus has shifted [187], carboxyl [157,188] and sulphite groups [189,190], or cationic towards improving the material life cycle to balance the need for quality ammonium [191,192]. Ozonation has also been reported to be a cheap as well as the product specificationof plastics produced from renewable method to introduce carbonyl groups [193]. Soft mechanical treatment biomass [213]. In addition to this, the biodegradability, compostability, could also be used as pre-treatment in addition to biological or chemical benignity towards animals and environment is also a metric considered pre-treatment or as a unique pre-treatment step. This includes refining, towards the progressive shift, as pointed out by Sidek et al. [213]. Thus, blending, and grinding [194–197]. novel materials are required to meet the established international ma­ The mechanical treatments generally achieve total fibrillation to terial standards tested by officialcertification bodies (e.g., DIN CERTCO individual cellulose fibres (i.e., CNF) using high shear force processes. (Germany) TÜV Austria (Austria), etc.) in order to guarantee a uniform Conventional processes include high pressure homogenization (ho­ certification representative of the standard specified. These standards mogenizer, microfluidizer)[ 198,199], ultra-finefriction grinding [200] are based on conventional packaging material assessments which are and refining [201]. These processes are considered to be the most effi­ recoverable through composting and biodegradation, e.g., EN 13432. cient to delaminate cellulose bundles for CNF production, and are suit­ Unfortunately, the scientific results show that the established industry able for upscaling [168]. Other emerging processes, as shown in Fig. 6, standards for such certifications might not be fully applicable to can also be used for mechanical disruption [202–208]. degradable bioplastics. They do not project a realistic lifetime and therefore have to be adapted, as stipulated by Harrison et al. [214], Van 5. Past, current and future obstacles in End of Life scenarios den Oever et al. and Haider et al. [215,216] address the issue to inves­ linked to bioplastic tigate the special certification requirements for biodegradable plastics, as well as present drawback and improvement potential of present This section provides some insight into the current research activities testing scenarios. Haider et al. point out “the necessity [for tests under] and state of the art in relation to the possibilities and the difficulties environmentally authentic and relevant field-testing conditions”, thus, con­ encountered towards the various end of life scenarios of bioplastic ma­ firming that the current standard certifications do not embody realistic terials (Fig. 7). Although it is not directly linked to the production of CNF parameters. from algae and materials produced from it, the discussion herein is Furthermore, Napper et al. [217] showed the low degradability of necessary to identify, name, and ultimately prevent the issues evolving biodegradable certified plastic shopping bags, but with non-official la­ currently within this domain. It is understood that highlighting the bels. The study led to widespread media attention, potentially resulting present situation will ensure that algal-based materials do not suffer in a loss of consumer confidencein biodegradable materials as shown by from the same uncertain end of life scenarios as current bioplastics from Blesin et al. [218]. These findingsraise another important question as to other sources. why uncertified and unsuitable bioplastics receive approval for distri­ Alternatives to petro-based plastics are primarily wanted by bution to consumers.

End of Life

Waste Disposal Liering

Problemac due to various product Separaon Land Environments Aquac Environments materials & mixtures ISO 4582, ISO 4892, ISO 15851, 14852, ISO 10640, ... 14853, .. Recycling: ISO 15270, … (mechanical, chemical) Problems:

CH4 Industrial Composng: EN 13432, - No uniform standards and protocols for tesng available Ferle Substrate (aerobic, anaerobic) EN 14995, … - Convenonal plasc standards are being used

CH4 - Detecon of degradaon products: Micro(nano)plascs Home Composng ISO 14855, … Ferle Substrate - Analycal methods not established yet Ash Incineraon - Effects on microbial community, flora and fauna Heat/Electricity

CH4 Landfill

Fig. 7. Possible End of Life scenarios for bioplastic materials.

15 E. Zanchetta et al. Algal Research 56 (2021) 102288

Standard protocols like DIN ISO 4892 [219,220] exist to assess and heavily focused on the cultivation steps where a tradeoff between high indicate the lifespan of plastic materials under accelerated weathering productivity (and control) in a photobioreactor (PBR) versus a lower conditions. Friedrich et al. [221] showed that by using Wood-Plastic- energy input and environmental footprint in an open-pond reactor Composite in accelerated weathering tests and under real conditions, provided interesting results. However, very limited amount of research there was no correlation between results obtained from laboratory ex­ exists on algae bioplastics [235], as most of the focus has thus far been periments and under real conditions. Shimizu et al. [222] reached a on wastewater water treatment using algae for biofuels production. similar conclusion while trying to link polypropylene degradation from To this effect, the most relevant study towards developing an LCA for accelerated to natural weathering, but indicated that the carbonyl index algae-based bioplastics to date was conducted by Beckstrom et al. [236]. is an applicable measure. Different locations and therefore varying pa­ Based on cultivation in an open pond reactor (OPR), circular flow rameters like yearly rainfall, average temperature, soil pH and structure, photobioreactor (CF-PBR) and combined OPR/CF-PBR, as well as three aeration, prevailing enzymes, humidity etc. could have a major impact processing routes entailing fractionation, lipid extraction and drying on the degradation process, but these cannot be fully replicated in a only, bioplastic feedstock preparation was considered within the system laboratory environment, and thus require fieldtrials at various locations boundary. Under different scenarios, the most promising bioplastic [223,224]. Additionally, the accumulation of plastic residues in soil can feedstock preparation route utilised the CF-PBR. The emissions associ­ result in poor harvest as well as affect the local wildlife. Hegan et al. ated with the CF-PBR and OPR accounted for 0.315 and 0.656 kg CO2, [225] investigated the degree of annual film mulching of cotton fields, eq/kg bioplastic feedstock via drying and fractionation processing where a clear trend was detected showing a constant decrease in cotton routes, respectively. The negative emissions accounted mostly from the yield from accumulation of plastic material during harvest cycles over presence of biogenic CO2 from the atmosphere during cultivation. the years. However, this credit was eroded via the fractionation route since one of According to Van den Oever et al. [226], composting is currently the the by-products was fuel, resulting in emissions during its combustions. best solution to fully decompose biodegradable plastic material, fol­ It is well understood that in order to truly support advancement of algae lowed by recycling and/or landfilling. Kern et al. [227] tested com­ as a source of commodity chemical, material or fuel, the vision of an posting bag materials “in-situ” at four German composting facilities, and algal biorefinery should be realised. To maximise sustainability and showed that all tested materials decompose visually within the standard commercial viability, such a process would be expected to create mul­ process time of the facility but without analysing the chemical debris of tiple products, which could in theory also be fuels. As presented in the the degradation process. Even if the permitted proportion of materials study above, this could in fact make the algal to bioplastics vision other than biomass (e.g., additives, plasticizers) in bioplastics is below environmentally susceptible. It is known that resins such as nylon and 10 wt%, they can still pose a detrimental affect in the compost as well as polypropylene hold environmental burdens of 8.03 and 1.98 kg CO2,eq/ the soil, potentially resulting in negative effects on microbial life, plant kg bioplastic [237], respectively, which is approximately three times growth [228,229], or even human health [230]. greater than that computed by Beckstrom et al. [236]. It could be argued As such, the question arises as to what is really happening to all the that the environmental burdens associated with algal processing can be additives and plasticizers once the material decomposes. In particular, improved via the use of renewable energy sources in the energy input monitoring and determining degradation in aqueous environments is stream, but such a hypothesis is not unambiguous [238]. especially challenging given the variations and conditions involved. It Further to this, any bioplastic material designed with a view of could be suggested that this is primarily due to the lack of experience in sustainability should consider biodegradation. During this process, there open environment degradation, but could also be from the lack of will certainly be an aspect of CO2 being released to the atmosphere once established protocols and analytical methods to assess the whole end of the material starts to decompose. Thus, any positive effects computed life. Approaches have been made to identify suitable analytical methods during the bioplastic resin manufacturing boundary would be lost as the but the nature of biodegradable material presents a special challenge to material reaches its grave. Such cradle-to-grave approach as opposed to laboratory equipment and procedures [231,232]. Even though labora­ cradle-to-gate or other such variant can yield misaligned environmental tory tests lead to reliable and reproducible results in a controlled envi­ footprints, or even not represent the hotspots appropriately. Especially, ronment, the outcomes do not represent reality as the conditions are not since the end-of-life aspect as well as the burden associated with supply sufficiently simulated [233]. Hence, the results are not transferable to chain remains unattended. Thus, an important point to consider is the real-life scenarios as weathering under natural environments and the reliability of the results presented from LCAs carried out in various material life and consequences cannot be precisely projected for various studies. Not only are there substantial differences from factors reasons, which is also mentioned in the international standard as pin­ mentioned above, but there are also inconsistencies present due to the pointed by Crewdson [234]. standard methodology applied to obtain the environmental burdens and Ultimately, the main research question that enables the circularity of or data-sets for biopolymers in the inventory [239]. Considering the new products and innovative processes should also be based on ques­ limited number of investigations currently available in the algal bio­ tions such as: what is the true End of Life scenario for bioplastics plastics field, it could be worth bearing in mind that a standardised currently? Can it be properly recycled and reintegrated into the supply methodology to ensure credibility and ease of comparison of the burdens ¨ chain without using new (or minimal) materials coming from renewable is respected. As such, Ogmundarson et al. [240] recommended the ISO resources or will it ultimately complicate conventional plastic recycling 14040 standard series as a benchmark. Similarly, ISO 14044 could also once it is introduced to the recycling stream? Van den Oever et al. [215] prove to be a successful means towards harmonizing results from future showed that biomaterials in conventional plastic recycling stream had studies since they are both based on following a cradle-to-grave very little problems for actual recyclability. However, concerns remain approach. about bioplastic materials to which chemicals are added to increase the desired material properties. These will eventually create problems in the 7. Conclusion long run [229], even though research is not yet able to identify, quantify and name all the end products sufficiently. The vision of an algal biorefineryoffers a promising alternative to the traditional petro-chemical industry. Although several algal products 6. Life cycle assessment of algal bioplastic have now been commercialised at scale (nutraceuticals, value products, etc.), the complete concept suggested by many researchers has not yet It should be noted that computation of the life cycle assessment been reached. At present, research on algal cellulose is limited, but (LCA) is very much dependent, but not limited to, the system boundary, rapidly growing, and the desirability of species with high purity and products considered as well process variables and route. Past research quantity is much sought after. Stress-induced methods to increase

16 E. Zanchetta et al. Algal Research 56 (2021) 102288 cellulose production biologically can be achieved, but this usually comes 6. Adrian Pulgarin ([email protected]) at the expense of the expense of productivity. To that effect, genetic engineering techniques such as gene editing or recombinant DNA could - Conducting a research and investigation process, data/evidence be beneficial, provided the regulatory organizations and governments collection allow modified species to be used commercially. Bioplastics are an - Writing: Review and Editing important and crucial element in this puzzle with demand in line with quantities of petro-fuels. Thus, the infancy of the algal bioplastic field 7. Prof. Christian Ludwig ([email protected]) has to be overcome as there is already a pull factor from the market resulting in producers/users finallypushing for commercialisation. The - Critical revision of the article for important intellectual content costs remain uncertain given the extraction and conversion process of - Administrative, technical, or logistic support cellulose when applied to algae, particularly for technologies that are - Supervision translated from existing biomass feedstock. However, the technical un­ - Conceptualisation derstanding, research and development as well as prototypes to show­ - Writing: Review and Editing case algae’s potential is severely lacking and has to be recognised as a bottleneck. To close the material loop for bioplastics (and realise an Declaration of competing interest algal biorefinery), it is of utmost importance to collaborate within and beyond the technical fieldsto produce a product that is at the forefront The authors declare that they have no known competing financial of innovation, both in sustainability and acceptability. Whether the algal interests or personal relationships that could have appeared to influence bioplastics vision will push forward the algal field into the commercial the work reported in this paper. realm or prove to be another potential by-product with limited eco­ nomic/environmental credential remains to be seen. Acknowledgment CRediT authorship contribution statement The authors would like to thank the Innosuisse - Swiss Innovation Agency, forming part of the Swiss Competence Center for Energy 1. Enio Zanchetta ([email protected]) Research SCCER BIOSWEET for their support. We also thank Dr. Michel Rossi for providing insights during his impartial reviewing of the article - Conceptualisation and Mr. Clement Derrien for his general inputs on alternative feedstocks - Data curation to produce bioplastic. - Methodology - Visualisation - Writing – original draft References - Project Administration [1] K. Gunaalan, E. Fabbri, M. 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