Microalgal biomass pretreatment for integrated processing into biofuels, food, and feed

Bioresource Technology 300 (2020) 122719

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Review

Microalgal biomass pretreatment for integrated processing into biofuels, food, and feed

T

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Júlio C. de Carvalho , Antônio Irineudo Magalhães Jr., Gilberto Vinicius de Melo Pereira, Adriane Bianchi Pedroni Medeiros, Eduardo Bittencourt Sydney, Cristine Rodrigues, Denisse Tatiana Molina Aulestia, Luciana Porto de Souza Vandenberghe, Vanete Thomaz Soccol, Carlos Ricardo Soccol Federal University of Paraná, Department of Bioprocess Engineering and Biotechnology, Centro Politécnico, 81531-990 Curitiba, Paraná, Brazil

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO

A B S T R A C T

Keywords: Drying Pyrolysis Cell disruption Proteins Lipids

Microalgae are sources of nutritional products and biofuels. However, their economical processing is challenging, because of (i) the inherently low concentration of biomass in algal cultures, below 0.5%, (ii) the high-water content in the harvested biomass, above 70%; and (iii) the variable intracellular content and composition. Cell wall structure and strength vary enormously among microalgae, from naked Dunaliella cells to robust Haematococcus cysts. High-value products justify using fast and energy-intensive processes, ranging from 0.23 kWh/kg dry biomass in high-pressure homogenization, to 6 kWh/kg dry biomass in sonication. However, in biofuels production, the energy input must be minimized, requiring slower, thermal or chemical pretreatments. Whichever the primary fraction of interest, the spent biomass can be processed into valuable by-products. This review discusses microalgal cell structure and composition, how it affects pretreatment, focusing on technologies tested for large scale or promising for industrial processes, and how these can be integrated into algal biorefineries.

1. Introduction Microalgae products are one of the frontiers of a sustainable bioeconomy. What could be better than using these fast-growing, photosynthetic microorganisms to produce biofuels, food, feeds, and specialty chemicals ranging from vitamins to biopolymers? And yet, despite projections of significant growth in the next decades (Ruiz et al., 2016), the market today is still modest. While the overall exports of

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food-grade seaweed products (algae and microalgae) was about 216,000 tonnes at a value of 568 million USD in 2017 (United Nations Statistics Division, 2017), and traded at roughly 2.6 USD/kg, it is estimated that the microalgae market is quite smaller, at about 40 million USD/yr. The global biomass market is dominated by Arthrospira (formerly Spirulina), Chlorella and Dunaliella, with production estimates ranging from 5 to 10, 2–4 and 1 ktonne/yr, respectively) (Benemann, 2013; Milledge, 2012; Spolaore et al., 2006; Vigani et al., 2015). The

Corresponding author. E-mail address: [email protected] (J.C. de Carvalho).

https://doi.org/10.1016/j.biortech.2019.122719 Received 29 October 2019; Received in revised form 29 December 2019; Accepted 31 December 2019 Available online 08 January 2020 0960-8524/ © 2020 Elsevier Ltd. All rights reserved.

Bioresource Technology 300 (2020) 122719

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Fig. 1. Phylogenetic tree of 25 industrially relevant microalgal species, showing the phylogenetic relationship through 16S and 18S rRNA gene sequences retrieved from the GenBank database. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. For more information about the sequences, see Supplementary Material S1.

microalgae encompasses genera not only of different order, classes or phylum, but diverse kingdoms: Eubacteria genera such as Arthrospira sp.; several eukaryotes – mostly Plantae such as Chlorella, Haematococcus, Dunaliella, and Botryococcus, but also Chromistae such as Phaeodactylum and Nannochloropsis, and even members from Protozoa (Euglena) and Fungi (Schizochytrium). The latter is not photosynthetic, thus not a microalga. However, it is often referred to as such and is of industrial relevance. This diversity in microalgae is reflected in the myriad products, and also in the quite different processing approaches, which are dependent on the cell composition and structure. Fig. 2 illustrates the cell wall structure of 10 selected microalgae, showing the difference in layer composition and disposition. The cell membrane is protected by relatively thick carbohydrate-based fibrillar walls. Cell wall permeation, disruption, or hydrolysis is an essential goal in microalgae biomass pretreatment. Algal biomass pretreatment is intertwined with harvesting and fractionation. Microalgae can serve as fuel, feed, food, or fractionated into fine chemicals with various uses—from additives to nutraceuticals to pharmaceutical ingredients. There is a marked difference in biomass processing relative to each use; however, three main steps are necessary after microalgal production, viz., 1) biomass recovery, which is usually done by flocculation followed by sedimentation, centrifugation or filtration, 2) biomass pretreatment, to enhance digestibility, or the access to molecules of interest in the next processing step, and 3) product isolation and purification, which is highly specific for the biomolecule or fraction of interest, and follows traditional bioprocessing strategies (Khan et al., 2018; Martinez-Guerra and Gude, 2016; Medipally et al., 2015; Vermuë et al., 2018). While steps 1 and 3 are somewhat similar to what is used in other bioprocesses such as fermented products and biofuel production, step 2 (microalgal biomass pretreatment) involves

industrial production of valuable fractions is in its early stage and still developing (Vigani et al., 2015), which makes it the object of intense research. This limited market tends to expand with the development of low-cost biomass production and efficient processing technologies. As with any biomass, microalgae must be pretreated for further processing. However, unlike lignocellulosic biomasses, size reduction is not a primary concern; the vast majority of microalgae—currently produced or considered for industrial purposes—is already microscopic, with size reaching about 5–50 μm (Kirnev et al., 2018). Nevertheless, each of these tiny cells is a complete, complex, and often robust structure. In cells, the molecules of interest—the very product—are protected by a membrane and cell wall. The pretreatment of microalgal biomass is essential to expose this material, either for further digestion or for extraction. However, microalgae are not all the same: these microorganisms are as diverse as trees are from grasses, or as different tissues are in a single plant. The term “alga” has not a taxonomic significance; algae are oxygenic photosynthesizers other than land plants (Cavalier-Smith, 2007), while “microalgae” describes microscopic algae, not necessarily unicellular (Molina et al., 2019). Fig. 1 illustrates the rRNA-based phylogeny of selected microalgae, showing the vast diversity of microalgae popular in research and industry. The 25 species presented in Fig. 1 are those of higher market volume or more intensively researched (Caporgno and Mathys, 2018; Chilton et al., 2016; Hemaiswarya et al., 2011; Rizwan et al., 2018), using currently accepted names (e.g., Arthrospira platensis instead of Spirulina platensis), and the most common species of the genus when more than one is relevant (e.g., Chlorella vulgaris instead of C. sorokiniana) (Borowitzka, 2016; Guiry and Guiry, 2019). The phylogenetic relationship was evaluated through 16S and 18S rRNA gene sequences retrieved from the GenBank database and built using the standard tree builder of Geneious® 10.2.3. The group commonly referred as 2

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Fig. 2. Cell wall structure of 10 important microalgae, illustrating the main components and the diversity of structure. Layers are not to scale – the cell wall is 10 to 100 times thicker than the cell membrane.

60%, and lipids from 5 to 30%. Biomasses subjected to stress conditions or heterotrophic growth can accumulate more of one of these fractions. Besides the classical protein-carbohydrate-lipid form of evaluating microalgal composition, three other aspects are essential: the cell growth rate, the content of specific, valuable molecules such as carotenoids, and the composition of the cell wall. High growth rate (=high productivity), and the high content of a desired fraction are both obvious advantages in industrial strains. However, as Fig. 3 shows, nutrient-rich biomasses—the ones with higher growth rates—have a high protein content; conversely, lipid-rich biomasses can be produced after a nutrient-depleted phase where biomass concentration increases only slightly, but lipids accumulate over time. The process changes the composition but reduces productivity. Finally, knowing the composition of cell walls of microalgal biomasses is vital because it shows the possible targets for lytic enzymes, which can be used to weaken or disrupt the wall. It also gives insights into what possible polysaccharides can be derived from its partial digestion. Table 1 illustrates the cell wall composition of important microalgae. One crucial advantage of microalgal biomass as a feedstock is that it does not contain lignin, making it easier to hydrolyze compared to lignocellulosic materials. But also contrary to lignocellulose, there is a high diversity not only in the proportion, but also in the types of polysaccharides that constitute the cell wall (Saratale et al., 2018). The popular Chlorella vulgaris, for example, has chitin-like polysaccharides

breaking the tough polymers present in the cell envelope of microalgae (Alhattab et al., 2019), which can be energetically expensive. It is also the first step that potentially changes composition, with complex transformations that affect the products. Therefore, biomass pretreatment is a contemporary concern in microalgal research. Most research in the area is aimed at biomass biodigestion, or conversion in liquid biofuels, or in drying and cell disruption for further processing in biorefineries, frequently with lipids as the product of interest—but also with more refined products such as vitamins, biopigments or peptides. This review discusses relevant aspects of the pretreatment of biomass and how it is affected by biomass recovery and further product isolation and purification. 2. Microalgal biomass composition Understanding the composition of biomass is essential for (i) selecting adequate microorganisms for production of a specific fraction (e.g., lipid- or protein-rich content), (ii) identifying biomass components that could be potential by-products, and can be integrated in a biorefinery process (Velazquez-Lucio et al., 2018), and (iii) evaluating processing aids, such as enzymes, that can target specific molecules. Fig. 3 shows the basic chemical composition of microalgal species of industrial relevance, mostly grown in nutrient-rich media. In general, its protein contents ranging from 30 to 75%, carbohydrates from 25 to 3

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Fig. 3. Ternary diagram for the biochemical composition of microalgal species of industrial relevance. Protein, carbohydrates and lipids are presented as % of the organic (CHNO) fraction. Bubble sizes are proportional to the mineral content (ash), averaging an extra 8%. Data compiled from (Brown et al., 1997; Giostri et al., 2016; Leow et al., 2015; Matos et al., 2016; Renaud et al., 1999; Santos et al., 2003; Shields and Lupatsch, 2012; Zhu et al., 1997).

inherently analytical, the flux of products necessarily integrates all steps: each process has requirements and outputs that affect the other processes. In this section, we take the integrative approach to briefly discuss the typical steps in microalgal biomass processing, showing where pretreatment is necessary. Fig. 4 shows typical operations required in biomass processing for selected products. After cultivation, microalgal biomass is typically very dilute, with concentrations ranging between 0.5 and 5 g/L (Kirnev et al., 2018). Concentrating the cell suspensions is essential to reduce the processing volumes and equipment sizes downstream. Filtration, centrifugation or sedimentation can be used to concentrate the culture, sometimes with the aid of flocculating agents. The resulting slurries or pastes reach concentrations of 2–3% solids (with simple sedimentation) to 10%

in its cell wall (Baudelet et al., 2017; Ortiz-Tena et al., 2016). Other genera, such as Thalassiosira and Cyclotella, also show chitin (Steinfeld et al., 2019). The presence of the genes for chitin synthase indicates the presence of this structural polysaccharide in the cells. Because of the diversity in polysaccharide composition, microalgal cells can respond quite differently to similar enzymatic cocktails. 3. Microalgal biomass processing basics Processing microalgal biomass into valuable products involves several steps (unit operations). The development of a new bioprocess requires an alternating view between integrative and analytical approaches. While the study and optimization of each processing step are

Table 1 Composition of the cell wall and selected fractions of industrially important microalgae. Polysaccharides and carbohydrates in cell walls (dry weight %), and glycosidic bond types

Arthrospira

Botryococcus Chlamydomonas a

Chlorella

Dunaliellab,c Haematococcus

b

b

Hemicellulose

Galactose

Rhamnose

Hexose

Mannose

Glucose

Xylose

–

5

–

–

50

35 β-1,4

8

–

–

–

–

–

–

–

–

–

–

–

–

56

22–25 α-1,2; β − 1,4; α-1,3 –

60–66 α-1,6; β − 1,3; β − 1,4 –

60–66 α-1,2; α-1,3

–

–

–

–

–

–

–

89 β-1,4

Arabinose

References Fucose

Glycocalyxtype

Glucans, possibly cellulose

2

–

–

–

–

–

–

–

–

–

–

–

–

–

–

β-1,4; β-1,3 32 β-1,3 –

–

–

–

–

–

89

6 β-1,4

1,3 β-1,4

1,6

–

Conditionspecific –

Kataoka and Misaki, 1983; van Eykelenburg, 1978 Borowitzka, 2018; Weiss et al., 2012 Baudelet et al., 2017; Grief et al., 1987 Acosta and Gross, 1995; Baudelet et al., 2017

–

Borowitzka, 2018

–

Baudelet et al., 2017

a Chlorellae have hemicellulosic and “rigid wall” fractions in the cell wall. The latter is made of galacto- and rhamnomannans for some groups, and glucosamine for other groups in a chitin-like polysaccharide. Linkages depend on specific growth conditions. b Cyst wall. c The cells do not have a cell wall. Sometimes are covered by polysaccharides.

4

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Fig. 4. Process outlines for selected microalgal bioproducts. Arrows indicate the unit operations that are most commonly used in the process. The processing steps sequence is from top to bottom. Pretreatment steps are highlighted.

components do not need to be dried initially, but rather conditioned—disrupted or otherwise modified. This step can enhance digestibility and accessibility to target biomolecules. Besides, cells may not necessarily need disruption per se: the cell wall is porous enough for the diffusion of small molecules if the cell membrane is permeated or solubilized (de Carvalho et al., 2017b). The biomass, thus conditioned, can be fractionated, e.g., by a preliminary lipid solubilization step, followed by a protein and carbohydrate solubilization step. The product of each extraction is a mixture of compounds that may require further purification; for example, the extraction of a lipid fraction with a low polarity solvent mixture such as methanol: chloroform gives a fraction rich in nonpolar and polar lipids that can be segregated in triacylglycerols and lipoproteins (Patil et al., 2018, Khanra et al., 2018); a more polar solvent such as hot ethanol may also extract carbohydrates and proteins, and processing disrupted biomasses followed by solids separation with water gives a protein- and mineralsrich extract. In some cases, the raw extract can be directly dried into a product—e.g., lipids from Isochrysis sp., astaxanthin from Haematococcus sp., or protein extract from Arthrospira sp. (Bhalamurugan et al.,

solids (for centrifuged pastes, such as that obtained by Xiao et al., 2011). Even vacuum- or pressure-filtered biomass cakes still have intracellular water; therefore, the solids content will generally reach at best 30%. The mineral content attached to microalgal biomasses can be high, and depending on the biomass use, the suspension can be washed one or more times during this step. The cell concentrate can be dried if required by the next processing step; water complicates the extraction of lipids or carotenoids with nonpolar solvents, for example, while direct transesterification is especially sensitive to water (Sorgatto et al., 2019). The drying process consumes energy for phase transition, and temperature can affect the biomass composition and structure; but ultimately, the process stabilizes biomass for further processing. Biomass suspension concentrates can be stored cold or frozen, with diverse viability of the cells after storage. Some pretreatment techniques such as hydrothermal liquefaction (HTL) or wet extraction can handle wet biomass (Neto et al., 2019), and others—especially for cell disruption—may indeed require the cells to be suspended in liquid. Biomass that is used for foods, feeds or for the extraction of specific 5

Bioresource Technology 300 (2020) 122719

6

Fast High-quality biomass Moderate High

High

Fast, generates powder.

Higher energy intensiveness, requires large air volumes Higher energy intensiveness, requires large air volumes Degrades biomass Slow, high CAPEX, Fast, generates powder. High

Can degrade biomass Practical Moderate

Fast, highly controllable

Slow, requires large areas, depends on the weather High CAPEX, higher safety concerns Low energy cost

Conduction or Contact (drum, tray) Lyophilization (freeze-drying)

Warm air heats the biomass and carries the water

Direct or Convective (oven, tray, tunnel) Spray

Warm air heats and carries the water, in a mixed suspended solid medium Warm surfaces heat the biomass Warm surfaces heat frozen biomass. Water sublimates.

Microwave heating Microwave drying

Warm liquid and air carry the water

Solar heating of the biomass Solar

Drying can be used as a pretreatment for further extraction or for simple biomass stabilization, because the low water activity reduces microbial and chemical decomposition. The process consists mainly of water removal, which depends on heat transfer and water diffusion (Biz et al., 2019). However, other phenomena can occur: the biomass components and the cell structure can be affected due to denaturation and even mechanical effects. This can have consequences in post-processing, from the enhancement of the digestibility to protein precipitation, enzyme inactivation, and cell collapse. When the dried biomass is later extracted for valuable products such as vitamins or

Principle

4.1. Drying

Drying method

Table 2 Methods applicable for microalgae drying.

Effect on biomass

The vast diversity of microalgae and derived products leads to a variety of pretreatments; there is no “silver bullet” that can be used for all biomasses. However, most methods can be classified into four categories according to the goal: 1) drying, 2) thermochemical processing, 3) cell disruption, and 4) targeted fractionation. There is some overlapping between categories, and different methods can be combined. Alkali or acid treatment, for example, usually includes thermal processing. Fig. 5 shows a bibliometric evaluation of terms appearing in the microalgal biomass processing literature, from 45 recent papers specifically covering microalgal biomass pretreatment. This keyword cloud shows only terms that appear 3 or more times. Most methods and biomasses depicted in Fig. 5 are well known but still investigated in aspects such as energy demand and recovery, process intensification, and the use of green solvents. The selection of appropriate pretreatment depends on their cost‐effectiveness (Kendir and Ugurlu, 2018). The evaluation of the integration of processes in biorefineries, and the life cycle analysis of these processes is growing in interest (Khoo et al., 2019; Rosen, 2018; Ubando et al., 2019). Keywords describing pretreatment methods appear frequently associated with bioenergy. The four classes of pretreatment are briefly described below.

Fluidized bed, spout bed

4. Microalgal biomass pretreatment

Moderate

2018). However, purified fractions or specialty products such as peptides may require further purification, similar to other bioproducts. The focus of the next section is on processes that can be seen as a pretreatment for further extraction or purification.

Low

OPEX (energy, air, vacuum)

Pros

Fig. 5. Keyword cloud and links showing typical algal species, products, and pretreatment techniques, reported in the last decade. Keyword size is proportional to their frequency of occurrence, and link thickness is proportional to how often keywords appear together. Papers are from a Scopus search for “ microalg* W/3 biomass W/3 *treatment”, from 2010 to 2019, in the fields TITLE-ABS-KEY, and excluding papers whose object is water treatment. 592 index keywords were translated into 127 representative keywords. Only those with a frequency equal or superior to 3 are presented.

Compaction, some degradation during slow drying Variable. Compaction, expansion or degradation can occur Compaction, degradation at higher temperatures Enhanced permeation and some degradation can occur Enhanced permeation and some degradation can occur Compaction, degradation likely Low, can maintain cell viability. Biomass cake very porous.

Cons

J.C. de Carvalho, et al.

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4.18 kJ.kg−1.°C−1, and that all the difference in temperature is used for evaporation of part of the water); and 2) biomass usually has external, surface-bound water that evaporates using part of the heat from warm biomass. Still, the process can cause some permeation of cells. Simple drying processes cause collapsing because water is the main filling of cells. Gradual water removal also causes the concentration of intracellular components and precipitation of proteins. Drying usually leaves residual water from 1 to 10% (de Carvalho et al., 2019; Ho et al., 2019). Adequate concentrations of residual water in the “dry” biomass must be evaluated considering the next processes downstream. The bound water may enhance the stability of some proteins and is not a problem in nutritional biomass. However, it can be a problem for storage or lipid processing. The presence of concentrated liquid water can allow degradation reactions to occur, even in seemingly frozen biomass (Livingston, 2007; Poiana et al., 2010). Furthermore, residual water affects lipid extraction, can be partly carried by the extraction solvent, and end up leading to lipid hydrolysis, forming free fatty acids during direct transesterification. This requires either higher temperatures or more alcohol and catalyzer for an effective reaction (Cao et al., 2013; Sathish et al., 2014)

enzymes, a gentle and slow drying process is needed, precisely because it produces viable, porous biomass with low alteration of the molecules (de Carvalho et al., 2013). Lyophilization can be a good choice. When the biomass is processed for the extraction of more resistant fractions, the drying temperature and rates can be higher. Recent research shows that classical processes such as spray drying can enhance cell permeability, possibly because of an expansion effect in the quick drying. Table 2 summarizes common drying methods used for microalgal biomass processing. Several effects occur during drying, which can be explored to modify the biomass structure. The process consists of heating the biomass through contact with a solid surface (conduction), hot air (convection), or electromagnetic radiation (infrared or microwaves). Heat is necessary for the vaporization of water, which is then removed by the air stream. A vacuum can be used to enhance the mass transfer, but energy is still required for phase change, and that is why lyophilization still requires heating of the frozen biomass. In Table 2, degradation ranges from loss of cell viability to protein precipitation, lipid oxidation, and even thermal decomposition, depending on the temperature and duration of the process. Thermokinetic analysis methods such as TG (thermogravimetry) and DSC (differential scanning calorimetry) useful for evaluating effects of temperature on biomass during drying and thermochemical processing (Sanchez-Silva et al., 2013; Supeng et al., 2012; Yang et al., 2014) Drying systems differ in both CAPEX and OPEX investments, especially in energy consumption. The choice of a method for drying algal biomass is usually based on the scale of production and the intended use of the dry biomass. In the drying process, energy costs exceed those of microalgae production mainly when the water content should be reduced to approximately 10%, as in the case of its use for oil extraction. It can consume up to 75% of the overall cost of algae processing (Show et al., 2015). Among the significant drying process, both spray-drying and freezedrying are expensive, but analyzing microstructure and other characteristics of dried algal biomass, they are commonly found to be superior to conventional methods such as drum drying. Lin (1985) observed morphological changes in Chlorella and Spirulina when submitted to spray drying and freeze-drying under different conditions. Spray-dried particles were individual spheres with a void space in the center. Each particle was composed of a few thousand cells in the case of Chlorella and several trichomes in the case of Spirulina. In contrast, freeze-dried algae formed sheets of cells that were no longer spherical, and which adhered together linearly. In terms of the energy requirements and thermal efficiency, the consumption of heat per kg water evaporated is high, 2260 kJ/kg (0.628 kWh/kg), and dryers tend to have inherently low thermal efficiency, from 40% to 85%. According to the report of Nappa et al. (2016), thermal efficiencies of different microalgae drying processes vary from 0.3 to 1.08 kWh per kg evaporated water. In another study on energy requirement for drying algae with a water content of 4%, a heat input of up to 15,700 kcal was needed for evaporating 18.2 kg of water per kg of dry algae product. Besides, an additional energy input of 1.4 kWh was needed to operate the dryer (Soeder and Pabst, 1975). On a different study drying food materials, Baeghbali and Niakousari (2018) compare the energy efficiency and energy consumption of drying methods. The calculated energy required for drying 1 kg sample is similar in spray drying (1.42 kWh) and freeze-drying (1.46 kWh) methods. However, the overall energy consumption is much higher in freeze drying (130.65 kWh). The overall energy efficiency is 1.12 and 12.92%, respectively Theoretically, quickly pumping biomass into a vacuum chamber could cause its expansion while drying. However, 1) the energy content of hot biomass is not enough for complete vaporization: a single cell with 90% of intracellular water, entering a dryer at 105 °C and exiting at 20 °C, loses at best around 14% of its water (considering the enthalpy of vaporization of around 2300 kJ/kg water, the heat capacity of water

4.2. Thermochemical conversion This pretreatment encompasses hydrothermal liquefaction (HTL) and pyrolysis. Both processes are destructive, and suitable for the production of biofuels, producing biochar, bio-oil, and syngas fractions. Hydrothermal liquefaction uses lower temperatures (about 250–350 °C) and wet biomass in pressurized systems to avoid phase change. The process gives solid and liquid fractions. Pyrolysis heats biomass up to 800 °C in an anoxic, atmospheric pressure, giving the three phases in proportions that vary with temperature. These fractions must be further processed before use as a fuel. Although thermochemical conversion deviates from traditional, low-temperature bioprocesses, these are quite efficient ways to access the energetic content of microalgal biomass, while giving a stable solid fraction. For example, while microalgal biodiesel production from biomass with 13% of extractable lipids may require drying, extraction, and desolventizing, leading to 130 kg of lipid extract/tonne of dry biomass, HTL of the wet biomass could lead to a solvent fraction carrying 400 kg of bio-oil, while the direct, slow pyrolysis of the same (dry) biomass could lead to 270 kg of a raw oil fraction (projections based on Jena and Das, 2011). This can be explained by the fact that not only the lipid fraction, but also proteins and carbohydrates, are converted to bio-oil in thermochemical processes, resulting in a complex composition of more than 300 compounds (Biller and Ross, 2011; Guo et al., 2015). Some authors recognize pyrolysis at very high temperatures (800–1000 °C) as a diverse process of gasification (Panahi et al., 2019). Both HTL and pyrolysis require considerable energy, but the net energy balance can be positive. It is estimated that pyrolysis and HTL could recover 78.7% and 89.9% of the energy content of microalgae (Biller and Ross, 2011). Fractions from the process can be used to produce energy in situ for further processing in a self-sustainable operation. In fast pyrolysis, the processing is done at high heating rates and low residence times, less than a minute for the volatile fractions, while slow pyrolysis has residence times of several minutes, and lower heating rates (Vargas e Silva and Monteggia, 2015). HTL has similar residence times to slow pyrolysis, of the order of 1 h. HTL uses lower temperatures and gives high conversions into liquid fractions, and may have a favorable energy balance compared to pyrolysis (Hognon et al., 2015). However, fast pyrolysis can also be attractive. Table 3 shows examples of thermochemical conversion with the energy content of the biomass and its fractions. As Table 3 shows, thermochemical conversion can have a variable energy recovery. It can be observed that the median value of energy balance for bio-oil from HTL and Slow Pyrolysis were similar: 7

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Table 3 Thermochemical processing of biomass, bio-oil yields, and energy balance. Microalga

Process

Temperature (°C)

Moisture (%)

HHV of the biomass (MJ/kg)

Residence time

Fractions yield (%)

HHV bio-oil

Energy balance (%)*

Reference

Unidentified consortium Chlorocystis sp.

HTL

350

–

–

120 min

41.6

58.5

Bravo et al., 2019

HTL

325

–

13.87

30 min

32.8

–

Das et al., 2019

Picochlorum sp.

HTL

325

–

17.41

30 min

33.4

76

Spirulina Porphyridium sp. Nannochloropsis sp. Chlorella sp.

HTL HTL HTL Fast pyrolysis

350 350 350 500

7.8 5.1 7.2 –

21.2 14.7 17.9 19.5

60 min 60 min 60 min < 5s

36.8 35.7 34.5 25.5

50.7 51.6 66.1 –

Biller and Ross, 2011

Scenedesmus sp.

Fast pyrolysis

440

4.59

21.10

3.8 s

29.8

73

Kim et al., 2014

Spirulina sp.

Fast pyrolysis

500

8.04

23.42

<5 s

27.6

54.2

Chaiwong and Kiatsiriroat, 2015

Spirulina

Slow pyrolysis

550

8.45

22.34

60 min

21.68

43.7

Nannochloropsis gaditana

Slow pyrolysis

600

2.5

21.5

30 min

12.6

–

Adamczyk and Sajdak, 2018

Chlorella sp.

Slow pyrolysis

450

6.5

21.2

30 min

27.0

43

Babich et al., 2011

Dunaliella salina

Slow pyrolysis

500

4.0

21.2

24.0

68.7

Gong et al., 2013

Chlorella vulgaris

Slow pyrolysis

500

4.4

19.3

Bio-crude: 18% Bio-crude: 34.8 Bio-crude: 39.6 Bio-crude: 30 Bio-crude: 25 Bio-crude: 38 Solid:29% Liquid: 53.9 Gas: 17.3 Solid: 26.4% Bio-oil: 10.3 Gas: 21.8% Solid: 31 Bio-oil: 29 Gas: 40 Solid: 31 Bio-oil: 14.5 Gas: 24 Solid: 45 Liquid: 38 Gas: 17 Solid: 30 Bio-oil: 56 Gas: 14 Solid: 31.5 Bio-oil: 55.4 Gas: 4.6 Solid: 28.0 Bio-oil: 49.2 Gas: 3.9

22.0

74

*Energy balance is in relation to bio-oil; ** calculated based on energy balance.

disruption method is transformed in heat, and that limits the process application or throughput. Sonication is commonly used in labs for complete biomass destruction—for nucleic acid extraction, protein characterization, lipid extraction, etc. However, the process is very energy-intensive and may not be economical, even if scalable (there are high power, high throughput sonicators available in the industry, such as those produced by Industrial Sonomechanics – www.sonomechanics.com – and Hielscher Inc. www.hielscher.com). There is a lack of scientific reports of large scale sonication – most papers that advocate the use of this process obtained promising results in small scale or worked with sonication coupled to extraction, e.g., Neto et al., 2013 and Prabakaran and Ravindran, 2011. High shear and impaction: High-pressure homogenizers, impingement, and microfluidization work on similar principles. The suspension is pumped at high velocities through a homogenizer valve, where high shear occurs, and the liquid decelerates. The steep velocity gradient in the system deforms and disrupts the cells. High pressures are required to generate these high velocities in narrow passages, typically 500–1500 bar. These systems are also used emulsification and milk stabilization (de Carvalho et al., 2017a; de Carvalho et al., 2017b). In these systems, the temperature rise ΔT can be estimated by:

55 ± 11.7% and 56.6 ± 16.3%, respectively, while fast pyrolysis resulted in slightly favorable energy balance: 63.6 ± 13.3%. Fast pyrolysis is promising for biofuel production coupled with nutrient recovery (Markou and Monlau, 2019; Santos and Pires, 2018). The solid, liquid, and gaseous fractions produced are complex and may require further processing such as filtration, neutralization, or fractionation for specific uses. 4.3. Cell disruption This pretreatment step encompasses several methods that permeate, disrupt, or dissolve cell walls or the cell membranes. There are several techniques for cell disruption, loosely divided into mechanical, physical, and chemical. The cell membrane (CM), a fluid, lipid bilayer with roughly 4 to 5 nm (Andersen and Koeppe, 2007; Manrique et al., 2014), is a selective barrier that protects internal components and can be dissolved or thermally destroyed with relative ease. The cell wall (CW) is comparably permeable for small molecules, absent for D. salina (Xu et al., 2015), but typically thick and strong for most microalgae, e.g., 200 nm for N. oleoabundans (Rashidi and Trindade, 2018). The mechanical disruption of the CW exposes internal macromolecular components and organelles, and its hydrolysis is required if CW components are to be used. Table 4 lists cell disruption methods and estimated energy intensiveness and scalability, for a 10% biomass suspension or dry biomass when applicable. An important distinction exists between physical methods popular in small scale, and methods of industrial scalability. Chemical methods are generally scalable, while not all mechanical or physical methods are. The methods depicted in Table 4 are generally scalable, even if costly, in some cases. Energy intensiveness is one of the keys to evaluate process suitability, because most energy applied in a mechanical

Δ T= P/cp. ρ

(1)

where P is the pressure, cp is the specific heat, and ρ is the density of the solution (Floury et al., 2003; SPXFLOW, 2009). For a typical aqueous suspension, Eq. (1) shows that a pressure drop from 800 atm to 1 atm causes the temperature to rise by 19.4 °C quickly. Therefore, processes using multiple passes may require refrigeration. Fluid agitation usually is not enough to efficiently disrupt microalgal cell suspensions. The efficiency is increased using: i) high velocity, ii) 8

9

CW

CW

CW

CW

CW CW, CM

CW CW

CW, CM CW

CM CM

CM

CM

CM

CM

Sonication

High-pressure homogenization, Impingement, and Microfluidization

Microbead wet milling

Colloidal mills, rotor–stator homogenizers

Grinding (microbead, ball or jet mills) Freeze-thawing

Autolysis Enzymatic hydrolysis

Alkaline treatment, energic Acid hydrolysis

Alkaline treatment, mild Osmotic shock

Thermolysis

Detergent solubilization

Solvent solubilization

Pulsed electric fields (PEF)

Electroporation: High intensity, short duration electric fields (pulses) induce ion migration and membrane rearrangement, forming pores

Solvent destabilizes the cell membrane

Detergents dissolve the cell membrane

Heating the suspension increases membrane fluidity, destabilizing it

High pH saponifies lipids. Milder temperature than the energic process Immersion of cells in an hypotonic solution causes osmolysis

Very High pH catalyzes the hydrolysis of CW and CM components Low pH catalyzes the hydrolysis of CW components

Endoenzymatic lytic activity triggered by specific conditions Enzymes target structural components of the cell wall, such as cellulose, weakening, permeating, or dissolving it.

Solid grinding: collision and attrition Ice crystals damage the cell wall and membrane

High shear by a high-speed mixer

High shear and impact, from pressure transformed in a steep velocity gradient in a homogenizer valve (HPH), impact chamber (impingement) or interaction chamber (fluid collision) High shear generated by the agitation of fluid with a high content of hard solids

Cavitation of microbubbles causes high shear in cell walls

Principle

Detergents such as SDS or bile salts Solvents, such as toluene or xylene –

Base, KOH or NaOH Acid, H2SO4 or another lowcost, strong acid Base, KOH or NaOH Buffers, sometimes enzymes to weaken the cell walls –

– Enzymes such as cellulase, xylanase, lysozyme

Balls in some types –

Beads; diameter 100 to 2000 μm, typical load 40–60% V/V –

f

0.17–1.47

Very low

Very low

Low

Low Very low

Low Low

Very low Very low

Depends on efficiency. Generally, lower energy per volume than HPH or microbead. 11.8d 0.92e

0.23 operating at 800 atm; not accounting for motor/drive/pump losses 3.1 to 0.31c

– b

6

a

Energy input, kWh/kg dry weight

–

reagents

Generates heat

Can denature enzymes

Works only for cells with previously weakened or absent CW Depends on species, can denature enzymes Can denature enzymes

heat generation, slow process Slow process; depends on freezing rate. Gentle on the biomolecules Slow process Enzymes are relatively expensive, but the process is gentle on non-target biomolecules Energic, can degrade cell components Energic, can degrade cell components

Lower heat generation, inefficient for hardy biomasses

Severe heat generation, requires cooling, harder to scale up Heat generation, roughly 0.024°/atm of pressure applied. Feasible for most biomasses. Heat generation, feasible for most biomasses

Note

a: Industrial Sonomechanics ISP-3000 (EUA); b: from Bernoullís equation with P converted to energy for water. Power input based on nominal power and throughput of various equipment: c Netzsch LME300 (Germany), d: average for several microalgae, (Garcia et al., 2018); e: phase change energy, based on water enthalpy of fusion, 6.02 J/mol; f: Estimated from references (Eing et al., 2013; Grimi et al., 2014; Straessner et al., 2016).

Target

Cell disruption method

Table 4 Cell disruption methods applicable for microalgal biomasses, and their primary targets, CW (cell walls) or CM (cell membranes). Mechanical methods have high energy inputs, while chemical and physical methods use basically agitation but require specific reagents or heat exchange. All methods are described for wet pastes or suspensions, except grinding.

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Supercritical CO2 + ethanol

Essential oil (e.g., orange or citrus

n-hexane

Astaxanthin

β-carotene

Lipids for biodiesel production

Monoraphidium sp., freeze-dried

Dunaliella salina, fresh paste from centrifugation

Chlorella protothecoides, after heterotrophic cultivation phase, freeze-dried Scenedesmus sp., defatted dry biomass with 92% solids

10

Starch hydrolysis for ethanol fermentation

Carbohydrate dissolution for ethanol fermentation Liquozyme® Supra 2.2X and amyloglucosidase AMG® 300 L, from Novozymes

No separation

3 h at 65, 95 and 55 °C at 400 rpm Up to 10 h at 55 °C under mechanical stirring at 100 rpm

PHB, Polyhydroxybutyrate

Nostoc muscorum, filtered and dried at 55 °C Mixed, nitrogen-depleted microalgal biomass, fresh or dried Arthrospira platensis filtered, dried at 55 °C for 24 h and freeze-thawed

Precipitation with diethyl ether, drying No separation

Water washing

Stirring for 24 h at 28 °C

aDES, aqueous deep eutectic solvents: choline-oxalic acid, choline-ethylene glycol, and urea-acetamide. Water reduced viscosity to 20cP. Chloroform after pre-extraction of pigments β-Glucosidase, α-Amylase, Amyloglucosidase

Cell-wall carbohydrates, enhancing residual biomass extraction of lipids

Chlorella sp. produced in pilot scale raceway ponds, centrifuged and spray dried.

Soxhlet extraction for 30 h

No separation

Ambient temperature or 100C, 0.8% NaOH with a solid load of 36 g/L, 8 h.

Hydrolysis of 95.1% of total sugars. Similar results with suspension (fresh and dry biomass. Hydrolysis of 50% of the starch after 2 h, and 85% after 10 h.

26.37% PHB content

Rempel et al., 2019

Ansari and Fatma, 2016 Shokrkar et al., 2017

Lu et al., 2016

Yang et al., 2010

Xu et al., 2006

–

Carbohydrate and protein dissolution improved from 22 and 2% to 40 and 29%, respectively, with alkali. Thermal processing improves extraction rates but not the overall dissolution aDES dissolved on average 45% of the carbohydrates, increasing posterior lipid extraction from 52 to 74.36%

Laurvick, 2017

Commercial route

Fujii, 2012

–

Evaporation after chlorophyll precipitation with H2SO4 Sedimentation, saponification, centrifuging, evaporation Evaporation

Garcia et al., 2018

–

–

Reference

Conclusions

Solvent/solute separation

Water, 0.8% NaOH

Soxhlet extraction

Direct (stirred tank extraction

Biomass centrifuged, stored at 20% w/w under refrigeration, then disrupted with a ball mill 20 MPa, 30 °C, 15 min

Conditions

Carbohydrates and lipids

Water

Proteins

Testraselmis suecica

Solvent

Target

Biomass

Table 5 Examples of targeted fractionation of biomass, with solvents and digestive reagents used.

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cell wall component, and used in lower concentrations as a supporting method that can facilitate and reduce the energy input in other cell disruption methods. The processes with highest saccharification yields using commercial enzymes such as cellulases, xylanases, and amylases, as compiled by Demuez et al. (2015b), take 24–72 h, or less time when combined with another preprocessing such as milling and autoclaving.

carefully machined rotor–stator systems, and iii) using abrasives or microbeads; in all cases, high turbulence and shear is created in the liquid by agitation, and the process is energy-intensive but scalable, with manufacturers such as the Wab group – wab-group.com, and GlenMills – glenmills.com. The latter also produces dry grinding equipment such as jet mills. Grinding of powdered biomass: previously dried algal biomass can be pulverized using methods common for other biomasses or minerals. In this process, the energy consumption and transfer to the product vary with the specific biomass, the use of grinding aids, and the final size desired for the powder. The energy input follows a power law, with the specific energy requirements growing with the inverse of the particle size. Temperature rise is even more critical than in wet grinding: without the water to absorb energy, dry biomass heats quickly to denaturing temperatures unless the process is extended, and the throughput reduced. Freeze-thawing is a process that can be used for some microalgae, most notably Arthrospira (Moraes et al., 2011; Zhu et al., 2007) for phycocyanin production and intracellular starch exposure (Rempel et al., 2019), and despite the relatively high energy input for freezing, is very gentle on the biomolecules. Chemical disruption methods, such as acid or alkali treatment, tend to be scalable, although an economic analysis is still necessary to evaluate the process. These processes overlap with targeted fractionation: the complete hydrolysis of cell walls using acid or alkali ends up producing a carbohydrate-rich fraction, besides intracellular compounds. Acid or basic hydrolysis is similar to processes used for pretreatment of other biomasses, with some fractions such as hemicellulose and the cell membrane being easily hydrolyzed, and fractions such as cellulose or chitin (in some species) resisting in milder conditions. Membrane dissolution with detergents or solvents may be enough for cell permeation and further processing. Autolysis is a less explored, but possible approach for cell disruption. It is species-dependent, and can be triggered by diverse environmental cues such as anoxia with temperature increase (e.g., Halim et al., 2019, working with Nannochloropsis gaditana at 38 °C, and Kightlinger et al., 2014 with Chlamydomonas reinhardtii at 50 °C, both processes with 24 h of incubation). When it works for a selected species, it is attractive because of the mild conditions and low-cost process, although somewhat slow. Pulsed electric fields (PEF) – this is supercharged electroporation: high voltage, short time (pulses) electric fields are applied to cell suspensions. The process uses intensities of the order of 10 to 100 kV/cm and pulse durations of 1–10 ms, charging and destabilizing the cell membrane in a few consecutive pulses. The currents can be high depending on the suspension conductivity, and that causes heating. However, the energy input is comparable to some mechanical methods, because of the short duration of pulses relative to treatment time: (Grimi et al., 2014) used 13.3 to 53.1 kJ/kg for a 1% suspension of Nannochloropsis sp., which translates into 0.37 to 1.47 kWh/kg dry cells, while (Eing et al., 2013) applied 0.4 kWh/kg dry cells for Auxenochlorella protothecoides, and (Straessner et al., 2016) applied 0,17 kWh/kg dry cells for A. protothecoides and Chlorella vulgaris suspensions. This group used 50 kJ/kg of cell suspension but reduced the overall energy input by using pre-concentrated suspension at 8% w/w. PEF is a technology already marketed in the food industry for sterilization (Kempkes, 2017) with several vendors. For use with microalgae, it is still relatively recent, and under development. Enzyme fractionation – Cell walls of microalgae can be permeated and respond diversely to different enzymes, as is exemplarily shown by Gerken et al. (2013), who tested 16 enzymes and selected combinations and evaluated the increase in cell wall permeation for Chlorella. Lysozyme, combined with other enzymes, showed high efficiency. The method has an inherent cost of enzyme; for example, lysozyme, one of the most common lytic enzymes, costs 100–500 USD/kg (de Carvalho et al., 2017a; de Carvalho et al., 2017b), making it inadequate for lowcost products. However, enzyme cocktails can be targeted to a specific

4.4. Targeted fractionation or solubilization This pretreatment encompasses methods that dissolve components of the biomass, similarly to some cell disruption methods, but generating extracts that can be further processed for fermentation or purification. This step may require a preliminary biomass disruption process, and it is quite variable because of the diversity of molecular targets. Fig. 3 and Table 1 detailed the composition of selected biomasses. As shown in Table 5, these components can be targets for selective extraction: lipids with nonpolar solvents, proteins with aqueous buffers, and other molecules with specific solvents. Targeted fractionation is the most critical step in the production of high-value products such as nutritional bio-oils and other components of low polarity, including carotenoids, xanthophylls, and vitamins. Another vital aspect of fractionation is changing from an ideal, focused pretreatment to a byproduct-generation step—e.g., instead of using mechanical cell disruption to expose cell contents, slower hydrolysis generates a fermentable fraction. The fractionation step is slower but may enhance the profitability of the whole process in a biorefinery. For protein-rich fractions, the removal of lipids can be beneficial; as shown by Montalvo et al. (2019), and Ovando et al. (2018), the dissolution of the cell membrane and lipid fractions in Arthrospira sp. is simple and can lead to a useful mixture of low polarity components. The subsequent dissolution of proteins with adequate buffers leads to another fraction of spent biomass that can be further processed into bio-oils, biodigested, or used as feed. For foodstuffs and food ingredients, there is a limited set of extracting solvents permitted by regulations. Examples are propane, butane, ethyl acetate, ethanol, carbon dioxide, acetone, and nitrous oxide. Thus, legislation must be checked regarding market regulations. The Directive 2009/32 from the European Commission, for example, describes permitted solvents for extraction. Other solvents such as hexane, dichloromethane, and methanol, must be verified against the intended use; for example, defatting algal biomass for further anaerobic treatment should not use chloroform, which inhibits methanogens (Neves et al., 2016) 4.5. Harvesting, storage, and pretreatment synergy The scientific literature brings focus to pretreatment processes without necessarily evaluating the effects of preliminary processes on microalgal cells. That is natural, in the sense that pretreatments are compared among themselves using the same (preliminarily concentrated) biomass to answer a research question or hypothesis. However, biomass from laboratory and pilot-scale algae cultures is usually previously centrifuged and dried, or frozen for storage as concentrates, or even freeze-dried, as shown in Table 5. Concentrates or dry biomass are often resuspended in hypotonic media before pretreatment assays. When trying to reproduce or improve these results, it is essential to evaluate if these preprocessing steps affected pretreatment and had effects that may not appear when fresh biomass is used—a desirable situation in industrial plants. Examples of good processing results obtained with pre-frozen biomass are alkaline treatment of frozen and thawed Chlorococcum infusionum (35 %w/w of glucose, by Harun et al., 2011); high protein-content extracts from frozen and thawed Nannochloropsis paste (Gerde et al., 2013), supercritical CO2 extraction of astaxanthin from Monoraphidium sp. from with freeze-dried biomass, (Fujii, 2012); and specific comparisons of methane production from three microalgae previously frozen or cold-stored, by Gruber11

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the harvested, concentrated biomass and facilitate further processing (Demuez et al., 2015a; He et al., 2017; Zhu et al., 2007). Careful control of the process is mandatory to avoid contaminating growing cultures or creating resistance from processed cultures. With its potential and complexity, this is a promising area of research. Sonication, pulsed electric fields (PEF), microwaves, and energy intensiveness – Processes such as sonication, PEF, and microwave disruption are intensively researched at a small scale and could be used in largescale processing if the energy intensity is reduced. This can be done by careful design of continuous equipment, such as flow cells where the disruption takes place, but also, by developing processes with higher cell concentration in the suspensions. Much of the research reported uses suspensions where most of the sonic or electrical energy is dissipated as heat and, thus, the effective energy load for disruption can be reduced if the concentration is increased. Although lower ultrasound frequencies (near 20 kHz) are very effective and easy to generate, higher frequencies could be better for the disruption of specific biomasses (Kurokawa et al., 2016), and this requires further investigation. Diversely of ultrasound (which can create spots with so much energy, that free radicals can form), microwaves have only a thermal effect. Several studies show effective microwave-assisted pretreatments (Du et al., 2011; Passos et al., 2013; Qv et al., 2014). Radiofrequency in the range of commercial magnetrons causes molecular agitation and rapid heating, but the process must be addressed as such, focusing on thermal and rate effects such as superheating. Non-thermal effects are a possibility, e.g., in protein denaturation (Chen et al., 2017; Porcelli et al., 1997), but there is no consensus yet. Pulsed electric fields, however, are quickly becoming popular, but still require investigation. The adaptation of commercial systems, the improvement of flow cells, and the use of concentrated suspensions are promising directions to investigate. Wet disruption – High-pressure homogenization and microbead mills continue to be vital equipment in cell disruption, capable of handling robust cell walls. However, microalgal cell walls are quite diverse, and high shear fluid mixers, which are being popularized through nanobiotechnology research, are a promising method for either thin or enzyme-weakened cells. Novel solvents – The physical chemistry of fractional solubilization is well known, as are the common solvents that can be used. But among hundreds of industrial solvents, some are renewable and worthy of consideration in the development of novel processes. Also, and despite some hype, ionic liquids such as deep eutectic solvents are promising for targeted extractions (Kumar et al., 2017; Lu et al., 2016; Paiva et al., 2014). For these solvents, it is essential to perform engineering analysis, including not only the solvent capability but the recovery and effluent treatment costs; precisely because of their low volatility, ionic liquids adsorbed in solid matrixes can be challenging to remove, requiring either an extra washing step or leaving residual solvents.

Brunhumer et al. (2016). Examples of pretreatments using previously dried biomass abound, perhaps because of the option for using commercial biomass, or of long-term storage independent of cultivation. Most of these works focus on the pretreatment, such as alkaline pretreatment for biohydrogen production (Yang et al., 2010) and lipid extraction with deep eutectic solvents (Lu et al., 2016), but some also evaluate the synergic effect of drying on pretreatments, such as (Eing et al., 2013) for lipid extraction, compared to pulsed electric fields, and (Rizwan et al., 2013) for biodiesel in an evaluation from both economic and environmental points of view. 5. Pretreatment selection and process integration Pretreatments and other processing steps (from biomass harvesting to final processing) should be selected regarding the biomass intended use. There are countless options for processing, and a few guidelines can help in the development of new processes in the selection of a pretreatment operation: 1. Processes development is iterative: a new process must be evaluated and improved regarding yield, throughput, cost, and environmental impact at each iteration. 2. Steal like a scientist: a process should be built on previous, successful approaches, and then improved with the most modern techniques. 3. Some rules of thumb apply when there is no precedent in processing specific microalgal biomasses: - For any pretreatment that requires low water activity or solid material: preliminary drying must be used; - For direct use as fuel: lipid content is not mandatory. The overall yield of biofuel is higher in HTL, followed by pyrolysis and lipid extraction and conversion in biodiesel. The oil quality and mineral content follow the inverse order. - For biodigestion: most recent research shows improved biogas and biohydrogen production after cell disruption (Kendir and Ugurlu, 2018). There is no need to preserve high biomolecule integrity. Therefore, cheap alkaline disruption can be better than mechanical methods. - For dietary lipid fractions: solvent extraction, if possible, without disruption - For feed and food: cell disruption, for enhanced absorption, or further fractionation 4. Cost and energy are crucial, but integration and valorization of coproduct streams are also essential and can increase the profitability of a process. 5. Legislation and safety are also relevant, especially when it comes to the use of solvents or reagents, and residual amounts permitted in the products.

7. Conclusion 6. Development of new technologies for microalgae pretreatment Microalgal biomass pretreatment is essential for further processing, especially in a biorefinery approach. However, pretreatment depends on microalgal cell structure and composition and can be energy-intensive. Processes reliable at small scale, such as sonication and lyophilization, may prove impractical or too expensive in industrial scale, while uncommon steps, such as freeze-thawing and pulsed electric fields, can end up having a positive energy balance. Classical processes such as high-pressure homogenization and acid hydrolysis remain economically competitive, but can still be improved. The central point is to remember that process development is iterative and must be looked at as a whole.

Throughout the microalgal biomass pretreatment chain, there are several technologies in development, some with a still limited largescale application. Promising approaches are: Strain development – It is essential to develop appropriate strains for easy pre-treatment through existing information on microbial physiology and metabolism, selection tools, and genetic modification approaches. This ranges from the selection of wild or mutant strains with thinner cell walls to the modification of organisms to enhance excretion or autolysis. Biological approaches for disruption – while mechanical and chemical methods are ubique for cell disruption, it is possible to use the antagonistic organisms or targeted enzyme cocktails to disrupt cells. The obvious advantage is the low energy intensity: lytic “microorganisms”, targeted enzyme cocktails, viruses and predators can be used to attack

Declaration of Competing Interest The authors declare that they have no known competing financial 12

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interests or personal relationships that could have appeared to influence the work reported in this paper.

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