We found a total sugar TS and lignin TL content of Conditions for hydrolysis with H 2 SO 4 were analyzed using an experimental design Figure 1. Results showed that, by increasing acid concentration, the release of total sugars increases during the chemical hydrolysis, reaching a maximum concentration of 0. The results obtained with regard to the generation of total sugars could be because cellulosic biomass is partially hydrolyzed, increasing the surface and exposure of the cellulose fibers [ 19 ], that is, significantly increasing the solubilization of cellulose for obtaining total sugars, generated in a process in which CBM is treated.
Time reaction was also studied; however, there was not any significant difference ; therefore, total sugars generation consistently increases at minute There is a maximum in the production of reducing sugars Figure 2 at elevated temperatures and the highest dilution of acid.
Chemical hydrolysis showed a reducing sugar concentration of 0. These factors have a positive effect on the amount of hydrolyzed sugars, playing an important role in separating the cellulose-hemicellulose complex [ 20 ]. The polymerization degree was analyzed in order to assess the average number of units of monosaccharides existing in the extracted polysaccharides [ 14 ]. A good efficiency of chemical pretreatment increases the accessibility of glycan to saccharification and therefore obtaining a value of polymerization degree close to one, so that they can be converted to ethanol [ 21 ].
According to the results, a degree of polymerization of During chemical hydrolysis, F is generated from pentoses and HMF from hexoses [ 22 ]. In experiments, degradation products increase in concentration as temperature and H 2 SO 4 concentration increase, reaching a maximum concentration of 2. Therefore, it can be said that 2. After applying the chemical hydrolysis to the CBM, liberated cellulose is degraded to glucose by the action of dilute H 2 SO 4.
Low concentration of glucose generally indicates that cellulose degradation suffered poor or incomplete hydrolysis; hence, it follows that the chemical hydrolysis is quite selective since the chemical hydrolysis conditions are not so severe as to cause cellulose solubilization, because its crystal structure makes it difficult to complete the hydrolysis [ 25 ]. Fructose concentration is not significant when compared with five-carbon sugars such as xylose, yet it is capable of being fermented in smaller molecules and more easily because they are sugars of six carbon atoms, and they are those with greater availability.
Xylose formation is increased with increasing temperature and acid concentration, thus creating a maximum concentration of 0. The direct application of chemical hydrolysis to CBM also reduces aggregation between cellulose microfibrils, as well as making direct hydrolysis obtain good yields 0. Chemical hydrolysis may be used then as a pretreatment that generates soluble sugars but also facilitates further enzymatic hydrolysis as seen by a higher yield 0.
Factors included in the analysis of main components explain The temperature favors the generation of F, HMF, glucose, and xylose; the acid concentration increases the generation of reducing sugars, lignin solubilization, and increased sucrose. The factors relevant to production of soluble sugars are temperature and acid concentration followed by time.
However, we set a reaction time of 60 minutes since we observed maximum generation of soluble sugars. Results of the experimental design show that acid concentration was only significant for fructose, glucose, and HMF generation. The statistical study, based on the values, shows that only temperature has a statistically significant effect on the chemical hydrolysis, while the other two factors are significant but less significant than temperature.
Figure 4 shows the effect that different pretreatments had on the CBM with respect to the generation of total sugars. Acid pretreatment resulted in a higher concentration of total sugars having a concentration of 0.
In the other pretreatments, sugars release is also promoted, however in smaller amounts. Reducing sugars measured by the DNS technique showed that dilute acid pretreatment presented a concentration of 0. Possibly, the failure in finding reducing sugars from alkaline pretreatments was mainly due to how they produce swelling of cellulose, but not necessarily reaching hydrolysis of reducing sugars present or their chemical composition changes [ 26 ].
Although the applications of basic pretreatment with CaO, basic pretreatment with NaOH, oxidizing basic pretreatment, and ozonolysis are effective because they improve the opening of cellulosic fibers [ 10 ], they do not degrade sugars at this stage, that is, only making the material susceptible to enzymatic attack. Efficiency of different pretreatments with respect to lignin solubilization Figure 5 showed that basic-oxidant pretreatment solubilized 1.
This behavior was probably because treatment with diluted NaOH produces a swelling in the biomass, leading to an increased internal surface area and a decrease in crystallinity and structural separation joints between lignin and carbohydrates, causing a break in the structure of lignin.
The mechanism of the alkaline hydrolysis of biomass seems to be based on the saponification of intermolecular ester bonds linking the xylan of the hemicellulose and other components [ 27 ].
It is noteworthy that the effectiveness of the pretreatment depends on the lignin content of the material to be pretreated. Dilute sulfuric acid pretreatment with 1. However, lignin does not dissolve as well as other pretreatments but rather increases yields of enzymatic hydrolysis [ 12 ].
These spectra show structural differences in nonpretreated solid and treated solids. Comparison between spectra from pretreated and nonpretreated biomass shows similar patterns; however, bands from treated material have a stretching vibration in their OH bands; that is, the area is augmented.
The OH group initiates substitution reactions on the solid pretreated with dilute acid. This condition causes the transformation of the hydroxyl group to an allyl ether and ultimately the ether is substituted with an acid group. The benefit of this reaction is that the presence of the acid group in the molecule causes the lignin polymer to be soluble in water [ 30 ]. The ester linkage containing glucose monomers in a polymer chain is the most important lignin link.
Therefore, ether bond cleavage can lead to separation of lignin from the polysaccharide matrix and degradation of the polymers of monomeric sugars and lignin fragments. The cleavage of this bond occurs through solvolytic reactions, which may take place under acidic or alkaline conditions or by different mechanisms.
In the case of lignin, under acidic conditions, the ether bond is converted to OH and then converted to carbonyl or carboxyl before being fragmented into molecules of C3 and C2. Under alkaline conditions, the mechanism is different and the end result is not a fragmented side chain, but the separation of the aromatic rings.
In the case of cellulose cleavage of ether bonds, this may be contained either in acidic or in alkaline media. When acidic media are used, the acid acts as a catalyst for the protonation of the oxygen atom.
The charged group leaves the polymer chain and is replaced by the hydroxyl group of water. The reaction that occurs is a first-order reaction. In order to determine which pretreatment is more efficient for delignified cellulosic material, a comparative analysis was performed before and after pretreatment. In the CBM, nonpretreated well-defined peaks are shown Figure 6 with vibration of OH groups, in addition to their CH, CH 2 , carboxylic acids, phenolic ethers, aromatic groups, and characteristic guaiacyl lignin groups.
However, treatment with dilute H 2 SO 4 shows similarity in their spectra with respect to the original sample, but functional groups are present in stretching vibrational peaks due to the effect of the pretreatment that was applied. For basic pretreatments, likewise, a change in the size of their peaks occurred, vibrational stretching was present, and lignin signals remained apparent via, as mentioned above, characteristic groups of lignin aromatic compounds, phenol groups, ethers present, and carbonyl groups.
With ozonolysis, some lignin is degraded causing a variety of compounds, among which are aromatic groups, phenols, alcohols, ethers, and carbonyl groups. Table 3 shows the results of enzymatic activity of Celluzyme XB, expressed as hydrolyzed filter paper units FPU per milligram of enzyme. An FPU is defined as the amount of enzyme necessary to produce a mole of reducing sugars per minute per gram of substrate [ 31 ].
The material being pretreated with the oxidizing basic treatment, for which a concentration of 0. This was due to applying the basic oxidizing pretreatment, whereby the materials increased the size of their pores, giving greater access penetration of enzymes and therefore increased enzymatic breakdown of carbohydrates.
The significant increase in the generation of reducing sugars was due to an enzymatic cocktail of cellulases and hemicellulases The generation of total sugars was favored in all pretreatments but significantly in the basic-oxidant 0. Second, algae grow in aquatic habitats and thereby do not compete with food crops on agricultural land, or cause deforestation. Third, algal biomass can be used to produce two types of biofuel bioethanol and biodiesel since they accumulate high amounts of carbohydrates and lipids.
Finally, the fresh water requirement for algal growth is significantly lower than plant demands to produce the same volume of biofuel. Nevertheless, there are several constraints that restrict the production of biofuel from algae [discussed by Hannon et al.
The hydrolysis of algal carbohydrates to basic sugars is primarily carried out using chemical and enzymatic methods. Although the chemical method yields high concentrations of fermentable sugars in a short time, this method requires harsh reaction conditions producing byproducts, which might inhibit the fermentation process and require costly disposal processes.
In contrast, enzymatic hydrolysis produces high amounts of fermentable sugars under mild conditions without producing inhibitory byproducts Chen et al. Algae produce a wide spectrum of polysaccharides that are specific to an algal group, family, or species.
The enzymatic hydrolysis of algal polysaccharides requires a wider range of enzymatic mixtures, compared to plants. This review focuses on the enzymatic hydrolysis steps of the major algal carbohydrates and their fermentation process to ethanol. Since the scope of this topic is broad, only the fundamental concepts of the field are addressed in this review.
Nevertheless, we will refer the reader to other reviews that are complementary to this topic. Algae are photosynthetic eukaryotes that are distinguishable from cyanobacteria, which are photosynthetic prokaryotes Brodie and Lewis, Because of their importance for biofuel production, this review will cover cyanobacteria as well.
Algae vary dramatically in size and morphology from microscopic unicellular phytoplanktons to m long seaweeds. Based on their morphology and size, algae are classified into microalgae and macroalgae. Currently, microalgae are the major source for third-generation biofuels. In contrast, only small amount of cyanobacterial biomass are utilized for bioethanol production. Additionally, development of methods that overcome obstacles in using macroalgae would greatly improve harvesting bioethanol from natural, renewable biomaterials.
The advantages and disadvantages of relevant algal sources are summarized in Table 1. Table 1. Comparison between relevant algal sources and the advantages and disadvantages of employing each for the production of third-generation biofuels.
Microalgae are microscopic in size measured in micrometers and exist as single cells; or unspecialized multicellular filaments and colonies Satyanarayana et al. They are highly diverse including 40, species that belong to nearly all major algal groups with the exception of brown algae [reviewed by Metting , Dahiya , and Kim ].
Microalgae exhibit several features that favor using them for industrial production of biofuel. First, they lack specialized tissues and structures, which simplify the cultivation and harvesting processes. In addition, microalgae exhibit high rates of asexual growth and yield huge amount of biomass from low inoculum Packer, ; Chen et al. Furthermore, microalgae accumulate large amounts of polysaccharides and triacylglycerols — storage lipids and energy sources, and thereby they are suitable for simultaneous production of bioethanol and biodiesel Mata et al.
The commercial production of microalgal biomass is obtained from cultivating the freshwater algae Chlorella and Haematococcus , and marine algae, such as Dunaliella, Phaeodactylum , and Tetraselmis Lee, ; Wikfors and Ohno, ; Carlsson et al. Additionally, other microalgae have been shown to be a potential source for third-generation biofuels due to their high oil and carbohydrates contents Singh et al. One of the challenges for commercial cultivation of microalgae is the economic feasibility.
Such low yield of microalgal biomass is not sufficient for the industrial production of bioethanol. The most common two methods for the cultivation of microalgae are the outdoor open pond system and the closed photobioreactor [for reviews, refer to Brennan and Owende and Benemann ].
The photobioreactor system, which produces high biomass under controlled growth conditions, requires high capital and operating costs Pruvost et al. In contrast, cultivation of microalgae in open ponds involves lower capital and operating costs but offers low productivity.
Additionally, microalgal cultures growing in open ponds are exposed to contaminants and affected by seasonal variations Chisti, In both systems, microalgal density must be controlled to maintain a viable culture Wang et al.
Other challenges associated with biofuel from microalgae have been discussed in detail elsewhere Hannon et al. Macroalgae refer to the macroscopic seaweeds. They are characterized by forming multicellular specialized tissues and defined structures that are comparable to plant leaves and roots John and Anisha, ; Murphy et al. Macroalgae are less versatile than microalgae and are distributed primarily over green, red, and brown algae Jung et al.
In comparison to terrestrial plants, macroalgae grow faster and produce more biomass per area due to their high photosynthetic efficiency Murphy et al. Although commercial third-generation biofuels are derived from microalgal biomass, seaweeds specifically red and brown macroalgae serve as an unexploited potential source for bioethanol production due to two facts.
First, macroalgae combine high biomass productivity with low capital and operating costs owing to the fact that macroalgae are harvested from naturally occurring stocks or aquacultured sea farms. Such cultivation systems require capital and operating costs that are significantly lower than the microalgal open ponds, nevertheless they provide high biomass productivity Carlsson et al. Second, macroalgae are cultivated worldwide on a large scale for non-biofuel purposes.
The remainder of the biomass, which is rich in carbohydrates, can be hydrolyzed to produce ethanol. In fact, the worldwide biomass production from macroalgae greatly surpasses that of microalgae. For example, in , approximately 9 million and 6. In comparison, a total of only 6. The potential application of macroalgae for biofuel production has been reviewed by others Murphy et al. The production of biofuels from macroalgae has several environmental advantages [discussed by Hughes et al.
First, in contrast to microalgal feedstocks, which are used for simultaneous production of bioethanol and biodiesel, macroalgae accumulate considerable amounts of carbohydrates, and thus can be used to produce bioethanol only [see Table 1 in Singh et al. Third, the macroalgal carbohydrates content varies depending on the alga growth stage and seasonal variations Suutari et al. Fourth, macroalgae accumulate lower amounts of glucan food reserves i. Therefore, the industrial production of bioethanol from macroalgae requires fermentation of both glucose- and non-glucose-based sugars Yanagisawa et al.
Spirulina sp. Its biomass is used primarily for human and animal consumption; however, only a small portion is directed toward biofuel production Ciferri, ; Wikfors and Ohno, ; Habib et al. Additionally, several cyanobacterial strains of Synechococcus species have been genetically modified for enhanced commercial production of bioethanol [reviewed by Dexter et al.
The production of biofuel from cyanobacteria has several advantages [discussed by Quintana et al. Among these advantages is the fact that many cyanobacteria, e. However, there are several disadvantages of using cyanobacterial biomass for biofuel production.
For example, in contrast to microalgae, which store high amounts of lipids and carbohydrates, cyanobacteria do not accumulate significant amounts of lipids, and therefore they are not suitable for biodiesel production Quintana et al. Other challenges that constrain bioethanol production from cyanobacteria have been discussed by other reports Nozzi et al.
Similar to plants, photosynthesis in algae is divided into two steps: the light-dependent reactions and Calvin cycle. In the light-dependent reactions, light energy is absorbed at the thylakoid membranes in the chloroplasts, where it is converted into adenosine triphosphate ATP and the reduced form of nicotinamide—adenine dinucleotide phosphate NADPH.
For reviews on algal photosynthesis, we recommend the reader to refer to Moroney and Ynalvez Algae produce a wide range of polysaccharides depending on the algal species Table 2. This diverse collection of polysaccharides functions primarily as food reserves or structural material Figure 1.
Here, we describe the most economically important algal sugars, which have received considerable amount of research interest. The advantages and disadvantages of employing these sugars for bioethanol production are highlighted in Table 3.
For more information about algal polysaccharides, we refer the reader to previously published reviews Peat and Turvey, ; Percival, , ; Avigad and Dey, ; Grant Reid, ; Synytsya et al. Table 2. Chemical structure and distribution of food reserves and structural polysaccharides among different groups of algae. Figure 1. Overview of ethanol production from major algal carbohydrates. A Algae store simple sugars in the form of simple and complex food reserves See Food Reserves and as structural polysaccharides See Structural Polysaccharides.
The chemical structures of the listed polysaccharides are presented in Table 1. DEHU, 4-deoxy- l -erythrohexoseulose uronic acid. Table 3. The advantages and disadvantages of employing different algal sugars for the production of third-generation biofuels. Food reserves are easily fermented into ethanol and thus are the primary source for industrial third-generation bioethanol.
In contrast, the hydrolysis of structural carbohydrates is challenging due to their rigidity. Therefore, optimization of the hydrolysis process of structural carbohydrates carries the promise of maximizing ethanol yield. In this section, we will first review the major algal food reserves. Additionally, we will discuss major algal structural polysaccharides because of their potential in enhancing the yield of bioethanol from algal feedstock.
The majority of algae store their food reserves in the form of starch-type polysaccharides such as starch, floridean starch, and glycogen Viola et al. Additionally, brown algae accumulate large amounts of mannitol, which functions as an antioxidant and regulator of cell osmolarity Davis et al.
In contrast to plants, which store starch granules in the amyloplast, most algae lack the amyloplast, and therefore store starch grains in the chloroplast Busi et al. Exceptions to this are the red algae, Dinophyta Dinoflagellates , and Glaucophyta, which store their food reserves in the cytosol Radakovits et al.
Floridean starch is another main food reserve polysaccharide Table 2. It is a starch derivative that is synthesized by red algae Rhodophyta. Its granules differ from starch by lacking amylose and thereby are composed completely of amylopectin Viola et al. The granules are similar in structure to plant starch but more variable in size diameter: 0.
Laminarin and chrysolaminarin are the third major food reserves. Laminarin is synthesized by brown algae, and it forms either a G-chain — with glucose molecule at the reducing end — or an M-chain — with mannitol at the reducing end Kadam et al. Chrysolaminarin is the food reserve polysaccharide in diatoms, and it is comprised only of glucose molecules G-chains at the reducing end Beattie et al.
Glycogen is the food reserve form in cyanobacteria. Glycogen and amylopectin one of starch granule constituents are similar in structure; however, glycogen is more branched and forms smaller granules diameter is 42 nm in comparison to starch granules diameter —, nm Ball et al. In addition to the previously described major polysaccharide forms, other granule forms exist among algae but to a lesser degree. For instance, algae of the class Euglenophyta store their food reserves in the cytoplasm as paramylon.
Mannitol is a sugar alcohol of the aldohexose d -mannose. In brown algae, it serves as a storage sugar, and an antioxidant, and protects against osmotic stress. It accumulates in the cell as a monosaccharide i. Mannitol is produced in brown algae from fructosephosphate, which is reduced to mannitolphosphate via Mannitolphosphate 5-dehydrogenase EC1.
In the second step, mannitolphosphate is converted to d -mannitol by mannitolphosphatase EC3. Structural polysaccharides are another putative source to increase bioethanol yield from algae.
Their main function is to confer rigidity to the algal cell wall. In contrast to plants, which usually have a lignocellulosic cell wall, the composition of algal cell wall varies among algal groups. Cellulose is the major algal cell wall polysaccharide, and it is present in most algal groups. In addition to cellulose, algae incorporate significant amounts of other polysaccharides into their cell wall, which can be converted into ethanol.
Such polysaccharides can be specific to an algal group, such as the red algae, which contain agarose and carrageenan; and the brown algae, which are rich in alginate Vreeland and Kloareg, ; Murphy et al. Variations in algal cell wall contents can also be found within families and genera of the same group. For example, the cell wall of the green seaweeds Ulva and Enteromorpha sp.
Cellulose chains aggregate together by intra- and inter-molecular hydrogen bonds to form cellulose microfibrils. Microfibrils are packed together to form fibrils, which in turn aggregate to form cell wall fibers Brown and Saxena, ; Pu et al. With exception to diatoms, cellulose is found in the majority of algal cell walls.
Agarose is a non-sulfated, non-water-soluble linear galactan that is composed of repeating disaccharide units of d -galactose d -Gal and 3,6-anhydro- l -galactose l -AnGal.
The repeating disaccharide unit is called agarobiose or neoagarobiose depending on 1 the position of each sugar in the disaccharide, 2 the bond that links the monomers within the disaccharide, and 3 the bond that links the disaccharides to form agarose. Carrageenan is a sulfated water-soluble linear galactan of carrabiose or neocarrabiose subunits Table 2. Carrabiose and neocarrabiose are similar in structure and linkage to agarobiose and neoagarobiose, respectively De Ruiter and Rudolph, ; Renn, ; Delattre et al.
The main function of alginate is to provide the cell wall with elasticity and rigidity to survive aquatic habitats Dornish and Rauh, Additionally, alginate is found in the matrix of some bacterial biofilms. Although its function in bacterial biofilms is not yet fully understood, alginate has been shown to play a role in bacterial pathogenesis and epiphytism Halverson, Ulvan is a water-soluble cell wall polysaccharide, which is found in green seaweeds, such as Ulva and Enteromorpha sp.
Jiao et al. It is comprised of sulfated rhamnose, glucuronic acid, iduronic acid, xylose, and sulfated xylose. The ratio and linkage of ulvan constituent monosaccharides vary among species [refer to Lahaye and Robic ]. Fucoidan is a cell wall polysaccharide that is found in the members of family Laminariaceae of brown algae.
Fucoidan structure is heterogeneous and varies among algal species. The process of bioethanol production from algal polysaccharides consists of three major steps: biomass pretreatment, enzymatic hydrolysis of algal polysaccharides, and fermentation of sugar monomers to ethanol.
The pretreatment step disrupts algal cell and releases intracellular sugars. Additionally, the pretreatment step reduces algal cell wall crystallinity making its polysaccharides accessible for enzymatic hydrolysis. The three most abundant polysaccharides are starch, glycogen, and cellulose. These three are referred to as homopolymers because each yields only one type of monosaccharide glucose after complete hydrolysis.
Heteropolymers may contain sugar acids, amino sugars, or noncarbohydrate substances in addition to monosaccharides. Heteropolymers are common in nature gums, pectins, and other substances but will not be discussed further in this textbook.
The polysaccharides are nonreducing carbohydrates, are not sweet tasting, and do not undergo mutarotation. It occurs in plants in the form of granules, and these are particularly abundant in seeds especially the cereal grains and tubers, where they serve as a storage form of carbohydrates.
The breakdown of starch to glucose nourishes the plant during periods of reduced photosynthetic activity. Commercial starch is a white powder. Starch is a mixture of two polymers: amylose and amylopectin. When coiled in this fashion, amylose has just enough room in its core to accommodate an iodine molecule. The characteristic blue-violet color that appears when starch is treated with iodine is due to the formation of the amylose-iodine complex.
This color test is sensitive enough to detect even minute amounts of starch in solution. The helical structure of amylopectin is disrupted by the branching of the chain, so instead of the deep blue-violet color amylose gives with iodine, amylopectin produces a less intense reddish brown. Dextrins are glucose polysaccharides of intermediate size. The shine and stiffness imparted to clothing by starch are due to the presence of dextrins formed when clothing is ironed.
Because of their characteristic stickiness with wetting, dextrins are used as adhesives on stamps, envelopes, and labels; as binders to hold pills and tablets together; and as pastes. When only comparing the values found at the beginning and at the end of the fermentation, the same trend as for the other data was found: an increase of the total carbohydrate content. It should be noted that the standard deviations of experimental data for FT-IR method are lower than those of the two other methods.
The low reproducibility of values obtained by the HPLC-RI and the phenol method, respectively, are most likely due to differences in cell disruption efficiency and the necessary but error-prone sample handling. Similar results were obtained for fed-batch fermentation 2 data not shown. FT-IR spectra of whole S. Samples were drawn from nitrogen limit fed-batch fermentation 1 at different times over the course of the fermentation.
Variations of normalized total carbohydrate content for real samples from nitrogen limited fed-batch fermentation 1 of S. Parameters and results of the PLS regression models for trehalose, glycogen, and mannan are summarized in Table 4.
Considering the concentration ranges used for calibration, these values show that FT-IR spectroscopy is a promising technique for fast, reagent-free measurement of intracellular carbohydrates. The analyte concentrations determined by cross-validated PLS regression and the reference concentrations found for the different samples drawn from the two fermentations are shown in Fig. The black squares indicate the values found by reference analysis and the red circles correspond to the values predicted from cross-validated PLS regression, i.
The values are in good agreement with each other and the expected changes in carbohydrate content of the yeast cells. These large increases and decreases in reference values are also the points where FT-IR data and reference data deviate strongly. Each single value obtained by reference analysis is prone to errors due to the necessary sample pretreatments and handling. When calculating a PLS regression model from these data, the erroneous values which are both positive and negative deviations from the true value are averaged over the whole calibration set and resulting PLS predictions show a smoother time course.
Relative trehalose, mannan, and glycogen content of the samples drawn from fermentations 1 and 2 at different times determined by reference analysis black squares and from FT-IR spectra by cross-validated PLS regression red circles. Most of the published articles to determine the carbohydrate composition of yeast cells describe time-consuming wet chemical assays requiring handling-intensive pretreatment steps.
The method determines the content of several polysaccharides such as mannan and glycogen and the disaccharide trehalose by cross-validated PLS regression. Results can be obtained within 30 min from sampling, including the most time-consuming step, i. Using the instrumentation and settings in this study, acquisition of spectra took approximately 3 min per sample spot, showing that significant improvement of measurement time is possible by using other drying methods, e.
The obtained results show that concentration values are in good agreement with reference values. Milic TV, Rakin M, Siler-Marinkovic S Utilization of baker's yeast Saccharomyces cerevisiae for the production of yeast extract: effects of different enzymatic treatments on solid, protein and carbohydrate recovery.
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