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Review

The Macroalga Kappaphycus alvarezii as a Potential Raw Material for Fermentation Processes within the Biorefinery Concept: Challenges and Perspectives

by
Adam Tabacof
1,*,
Verônica Calado
2 and
Nei Pereira, Jr.
2,*
1
Department of Chemical Processes Technologies, Federal Institute of Rio de Janeiro for Education, Science and Technology, Rio de Janeiro 20270-021, RJ, Brazil
2
Center of Biofuels, Petroleum and Derivatives, School of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro 21941-972, RJ, Brazil
*
Authors to whom correspondence should be addressed.
Fermentation 2024, 10(6), 283; https://doi.org/10.3390/fermentation10060283
Submission received: 7 April 2024 / Revised: 15 May 2024 / Accepted: 25 May 2024 / Published: 28 May 2024
(This article belongs to the Special Issue Algae—The Medium of Bioenergy Conversion: 2nd Edition)

Abstract

:
Seaweed is a fast-growing biomass source that is currently studied as feedstock for sustainable industrial production in a wide variety of markets. Being composed mostly of polysaccharides, macroalgae can be integrated in biorefineries for obtaining bioproducts via fermentation. Kappaphycus alvarezii has been introduced experimentally to Brazil’s south coastline in 1995 and is now cultivated on a large scale to keep up with the high carrageenan demand in various industrial sectors. In this review article, an introduction is given on renewable biomass and environmental issues, focusing especially on third-generation biomass and its promising features and use advantages. Later on, the processing of K. alvarezii for the use of its saccharide portion for fermentative processes is approached. The current state of research conducted alongside challenges and hurdles in K. alvarezii hydrolysate fermentation processes provides insight into future studies needed to make new fermentation processes viable. Next, some fermentation products are discussed, and the metabolism of galactose in microorganisms is also presented to bring to light other possible fermentation products that are not yet, but can be, obtained from K. alvarezii. Finally, a simple and comprehensive scheme for K. alvarezii fermentation biorefinery is presented to demonstrate a generic example for a possible configuration for obtaining valuable bio-products. In the literature, production of ethanol and lactic acid were already reported from K. alvarezii. This review aims to help envision new industrial processes that can be developed for this most valuable macroalga.

1. Introduction

Over the last decades, finding new biomass sources for chemical compound and commodity production is being researched to eventually substitute similar fossil-based products. This intense search emerged along with the rise in public awareness of global warming, which, in turn, could be directly related to large carbon dioxide emissions caused by the need to meet mankind civilization’s energy requirements.
Large-scale chemical industries usually have high energy requirements, leading to a relatively high carbon footprint. The carbon footprint theme is relatively new, being studied as an index for the contribution of individuals, companies, and government entities to global warming [1,2]. Sustainability is the measure of human production in harmony with nature and maintenance of planet Earth’s climate stability [3].
Carbon dioxide emissions have increased from 2600 Mt in 1960 to 3500 Mt in 2018 in the European Union alone [4]. Globally, there is still an increase in CO2 emissions. Liu et al. [5] reported that the emissions in 2022 were 1.5% higher than the prior year. The use of alternative energy sources can mitigate the increase in carbon emissions in order to reach emission goals set by international agreements.
Renewable raw materials are natural biological resources, continuously available for human consumption with no risk of running out if not used excessively [6]. The environmental impact of a specific bio-based industrial process can be evaluated by a life cycle assessment (LCA), a quantifying tool that includes the raw materials, production process, product distribution, use, and waste treatment as factors for measuring negative environmental effects [7]. Carbon emission mass balances can be used as an indicator of the carbon footprint of specific bioprocesses. As shown by Terlouw et al. [8], mass balance for carbon dioxide emissions should include farming, plantation, land use, logistics, industrial process, and distribution of products to accurately calculate the carbon input or output of a given bio-based process.
Algal biomass is estimated to be the most probable focus on biorefinery study investment for the next decades in both the US and European Union [9,10]. One of the reasons algal biomass is an attractive feedstock is its fast growth rates and carbon dioxide sequestration, which lead to a lower carbon footprint. Moreover, seaweed requires less land use for cultivation compared to terrestrial crops and can even be grown in artificial bodies of water when given a circulation of seawater to provide nutrients [11].
Macroalgae are composed mainly of polysaccharides, making them adequate as feedstock for producing fermentation products [12]. Most species are rich in valuable minerals and salts absorbed from seawater, which can be extracted alongside proteins and antioxidants with minimal environmental impact [13,14,15]. In the last decade, interest in seaweed refining processes has increased, with a current number of academic publications using the terms “biorefinery” and “macroalgae” in the same document larger in a two-order magnitude than the quantity published in the beginning of the second decade of the 21st century (Figure 1).
Kappaphycus alvarezii is a red seaweed which consists of carrageenan, a galactan polysaccharide, and linear cellulose. These natural polymers can be degraded mainly into two fermentable monomers, glucose and galactose. Carrageenan content can reach up to 49% of the algal biomass [16], and the exploitation of the species for its refining products has more than doubled over the last two decades [17].
The implementation of a K. alvarezii fermentation biorefinery process presents some critical hurdles, first of which is economic viability due to the high raw material price which is about four times higher than the sugarcane bagasse acquisition cost [18,19]. Government policies that provide incentives for farming, the development of new products, and scientific research could make using K. alvarezii more profitable, the same way as first-generation ethanol production was made more efficient in Brazil [20].
Fermentable saccharides from algal biomass can be obtained via dilute-acid and enzymatic hydrolysis, both of which can be used in sequence [21,22]. High-temperature dilute-acid hydrolysis of carrageenan leads to solutions containing high concentrations of 5-hydroxymethylfurfural (HMF), a compound that has inhibitory effects on fermentation processes [23]. In some cases, HMF should be removed from fermentation media prior to the microorganism inoculation. The removal of HMF could be carried out using activated charcoal [22,24], and overliming processes [24,25,26,27]. Alternatively, HMF can be removed directly from the hydrolysates, applying reactive extraction via zeolites as catalysts or using absorption in an organic phase in slug flow reactors or stripping columns [28]. It should be noted that HMF is a valuable molecule for some chemical industries, and it could be used as a precursor of levulinic and formic acids [13], as well as a building block for more complex added-value products [29].
Enzymatic hydrolysis can be applied to the cellulose content of K. alvarezii biomass using commercial enzyme cocktails [21], and the carrageenan itself can be reduced to monomers enzymatically with enzymes obtained from marine microflora [30]. Enzymatic hydrolysis provides specific chemical manipulation and bond cleavage, ensuring the preservation of other valuable compounds present in algal biomass, such as pigments, proteins, and phenolic compounds [31]. Application of marine-sourced enzymes is at the forefront of algae-polysaccharide degradation processes. In the case of K. alvarezii, the carrageenan and linear cellulose content can be reduced to oligomers with antioxidant properties and fermentable monomers.
Some K. alvarezii biorefinery schemes are available in the literature. Álvarez-Viñas et al. [13] show four different configurations for K. alvarezii processing, three with bio stimulants, ethanol, fertilizers, and biogas products in addition to carrageenan extraction. The other biorefinery presented is for a thermochemical process that produces HMF, levulinic and formic acids. This last configuration uses the biomass residue for energy generation via combustion. Torres et al. [12] present a K. alvarezii biorefining process where ethanol is produced from algal biomass residue after carrageenan extraction.
K. alvarezii is one of the most widely cultivated seaweeds on the planet, a fact that makes it readily available as raw material for obtaining bioproducts. This paper aims to relay information about the processing of K. alvarezii biomass for fermentation processes, including acid hydrolysis, detoxification and enzymatic hydrolysis. A short review of galactose metabolic pathways brings to light potential products that can be obtained from the algal biomass via fermentation.

2. Classification of Renewable Raw Materials

Biofuels are popularly classified in generations according to the raw materials used as feedstock to produce them. This “generation” term is also extended in the literature to biorefineries dividing them into generation groups by the same criteria [32,33]. Biomasses, like biofuels, can be divided in the same manner. First-generation (1G) biomass is essentially food products, extracted from agricultural crops such as sugarcane, wheat, corn, etc. Second-generation (2G) biomass is lignocellulosic material obtained from agricultural product waste, or other biomass not used for human consumption. Biofuels derived from algal biomass, being it microalgae or macroalgae, can be defined as third-generation (3G) biofuels [3,34]; thus, algal biomass can be defined as third-generation biomass. The literature mentions also a fourth-generation biomass, which consists of microorganisms, such as cyanobacteria, with genetically engineered metabolic pathways, that can convert sunlight directly into biofuels and other desired products [34,35,36].
Alternatively, raw materials can be classified according to their composition as suggested by McKendry in 2002 [37]. The humidity, caloric value, and the proportion of carbon, ash, alkaline metals, as well as the cellulose-to-lignin ratio can determine the potential products that could be obtained from any given biomass. This analysis helps to evaluate if a certain biomass can be used as a drop-in material in already-functioning industries. In any case, the choice of an appropriate raw material is a cumbersome task that requires considering logistics of transportation, plantation, and harvest. Moreover, all process technological aspects should be taken into account for the creation of a successful industrial endeavor.
It is worth mentioning that all renewable sources, especially for the energy sector, should be explored in roadmap plans for a sustainable future. This idea has been stressed in the literature for a long time [3,34,38]. Improvement in technology for 1st-, 2nd-, and 3rd-generation biofuels, making them more economically viable, will lead to the gradual substitution of fossil-based fuels. Eventually, promising technology such as cultivation of microalgal biomass would be made viable, enabling the conversion of sunlight directly into valuable bioproducts in photoreactors [39,40].

2.1. Raw Material of the 1st and 2nd Generations

1G bioprocess technologies are well consolidated, and their products are commercialized worldwide. Although renewable, this type of biomass cannot, on its own, guarantee sustainability in the long run, for being food products further converted to valuable products, the increase in world population could strain the food and energy market. Various authors have alerted about possible direct food market and bioproduct conflicts, which can cause price oscillations and consumer goods shortages [41,42,43].
In contrast to 1G biomass, 2G biomass is inedible, unaffecting the food market, and not creating a socioeconomic debate about nutritional resource division and competition. The cultivation of raw material for 2G industries creates opportunities for companies, generating jobs that boost economic development. Major players in the chemical industry are already investing in 2G biorefineries [42,44,45].
The use of renewable raw material promotes sustainability better than non-renewable fossil-based resources; however, obtaining products with no negative environmental impact is extremely hard to achieve. Most 1G and 2G biomasses require in-land cultivation, which can cause a delay in carbon gas sequestration from the atmosphere [46].
Replacement of wild flora by cropland could potentially create a long deficit in carbon fixation. The lack of carbon sequestration could take long periods of time to compensate, from 40 to 120 years with high-yield crops, and reach up to 1500 years in the case of low-yielding crops such as maize and soybeans [47]. Harper et al. [46] stress that forestation mitigation plans can be used to achieve environmental global warming goals.
Even given that the CO2 fixation of crop replantation and growth can account for some of the industrial process emissions, the destruction of local flora when preparing land for farming exploration can force wildlife from its habitat and disrupt the process of evapotranspiration, responsible for the planet’s healthy water cycle [48]. Indirect land use by industries is also underestimated and marked as a potential, unaccounted-for, greenhouse gas emission cause [49]. Without using the appropriate boundaries for carbon life cycle calculations, processes could mistakably present negative emissions [50].

2.2. Macroalgae: Third-Generation Raw Materials

Technologies for 3G biomass processing in the bioindustry are at an initial stage in comparison to 1G and 2G biomass processing, which puts it in a disadvantage, mainly regarding the efficiency of farming, production, and transportation [51]. Nevertheless, macroalgae present a potential to have a role in future mitigation of climate change, either by the maintenance or reforestation of seaweed natural forests [52]. Seaweed partake in the global fixation and embedment of CO2 in ocean floors by farming it either on or offshore [52,53].
Cradle-to-grave LCA estimates for macroalgae have been conducted in the last few years, mainly for the production processes of biofuels such as methane and ethanol, with promising results. In 2012, Langlois et al. [54] concluded that even though, at the time, natural gas showed less impact than macroalgae biomethane, technological advancement and high production rates could make methane produced by seaweed biomass emit less CO2 than natural gas. Brockmann et al. [53] stressed in 2015 that although producing bioethanol from green macroalgae presents higher emissions than sugarcane ethanol, this is due mainly to logistics, infrastructure, and electricity consumption. The need to pump nutrients from the sea makes for high energy requirements. Even in the face of the present data, the authors believe that seaweed bioethanol presents long-lasting environmental benefits and could reach higher standards with time.
Seghetta et al. [55] showed how seaweed cultivation could lead to water restoration and reach negative emissions for the production of ethanol jointly with extraction of proteins and fertilizers. In a review by Duarte et al. [52], it was claimed that for every ton of dry seaweed used for biofuel production, 961 kg of carbon dioxide can be removed from the atmosphere. LCA tools were also used to assess the impact of production of polymeric packaging materials [56]. Rose [57] compiled LCA studies for various biofuels, ranking them for the quantity of carbon dioxide capture and permanence of its storage out of the atmosphere. In another compilation amassed by Pangestuti et al. [51] in 2021, 50% of biofuels LCA showed negative emissions, and some showed less emissions than 1G similar processes.
A comprehensive illustration for system boundaries in LCA for products obtained from macroalgae is presented in Figure 2, considering land use as negligible, but it should be advisable to take indirect land use into account for farming purposes.
Macroalgae are composed mainly of gel-forming polysaccharides and cellulose from which a variety of fermentable monomers are obtainable. Green macroalgae monosaccharides include d-glucose, l-rhamnose, d-psicose, d-tagatose, and l-idronic acid. Brown seaweed contain l-fucose, d-mannitol, mannuronic acid, and glucuronic acid. Red seaweed contain galactose, 3,6-anydro-d-galactose, and agarobiose. Some pentoses such as l-arabinose, d-xylose, and d-ribose can also be found in macroalgae [58]. This high saccharide content makes fermentation processes with seaweed as raw material yield-effective. Fasahati el al. [59] demonstrated that using fermentation processes for ethanol production is the best alternative for energy production from seaweed, with physical processing (i.e., grinding and chopping) and transportation (i.e., wet or dry transportation and pumping) as main contributors to negative environmental impacts.
The carbohydrates of seaweeds make up, on average, 45% of their total dry mass. Due to their high mineral content, macroalgae normally contain a high concentration of calcium, magnesium, iron, and potassium in comparison to most terrestrial biomass, making them attractive for human consumption. The protein content is species-dependent, with significant quantities of amino acids and peptides, ranging from 3 to 15% in brown macroalgae (Phaeophyta), about 25% in green macroalgae (Chlorophyta), and reaching 40% in red macroalgae (Rhodophyta). Seaweeds are also known to contain valuable extractable pigments [12].
Traditionally farmed for consumption as part of the human diet, many seaweeds are a nutritive and healthy substance source that can be consumed in natura, or in the form of extracts, added to food products. In more recent times, seaweed extracts are used as gels and hydrocolloids in the cosmetic and health-care industries [14,60]. From the beginning of the 21st century, seaweed extract has been used as a bio-stimulant and fertilizer in terrestrial plantations, and as feed and compliment nutrition for poultry, cattle, and swine [12].
For their high sugar content, macroalgae are extensively studied as raw material for biofuels, mainly ethanol and butanol via fermentation processes. In addition to liquid fuels, which also include biodiesel from the low lipid content in macroalgae [61], gaseous fuels, such as methane and hydrogen, can be obtained from anaerobic digestion and dark fermentation. Biochar and bio-oil can be obtained through thermochemical transformation as well [12].

2.3. Kappaphycus alvarezii

Named by the biologist Vincente Alvarez, K. alvarezii, a red (Rhodophyta) seaweed, is considered as a dominant source of carrageenan [62]. K. alvarezii is fast-growing, able to duplicate its mass in less than a 30-day period [63]. It is a tropical species, viable strictly within 20° S and 20° N latitudes [64].
Like most seaweeds, K. alvarezii is composed mostly of saccharides (57.2%), in the form of carrageenan that contains galactose, and cellulose containing glucose. K. alvarezii also has a high quantity of minerals (15.8%), proteins (2.6%), and lipids (5.2%) [13]. The elkhorn-shaped sea moss can be divided into two essential parts: an exterior hydro-soluble gelatinous wall composed of carrageenan and a crystalline cellulosic interior. A macromolecular composition of K. alvarezii cultivated in Brazil as reported by Masarin et al. [65] is given in Table 1.
The hydro-soluble fraction is made mostly of galactose, these concentrations can reach more than 60% of the plants’ exterior mass when adding up the quantities of galactose, 3,6-anydro-galactose, and 6-O-methylgalactose obtained from carrageenan which consists of a repetition of the two former monomers. The hydro-insoluble residue of K. alvarezii, made mainly of linear cellulose, has a 70% glucose content, 14% galactose, about 4% of mannose, and 1% of xylose [66].
In addition to the sugar content, carrageenan can have a high calcium cation content (140 mmol/100 g) [67] and proteins [68]. K. alvarezii has a complex and adaptive pigment content. A detailed pigment composition can be found in the review of Indriatmoko et al. [69].
Fermentation processes, using seaweed biomass as raw material (Figure 3), involve three main steps. First, the polysaccharides are hydrolyzed to obtain the fermentable monomers, which are then fermented. Fermentation can also be carried out jointly with saccharification, e.g., simultaneous saccharification and co-fermentation (SSCF). The products are then separated from the fermented media, prepared, and packed for commercialization.

3. Processing K. alvarezii Biomass for Fermentation Processes

Sugars from K. alvarezii biomass can be obtained either by dilute-acid hydrolysis or enzymatic hydrolysis (Table 2). In most cases in the literature, carrageenan content is turned into galactose in a low concentration solution of either sulfuric or hydrochloric acid under temperatures ranging from 100 to 140 °C. Meinita et al. [70] and Ra et al. [71] reached concentrations of about 38.5 g/L of fermentable sugars from a 10–12% (w/v) biomass concentrated mix with a 0.2 molar concentration of sulfuric acid at 130–140 °C. These hydrolysis procedures in relatively higher temperatures and lower biomass concentration resulted in the formation of about 5 g/L of HMF. When using higher biomass concentrations in more prolonged hydrolysis procedures, higher galactose concentrations can be obtained. As shown by Khambhaty et al. [72] and Tabacof et al. [24], more than 50 g/L of galactose can be reached in a mixture with about a 30% (w/v) biomass concentration. The hydrolysis conditions in these cases were 100–110 °C and 1% (v/v) sulfuric acid concentration, and the hydrolysis time was 45 to 60 min long. Using similar conditions with an even higher biomass concentration of 50% (w/v) mix, Hargreaves et al. [22] reached a galactose concentration of 82 g/L. The work carried out by Hargreaves et al. [22] and Tabacof et al. [24,73] showed that exposing K. alvarezii biomass to prolonged acid hydrolysis results in the formation of 1 g/L of HMF for every 4 g/L of galactose.
As can also be seen in Table 2, most enzymatic hydrolysis research has been carried out to investigate the exploitation of the crystalline linear cellulose content of K. alvarezii biomass using commercial enzyme cocktails such as Cellic CTec2 [21,22,65] and Celluclast® 1.5 L [74], obtaining more fermentable sugars, mostly glucose. Attempting to make most of the total K. alvarezii biomass, Abd-Rahim et al. [74] reported an increment of 15 g/L of reducing sugars to the 35 g/L obtained from dilute-acid hydrolysis reaching approximately 63% of hydrolysis efficiency from the combination of both methods in comparison to the 45% obtained from the acid hydrolysis alone. Hargreaves et al. [22] were able to simultaneously saccharify and ferment the cellulosic residue, as well as doing so while co-fermenting the galactose from a dilute-acid hydrolysate for ethanol production using Saccharomyces cerevisiae as the fermentation agent. The fermentation of the galactose from dilute-acid hydrolysis from 1000 g of dry seaweed resulted in 48.1 g of ethanol and the glucose from the 150 g of cellulosic residue being converted to an additional 43.7 g of ethanol, showing an almost double amount of product yield when combining chemical and enzymatic hydrolysis methods. Puspawati et al. [75] also successfully produced ethanol from the cellulosic residue.

3.1. Enabling Dilute-Acid Hydrolysate Fermentation

Processing seaweed for fermentation processes requires removal of elements that can potentially hinder cellular growth or inhibit the formation of the desired fermentation product. The macroalgae biomass must first be washed to reduce its salinity and remove debris that could have accumulated during cultivation and dehydration. The desalinated biomass is then grinded or finely chopped either at its wet state or, as more often reported, after being dehydrated once more [76]. Since furanic and furfural compounds can form as products of acid hydrolysis [26], in some cases the hydrolysate will need to be detoxified (Figure 4).
During dilute-acid hydrolysis in high temperatures, HMF is formed from the acid interaction with the carrageenan monomers, galactose and 3,6-anydro-α-l-galactose. As explained in the work of Oh et al. [77], high HMF quantities are formed mainly from the dehydration of the 3,6-anydro-α-l-galactose molecule via acid catalysis, and the HMF, in turn, can be rehydrated to levulinic and formic acid.

3.1.1. HMF Removal from K. alvarezii Hydrolysates

Removal of HMF from K. alvarezii hydrolysates can be performed via activated charcoal filtration, where HMF is captured within the charcoal’s pores [22,24,78]. Hargreaves et al. [22] determined that fine powdered charcoal is preferable to charcoal granules, and that approximately 1 g/L of charcoal should be used for each 1 g/L of HMF removed from the hydrolysate. Activated charcoal can also be regenerated via oxidation processes [79], desorption and decomposition methods [80], and thermal treatment [73,81]. Since HMF is a valuable chemical compound [29], its recuperation from the activated charcoal should be considered. An interesting technique is the removal of HMF from the charcoal pores using an organic solvent. Slak et al. [28] showed a similar process using carbon black that could be replicated for activated charcoal.
Another reported method for HMF removal is that of overliming, which consists of the addition of calcium oxide (CaO) [25] or calcium hydroxide (Ca(OH)2) [24,26,27]. A byproduct of HMF formation under acid-hydrothermal treatment are humins. Humin is a general term used for describing the bulk of insoluble organic compounds in soils [82]. Although the natural formation of humin in soil is not well defined in the literature, its formation mechanisms from HMF and carbohydrates have been proposed by various authors [83,84,85,86]. Humins are known to agglomerate and become insoluble under alkaline conditions [82,87]. Furthermore, aldol reactions, which are part of the mechanisms for humin polymerization, can be base catalyzed [88]. This way, the overliming alkaline conditions facilitate the condensation of HMF into the humin macromolecules, generating even larger humic substances, rendering them more insoluble.
Overliming methods vary. Xia et al. [89] reported that 50% of furans and HMF were removed when adjusting the pH of corn stalk hydrolysates to 10 at 60 °C for 2 h. Chi et al. [26] studied overliming processes for sugar mixtures obtained by pyrolysis. Preparing the solution for ethanol production by E. coli, 18.5 g/L of CA(OH)2 was added, and 8–16 h of treatment were carried out at 20 °C. The process was accelerated to a 1–4 h treatment period when temperatures were maintained at 60 °C. Mohgheghi et al. [27] used a shorter treatment period (30 min), showing that pH and temperature are key factors for efficient removal of HMF. In a recent study, K. alvarezii hydrolysates containing 24 g/L were reduced to 9 g/L using the addition of Ca(OH)2, elevating pH values to over 11 for 30 min [24].
Combining both methods, first applying overliming conditions and subsequently treating hydrolysates with powdered activated charcoal, can remove nearly all traces of HMF in K. alvarezii hydrolysates [24]. The charcoal pores absorb the soluble humins left over from the overliming treatment alongside the HMF that was not condensed into insoluble organic material.
The downside to using charcoal and calcium hydroxide is the material cost at large-scale production. Other efficient and cost-effective methods can also be proposed for HMF removal. such as nanofiltration and reverse osmosis [90]. It is worth mentioning that activated charcoal can be regenerated by chemical and thermal methods [73], and the calcium sulphate (gypsum) produced after neutralization of the overlimed solution can be put to use for construction purposes.

3.1.2. Cellular Acclimation to Hydrolysate

The HMF tolerance varies depending on the species of the microorganisms. Some microorganisms have extremely low HMF tolerance, and its presence can lead to a halt in cell growth, substrate consumption, and product formation [91,92]. Microorganisms are hardy and fast-evolving and can acquire tolerance to new, and potentially harmful, environments. Microbial cells can adapt to stress caused by harsh medium conditions or by differing carbon sources and nutrient availability. Short-term adaptation is more complex, including enhanced expression of certain genes. For instance, in yeast cells adapting to a galactose-rich environment, the GAL proteins were overexpressed, identifying change in expression of their transcription factors [93]. Long-term adaptation requires exposure to new mediums in which cells go through genetic mutations and obtain favorable phenotypes that help them better cope with their growth medium. A detailed explanation of adaptation can be found in a review by Tan et al. [94].
Yeast species, such as S. cerevisiae, can have a high HMF tolerance and, in some cases, convert up to 100% of furfural and HMF elements to less inhibitory components [95]. Adaptation to certain substrate can help to make more efficient use of certain metabolic pathways. Khambhaty et al. [72] and Hargreaves et al. [22] both used S. cerevisiae to produce bioethanol using K. alvarezii dilute-acid hydrolysates. The former did not report an adaptation process to a galactose-rich medium, while the latter acclimated the cells used for inoculum to galactose. After acclimation, the total consumption of galactose took place in less than 20 h compared to the 48 h fermentation period when the same species was not acclimated to galactose.
The presence of HMF can lead to metabolic shifts and different undesired fermentation products. Monlau et al. [96] reported that hydrogen-producing pathways during dark fermentation processes are turned to lactate, acetate, and ethanol pathways in the presence of HMF. An alternative treatment solution is biotransformation. HMF can be bio-transformed into 2,5-furandicarboxylic acid that can be used as a chemical building block [97].
Some bacteria, such as E. coli, are unviable in non-treated medium [26]. The Lactobacillus pentosus strain used in a study by Tabacof et al. [24] showed a threefold faster lag phase, two times the substrate consumption rate, and a higher product formation rate when exposed gradually to K. alvarezii hydrolysates. Due to the vast variety of fermentation agents, it is imperative to study the effect of fermentation media on the chosen microorganisms.

3.2. Enzymatic Hydrolysis Using Carrageenases-Type Enzymes

Enzymatic hydrolysis is on the forefront of algal biomass refining. Saccharification and extraction of intact compounds requires the use of enzymes capable of breaking down seaweed polysaccharides without degrading valuable organic material. Enzymes used for algal biomass are obtained mainly from marine microorganisms. By applying enzymes specific to the polymer chains of the soluble gelatinous polymers or the cellulose content of the algal biomass, valuable compounds such as pigments and proteins can be safely extracted from the biomass for commercial or industrial use [98]. Furthermore, biopolymers can be degraded to oligosaccharides and polyphenols with antioxidant, anticoagulant, anti-inflammatory, antiviral, and antitumoral properties [99].
The formation of oligomers via enzymatic hydrolysis has a high degree of homogeneity. Since enzymes function in relatively mild conditions, the degradation or undesired change in molecular structure of valuable compounds can be avoided. Moreover, obtaining oligomers from carrageenan through other methods has some disadvantages. Microwave degradation requires special equipment and operates in high pressures. Ozone depolymerization promotes an undesired chemical reaction, leading to carboxylic compounds. Ultrasound degradation is less efficient, resulting in non-homogenic oligomers. The use of gamma radiation to form oligomers is efficient, albeit the products could be toxic and need to be further investigated [100].
As observed in Table 2, up-to-date enzyme hydrolysis of K. alvarezii biomass focused on exploiting the seaweed’s cellulose for fermentation processes. Carrageenan contains a large amount of galactose that can be extracted via enzymes specific to its structure, the carrageenases. The study of marine vegetal biomass is at an early stage. Bäugman et al. [30] described in their review the state of the art for seaweed enzyme degradation of carrageenan, ulvan, agar, porphyran, and laminarian polysaccharide complexes. In K. alvarezii, µ-carrageenan turns into κ-carrageenan upon alkaline extraction. The enzymatic saccharification of κ-carrageenan includes four steps. First, the main chain is cleaved into four-monomer oligomers called neo-carrageenan by GH16 κ-carrageenase. These are then desulphurized to β-carrageenan by S1_7 and S1_19 κ-carrageenan-G4S-sulfatase. After desulphurization, β-carrageenan is further cleaved by GH16 and GH167 β-carrageenases into the basic carrageenan dimer, made of galactose and 3,6-anydro-d-galactose. Finally, d-galactose and 3,6-anydro-d-galactose are formed via the GH127 and GH129 3,6-anydro-d-galactosidase enzymes. Table 3 summarizes the steps for κ-carrageenan enzymatic saccharification, and Figure 5 illustrates the metabolic pathway for the enzymatic activity.
Carrageenan-degrading enzyme mixtures can be obtained from marine bacteria, the most cited being Pseudoalteromonas atlantica, Zobellia galactanivorans, and Pseudoalteromonas carrageenovora [30]. The marine microbiome is diverse and adapted to environments with extreme conditions. Extremophiles can produce enzymes that can function under high salinity, in alkaline or acidic media, and in extreme pressures and temperatures [101]. Such enzymes can favor parallel saccharification and fermentation processes. Pseudoalteromonas sp. and its variants are bacteria well adapted to extreme conditions as well as severely low temperatures. They are also cited as efficient degraders of galactose-rich polysaccharides other than carrageenan, e.g., agar, pectin, and alginate [102].
Aside from monomer formation for fermentation processes and oligomers for pharmaceutical and food additives, carrageenases can have other applications. Carrageenases can be added to detergents to degrade gums during cloth washing, or used in the textile industry, for their interaction with alginate and starch. Carrageenases can also be used to expose protoplast in algae cell walls, aiding genetic studies [103].
It stands clear that the efficient use of algal biomass as raw material within the biorefinery concept requires a combination of various extraction methods. The study of enzyme hydrolysis is key for reaching efficient extraction technologies that will lead to obtaining a large range of products alongside reducing sugars for fermentation processes.

4. The Fermentation of Rhodophyta-Type Saccharides

Rhodophyta algae are rich in galactose, a naturally occurring sugar which is processed by a wide range of microorganisms. Due to its abundance and potential industrial use, it has attracted academic interest [104] and is tested for various fermentation products (Figure 6).

4.1. Metabolic Pathways for Galactose Fermentation

d-galactose is metabolized through two types of pathways, the Tagatose-5-phosphate (TP) and the Leloir pathways (Table 4 and Table 5) [58]. The TP pathway involves galactose-specific, active transport by a phosphotransferase (Gal-PTS), the resulting galactose-6′phosphate is then turned into tagatose-6phosphate by lacAB, and further phosphorylated by lacC into tagatose-1,6-diphosphate. The enzyme lacD breaks tagatose-1,6-diphosphate into dihydroxyacetone phosphate and glyceraldyde-3-phosphate, which go through glycolysis.
In the Leloir pathway, d-galactose is transported passively through the cell membrane with a galactose permease galP. The intracellular molecule, β-galactose, gets phosphorylated by galM into galactose-1-phosphate. Next, the joint action of the enzymes galT, galE, and galU morph the galatose-1-phosphate into glucose-1-phosphate, which is turned into tagatose-1,6-diphosphate by pgmB and thereafter goes through glycolysis. A detailed explanation of both the TP and Leloir pathways can be found in the work of Nagarajan et al. [58].

4.2. Products from Galactose Fermentation

As can be seen in Figure 6, galactose has been shown to be fermented into a wide variety of products, including biofuels, organic acids, and bacterial cellulose. In order to assess the viability of each product, its specific production rate and yield from algal biomass sugars should be compared to those obtained with 1st- and 2nd-generation biomass. A summary of bioproducts from K. alvarezii or another galactose source is shown in Table 6.

4.2.1. Biofuels

Among biofuels, ethanol has already been shown to be producible directly from galactose-rich macroalgae. Aside from K. alvarezii, Rhodophytae such as Gelidium amansii, Gracilaria sp., Chondrus crispus, and Palmaria palmata were also proved to be promising raw material for bioethanol production via fermentation [76]. For K. alvarezii hydrolysates, S. cerevisiae was used by a number of research groups [22,72]. The highest production was recorded by Hargreaves et al. [22], yielding 48.1 g of ethanol produced from galactose obtained via dilute-acid hydrolysates.
Hydrogen is a zero-carbon emission energy source producing only water when reacting with oxygen, which makes it one of the cleanest fuels of the future [116]. Recent research has also been conducted to evaluate the fermentation of galactose into hydrogen, envisioning the use of galactose-rich algal biomass. Heat treatments are used to separate heat-resistant spore-forming bacteria such as Clostridium butyricum [105] and Clostridium beijerinckii [106] that can successfully convert galactose into hydrogen via acetic and butyric acid pathways. In 2014, Park et al. [107] showed the predominance of the Clostridium cluster for hydrogen fermentation from galactose, reaching hydrogen from galactose yield of 1 mol/mol. Xia et al. [105] and Fonseca et al. [106] more than doubled this yield in 2016, with production rates of 33.6 mL/(g galactose·h) and 117.5 mL H2/L·h, respectively. One of the main hurdles in efficiency in hydrogen fermentation is preventing it from being consumed. This can be achieved by controlling the carbon dioxide concentration in fermentation media. Moreover, genetic and metabolic methods can be used to enhance hydrogen production and inactivate its intake by microorganisms [105].
Butanol is another molecule that can be used as a fuel, although its main use is as a solvent and chemical intermediate. Butanol-producing bacterium, such as C. beijerinckii, are able to intake galactose and produce butanol [108]. Better expression of galactose metabolic pathways is needed to accelerate galactose catabolism, making it equivalent to the processing speeds of cellobiose and glucose in microorganisms.

4.2.2. Organic Acids

Organic acids have extensive use as additives in food, cosmetic products, and solvents. Some organic acids are used as chemical building block platforms for the production of added-value products and biodegradable polymers.
The growth of the poly-lactic acid market has led to the search of new raw materials for lactic acid fermentation. Different seaweed species are already being studied as raw material for lactic acid fermentation, obtaining fermentable sugars principally through dilute-acid hydrolysis. Lactic acid has already been successfully obtained from galactose-rich algae such as Gelidium amansii, Euchema denticulatum, and Gracilaria sp., with final concentrations reaching 30 g/L and yields of lactic acid from reducing sugars around 0.9 g/g [58]. Microorganisms used for lactic acid production from galactose include Lactobacillus strains, L. rhamnosus, L. acidophilis, and L. plantarum, as well as other strains such as Weissella paramesenteroides and Bacillus coagulans. In 2022, Sudhakar and Dharani [110] demonstrated that K. alvarezii biomass can be processed into lactic acid by L. plantarum. In the work of Tabacof et al. [73] in 2023, it was shown that 115 kg of lactic acid can be produced by 1 ton of dry weight K. alvarezii using L. pentosus.
Succinic acid is also a valuable organic acid that can be used to produce poly-butyl-succinate among other added-value products. Actinobacillus succinogenes is a popular choice for studying succinic acid production, and this bacterium can utilize galactose as a substrate [117,118]. Anaerobiospirillum succinoproducenes was also proven to be able to utilize galactose alongside glucose and therefore be an interesting bacterium for red algal biomass hydrolysate fermentation [119].
Algal biomass is a potential feedstock for succinic acid. Laminaria digitata [120] and Saccharina latissimi [121] hydrolysates were fermented by A. succinogenes. As for galactose-producing macroalgae, Palmaria palmata hydrolysates containing glucose and galactose were fermented by a genetically modified strain of E. coli by Olajuyin et al. [109], reaching a 22.4 g/L volume of succinic acid solution with a 1.13 mol/mol yield.
Other widely commercialized organic acids have been produced from galactose fermentation. The vinegar acid, acetic acid, has a wide range of applications and is also a precursor of valuable products such as vinyl acetate. Ehsanipour et al. [113] used the acetogenic bacteria, Moorella thermoacetica, to ferment lignocellulosic sugars, which include galactose. In this case, the galactose was consumed in a slower rate than other reducing sugars and was detected in the final fermented media in low quantities. The authors stressed that recombinant strains should be developed to make galactose intake more efficient.
Citric acid is used as a food additive for preservation and flavoring. The fungi Aspergillus niger can produce citric acid from galactose in whey protein [111]. Citric acid-producing yeast can also be used to convert galactose to citric acid. Lazar et al. [112] demonstrated that the commonly used yeast Yarrowia lipolytica can express all the Leloir pathway genes and efficiently uptake galactose for citric acid production. The yeast, Candida guillermondii, was shown to produce citric acid from galactose as efficiently as from glucose when each carbohydrate was used as a sole substrate [122]. Although in both cases Y. lipolytica and C. gillermondii showed a strong preference to glucose, uptake of galactose shows the potential of applying these microorganisms to ferment galactose-rich media like K. alvarezii hydrolysates.
Spent coffee hydrolysates contain galactose that can be used as substrate for bioproduction via fermentation. He et al. [114] utilized the fermenting agent Clostridium tyrobutyricum to demonstrate production of butyric acid in galactose-rich media, indicating the potential of the production of this valuable organic acid that is used to produce butyrate esters and cellulose acetate butyrate.

4.2.3. Bacterial Cellulose

Cellulose is an abundant natural resource from plant tissue, and it is traditionally used for the manufacture of paper and fibers. Bio-cellulose derived from cellulose-excreting bacteria has a higher purity and crystallinity, which leads to an elevated degree of polymerization, an increase in water-holding capacity, and a superior permeability to oxygen [123].
Gluconacetobacter xylinus has been shown to be able to process galactose for bio-cellulose production [115,123,124]. Al-abdalla and Dahman [115] used wheat straw hydrolysates for bio-cellulose production, showing close to 72% of galactose consumption. Dahman et al. [123] reported that consumption of galactose reached 90% when it was used as substrate for bio-cellulose production with a 6.79 g/g yield. Although galactose in some cases has been shown to be consumed less efficiently than other substrate [124], a resolution could be found in genetic engineering and acclimation methods for activating and overexpressing the Leloir or Tagatose-6-Phosphate pathways.

5. K. alvarezii Fermentation Biorefinery Scheme

A generic process flow diagram for a K. alvarezii fermentation biorefinery is shown in Figure 7. Given the current state of K. alvarezii processing, it is found that the reducing sugars used for fermentation processes can be obtained either by dilute-acid hydrolysis, preferably with sulfuric acid or by enzymatic hydrolysis with the use of carrageenases for processing the carrageenan, and cellulases for obtaining glucose from the algal cellulose component. The cellulosic residue from the dilute-acid hydrolysis can also be enzymatically processed to obtain more fermentable sugars. As discussed earlier, fermentation products can include bio-cellulose, biofuels such as hydrogen, ethanol, and butanol, and organic acids, i.e., lactate, succinate, citrate, butyrate, and acetate.
For dilute-acid hydrolysis, the overliming and activated charcoal methods were shown to be efficient in HMF removal for enabling fermentation. Gypsum can be obtained after the neutralization of the sulfuric acid with calcium hydroxide, and HMF can be extracted from charcoal via organic phase extraction, rendering another valuable refinery product.
Red seaweed is more cultivated than other types of seaweed, representing more than half of the macroalgae cultivated worldwide [13]. Furthermore, these macroalgae do not compete as much with food industries, as they are mainly used for their extraction of their industrial gums (agar and carrageenan). Some species are cultivated more than others in different regions or countries. K. alvarezii is mostly cultivated in Indonesia, the Philippines, and Malaysa [17]. In Brazil, production of K. alvarezii is on the rise as the market for carrageenan amplifies [31]. Development of fermentation processes for K. alvarezii in countries where it is efficiently cultivated on a large scale are relevant because they can create market opportunities never before explored.
In general, fermentation processes broaden the array of products a biorefinery can manufacture. A feasible economic strategy is to integrate fermentation processes for K. alvarezii in existing refineries for other products, using biomass surpluses (if available) for fermentation processes. As enzymatic saccharification technology matures, the extraction of high-value compounds such as natural pigments from K. alvarezii, in addition to fermentable sugars, will be possible. These product combinations with valuable merchandise can make fermentation processes more attractive for industrial investment.

6. Concluding Remarks

Fermentation processes are used to obtain a wide array of products and their use is highly consolidated, and most products are industrially manufactured. In this review, a current state of the art of processing the macroalgae K. alvarezii was discussed. Ethanol and lactic acid have already been produced from this widely cultivated seaweed, and more products that can be potentially derived from K. alvarezii were presented. The main challenges for efficient processing of the biomass and making the sugar-rich medium more fermentable, detoxification and microorganism acclimation were also detailed.
K. alvarezii is one of the most widely cultivated types of seaweed worldwide; its sugar content makes it a promising feedstock for the production of biofuels, organic compounds, and highly valuable chemical building blocks via fermentation. The use of third-generation biomass can become an important milestone in the roadmap for sustainable industrial processes. Thus, it is of great importance to bring to light all the possibilities for further research and elaboration of processes for obtaining bioproducts from this biomass.

Author Contributions

Conceptualization, A.T. and N.P.J.; Data curation, A.T.; Formal analysis, A.T. and N.P.J.; Funding acquisition, N.P.J.; Investigation, A.T.; Methodology, A.T. and N.P.J.; Project administration, N.P.J.; Resources, N.P.J.; Supervision, V.C. and N.P.J.; Writing—original draft, A.T.; Writing—review and editing, N.P.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Number of yearly publications of academic papers which use both the term “biorefinery” and “macroalgae” (data mined from Google Scholar search engine).
Figure 1. Number of yearly publications of academic papers which use both the term “biorefinery” and “macroalgae” (data mined from Google Scholar search engine).
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Figure 2. Illustration of a life cycle assessment (LCA) of carbon dioxide for bioprocesses using algal biomass as raw material.
Figure 2. Illustration of a life cycle assessment (LCA) of carbon dioxide for bioprocesses using algal biomass as raw material.
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Figure 3. Fermentation processes using algal biomass as raw material.
Figure 3. Fermentation processes using algal biomass as raw material.
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Figure 4. Algal biomass pre-treatment for fermentation processes.
Figure 4. Algal biomass pre-treatment for fermentation processes.
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Figure 5. Metabolic κ-carrageenan degradation pathway by carrageenases (Bäugman et al. [30]).
Figure 5. Metabolic κ-carrageenan degradation pathway by carrageenases (Bäugman et al. [30]).
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Figure 6. Possible fermentation products from galactose metabolism reported in the literature.
Figure 6. Possible fermentation products from galactose metabolism reported in the literature.
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Figure 7. Kappaphycus alvarezii fermentation biorefinery scheme with HMF and gypsum as subproducts integrating dilute-acid and enzymatic hydrolysis.
Figure 7. Kappaphycus alvarezii fermentation biorefinery scheme with HMF and gypsum as subproducts integrating dilute-acid and enzymatic hydrolysis.
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Table 1. Macromolecular composition of Kappaphycus alvarezii cultivated in Brazil (adapted from Masarin et al. [65]).
Table 1. Macromolecular composition of Kappaphycus alvarezii cultivated in Brazil (adapted from Masarin et al. [65]).
CarrageenanCelluloseAshProteinsInsoluble AromaticsSulfate Groups
31.3 ± 0.813.5 ± 0.116.0 ± 0.22.5 ± 0.31.5 ± 0.210.1 ± 0.3
Table 2. K. alvarezii dilute-acid and enzymatic hydrolysis conditions and sugar products.
Table 2. K. alvarezii dilute-acid and enzymatic hydrolysis conditions and sugar products.
Hydrolysis ConditionsProductsRef.
Acid HydrolysisEnzymatic HydrolysisSugars (g/L)HMF (g/L)
10% (w/v), 130 °C, 0.2 M of H2SO4, 15 min-38.5 Gal4–5[70]
12% (w/v), 140 °C, 180 mM of H2SO4, 5 min-38.3 Tot<5.0[71]
50% (w/v), 121 °C, 1.0% v/v of H2SO4, 60 minCellic CTec2
(45 FPU/g DW)
50 °C, pH 5 150 rpm,
24 h
81.6 Gal20.7[22]
26.2–30.6% (w/w), 100 °C, 0.9 N of H2SO4, 1 h-54.0 TotN/R[72]
8.0 g/100 mL, 110 °C 0.2 N of H2SO4, 90 minCelluclast® 1.5 L
(150 FPU/g DW)
55 °C, pH 5.5, 150 rpm,
48 h
50.0 TotN/R[74]
-Cellic CTec2
(10 FPU/g DW)
45 °C, pH 4.8, 120 rpm,
72 h
13.7 TotN/R[65]
-Cellulase
(36 AU)
50 °C, 100 rpm,
12 h
8.0 TotN/R[75]
Enzyme-processed hydrolysate at 60–80 °C, 0.5–2% H2SO4,
30–90 min
Cellic CTec2
(100 FPU/g DW)
45 °C, pH 4.8, 120 rpm,
72 h
16.0 Tot<0.01[21]
0.4 M of H2SO4,
100 °C, 3 h
-4.08 mg Tot/g biomassN/R[76]
30% (w/v), 111 °C, 1.0% v/v H2SO4, 45 min-54.2 Gal12.51[24]
Table 3. κ-carrageenan saccharification steps (summarized from Bäugman et al. [30]).
Table 3. κ-carrageenan saccharification steps (summarized from Bäugman et al. [30]).
StepEnzymes for κ-Carrageenan Processing *Products
Initial fragmentationGH16 κ-carrageenaseOligomer of neo carrageenan
Sulphate group removalS1_7/S1_19 κ-carrageenan-G4S-sulfataseβ-carrageenan
Disaccharide formationGH16/GH167 β-carrageenased-galactose and 3,6-anydro-d-galactose dimer
Monosaccharide formationGH127/GH129 3,6-anydro-d-galactosidased-galactose
3,6-anydro-d-galactose
* Enzyme family: GH—glycoside hydrolase; S—sulfatase.
Table 4. Tagatose-6-phosphate pathway (summarized from Nagarajan et al. [58]).
Table 4. Tagatose-6-phosphate pathway (summarized from Nagarajan et al. [58]).
StepSubstrateEnzymes InvolvedProducts
Cell wall permeationGalactoseGal-PTSGalactose-6-Phosphate
Intracellular metabolismGalactose-6-phosphatelacABTagatose-6-Phosphate
Intracellular metabolismTagatose-6-phosphatelacCTagatose-1,6-Diphosphate
Intracellular metabolismTagatose1,6-diphosphatelacDDihydroxyacetone phosphate, Glyceraldyde-3-phosphate
Table 5. Leloir pathway (summarized from Nagarajan et al. [58]).
Table 5. Leloir pathway (summarized from Nagarajan et al. [58]).
StepSubstrateEnzymes InvolvedProducts
Cell wall permeationGalactosegalPβ-Galactose
Intracellular metabolismβ-GalactosegalMGalactose-1-phosphate
Intracellular metabolismGalactose-1-phosphategalT, galE, galUGlucose-1-phosphate
Intracellular metabolismGlucose-1-phosphatepgmBTagatose-1,6-diphosphate
Table 6. Metabolites produced via fermentation of K. alvarezii biomass and other potential metabolites that can be obtained from galactose-rich media.
Table 6. Metabolites produced via fermentation of K. alvarezii biomass and other potential metabolites that can be obtained from galactose-rich media.
ProductMicroorganismFermentation TypeOrigin of GalactoseRef.
EthanolSaccharomyces cerevisiaeAnaerobicK. alvarezii[22,74]
Hydrogencluster I Clostridium
Clostridium beijerinckii Br21
AnaerobicSynthetic[105,106,107]
ButanolC. beijerinckii BA101
C. acetobutylicum 824
AnaerobicSynthetic[108]
Succinic acidE. coliAnaerobicPalmaria palmata[109]
Lactic acidLactobacillus pentosus
Lactobacillus plantarum
AnaerobicK. alvarezii[73,110]
Citric acidAspergillus niger
Yarrowia lipolytica
Candida guilliermondii
Aerobic and AnaerobicWhey protein
Synthetic
[111,112]
Acetic acidMoorella thermoaceticaAnaerobicLignocellulosic sugars[113]
Butyric acidClostridium tyrobutyricumAnaerobicSpent coffee ground[114]
Bacterial CelluloseGluconacetobacter xylinusAerobicWheat straw[115]
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Tabacof, A.; Calado, V.; Pereira, N., Jr. The Macroalga Kappaphycus alvarezii as a Potential Raw Material for Fermentation Processes within the Biorefinery Concept: Challenges and Perspectives. Fermentation 2024, 10, 283. https://doi.org/10.3390/fermentation10060283

AMA Style

Tabacof A, Calado V, Pereira N Jr. The Macroalga Kappaphycus alvarezii as a Potential Raw Material for Fermentation Processes within the Biorefinery Concept: Challenges and Perspectives. Fermentation. 2024; 10(6):283. https://doi.org/10.3390/fermentation10060283

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Tabacof, Adam, Verônica Calado, and Nei Pereira, Jr. 2024. "The Macroalga Kappaphycus alvarezii as a Potential Raw Material for Fermentation Processes within the Biorefinery Concept: Challenges and Perspectives" Fermentation 10, no. 6: 283. https://doi.org/10.3390/fermentation10060283

APA Style

Tabacof, A., Calado, V., & Pereira, N., Jr. (2024). The Macroalga Kappaphycus alvarezii as a Potential Raw Material for Fermentation Processes within the Biorefinery Concept: Challenges and Perspectives. Fermentation, 10(6), 283. https://doi.org/10.3390/fermentation10060283

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