Advances in Third-Generation Bioethanol Production, Industrial Infrastructure and Efficient Technologies in Sustainable Processes with Algae Biomass: Systematic Review

Abstract

The growing global concern about the environmental impact of fossil fuels’ greenhouse gas emissions has spurred the introduction of innovative, sustainable alternatives. Microalgae biomass holds substantial potential as a viable source material for producing environmentally friendly biofuels. Third-generation (3G) biofuels, specifically algae-derived bioethanol, have emerged as viable alternatives to traditional biofuels. The research provides an exhaustive analysis of the contemporary understanding of manufacturing 3G biofuels from microalgae and macroalgae. Additionally, the study provides an in-depth discussion of the identified gaps within these areas. By conducting a systematic literature review, the authors describe current knowledge of 3G biofuel production. The study addresses two key categories: (i) infrastructure and industrial technology, and (ii) the processes for obtaining third-generation biofuels. One highlights the need for efficient management in all stages of bioethanol production, including cultivation, harvesting, extraction, and conversion. Furthermore, leveraging technological advancements, such as selecting superior genetic strains and developing novel conversion technologies, is essential for improving the efficiency and profitability of the manufacturing process. The successful production of 3G bioethanol from microalgae requires a comprehensive approach that addresses various challenges and incorporates sustainable practices to achieve environmental and economic goals.

1. Introduction

The global market demands suitable energy resources, and there is a need to establish environmental protection strategies via promoting biofuel production. The latter provides viable options for these global challenges [1]. The European Union (EU) is transitioning from fossil fuels to biofuels, as they offer better performance than gasoline and diesel, particularly in the automotive sector. According to statistical data, excluding indirect emissions from fuel and automobile manufacturers, the transportation sector is responsible for 33% of all carbon dioxide emissions and 25% of all greenhouse gas emissions in the EU. Passenger cars are accountable for more than half of the EU’s greenhouse gas emissions recorded from the transport sector [2].

As an alternative, biofuels and their blends can reduce detrimental emissions, including carbon monoxide (CO), carbon dioxide (CO₂), and nitrogen oxides (NO_x). They also improve the performance of gas turbines [3]. Bioethanol reduces GHG emissions by the same amount. Research and development (R&D) efforts aimed at producing biofuels from microalgae are encouraging, owing to their high biomass content and lipid yield per hectare compared to other biomass sources [4].

Algae, as living organisms capable of photosynthesis, are a promising source of biofuel. Their carbohydrates are suitable for bioethanol production [5]. New directions in biofuel production are linked to sustainable needs [6]. In producing bioethanol from non-algal biomass, hemicellulose extraction with mild organic acids rather than sulfuric acid is a more environmentally friendly alternative [7]. Target future industrial applications and converge in attractive action schemes and mechanisms focused on algae, given their growth performance [8], and their potential for renewable energy in transportation [9]. According to Sarri and co-workers, seawater supplemented with commercial nitrate, phosphate, and micronutrient fertilizers is commonly used to cultivate marine microalgae [10]. Sindhu et al. defined third-generation biofuels (3G) as including bioethanol derived from microalgae and seaweed [11].

Converting solar energy into chemical energy is an efficient and sustainable process, as microalgae can grow on non-agricultural land and require relatively unsophisticated technological infrastructure [12]. Therefore, microalgae support food security, promote energy security, and cause only limited environmental issues [13]. 3G biofuels are produced by consortia of organisms, such as microalgae, macroalgae, and cyanobacteria (also known as blue-green algae) [14]. Cheng et al. considered that algae, in general, and green unicellular microalgae, in particular, offer an excellent opportunity for biofuel production, with high productivity of 3G biofuels [15]. Li et al. established that the high lipid content and fast growth of mutant microalgae added value to their utilization [16]. Mixotrophic culture of microalgae represents a promising approach to enhance biomass productivity and metabolite synthesis. Mixotrophic cultivation can be coupled with phycoremediation, a green technology for wastewater treatment that uses microalgae to remove or transform pollutants [17]. Therefore, microalgae-based wastewater treatment is a promising technology, owing to its efficient nutrient removal, biomass valorization, and carbon sequestration. Operational costs and the inefficiency of biomass harvesting restrict the large-scale implementation of this alternative. Recent advances in techno-economic assessments and bioprocess optimization have improved system efficiency, cost-effectiveness, and bioproduct recovery [18].

When cultivation conditions for microalgae become adverse, their cells undergo rapid metabolic rearrangements that enhance the accumulation of neutral lipids. Nonetheless, this rearrangement affects growth, thereby reducing the overall yield. To overcome this issue, one can utilize genetic engineering, modify algal nutrition, or engage in a two-phase cultivation approach [19].

Recently, a study evaluated the entire value chain of microalgal biorefineries to identify the key technological, economic, and environmental factors that enable or hinder scale-up from pilot-scale demonstrations to commercially viable circular economy applications [20].

Herbicides may affect photosynthetic organisms such as microalgae, and pesticides targeting other pests can also impact these communities. Such unwanted side effects are directly related to biodiversity loss and to human health, as well. Taking a systematic approach, one can exceed current yields from oilseed crops while requiring less water and without using herbicides or pesticides [21]. They reduce the emission of greenhouse gases, as 1.00 kg of dry algae biomass can sequester approximately 1.8–2.0 kg of carbon dioxide [22].

This systematic review offers a novel contribution compared with previous studies on third-generation bioethanol by articulating, within a coherent analytical framework, the entire value chain from micro- and macroalgal biomass to industrial infrastructure and the specific conversion technologies for 3G bioethanol. By focusing on the period 2019–2025 and applying a systematic literature review (SLR) methodology based on PRISMA, complemented by a modified PICO scheme, the review integrates previously scattered results in terms of productivity, energy efficiency, greenhouse gas (GHG) emissions mitigation, and scaling potential, thereby delineating concrete gaps between the experimental domain and industrial implementation.

The primary objective of this review paper is to analyze and describe the manufacturing processes for 3G biofuels, specifically bioethanol produced through biotechnologies, utilizing sustainable management practices. Two categories are emphasized: (i) the infrastructure and industrial technology, and (ii) the processes of obtaining 3G biofuels. The research reported here examines the state of knowledge reached in producing third-generation biofuels from microalgae and macroalgae. One discusses in detail the gaps found in the chosen categories.

The current study is divided into seven sections, each addressing a particular topic, as listed below. The introduction (Section 1) presents the global use of biofuels derived from biomass, emphasizing the need for sustainable environmental protection and highlighting the importance of selecting efficient alternative production methods that use microalgae as raw material. Section 2 presents a systematic review of the literature aligned with the selected categories. Section 3 outlines the methodological approach employed to gain insight into this topic. Section 4 focuses on infrastructure and industrial technology, emphasizing advancements in producing algae feedstock for biofuel production. Section 5 discusses the processes for manufacturing third-generation biofuels and the selection of the most efficient technologies. Section 6 discusses the global trends in producing biofuels from algae. Section 7 addresses the price comparison of third-generation bioethanol, and ends with the conclusions reported in Section 8.

2. Scope of the Literature Review

In this section, we discuss the potential integration of biorefineries that convert algae into other industries. Additionally, we scrutinize the processes involved in 3G biofuel production and their sustainability.

2.1. Industrial Systems and Sustainability

Biofuels, derived from renewable, biodegradable feedstocks such as agricultural residues and algae, offer a sustainable alternative to fossil fuels. To benefit from biofuels, countries must address deficiencies in industrial infrastructure encountered during their production. Algae are attractive feedstocks for biofuel production, as they possess high photosynthetic efficiency, rapid growth rates, and the ability to thrive on non-arable land and in wastewater. Biorefineries enable the conversion of biomass into biofuels and value-added products while reducing environmental impacts. Several challenges remain, such as land use, water consumption, and resource-intensive production [23].

Most countries have adopted strategies that focus on the costs of achieving sustainability in using microalgae as a feedstock for biofuels and other products. Current cost barriers to large-scale cultivation are harvesting and biomass concentration [24]. Therefore, they require relevant legislative regulations. Using three modified microeconomic models, Janda et al. investigated the impact of ethanol blending mandates on retail fuel prices in the United States. Their findings eliminate the hypothesis that increasing ethanol blend rates would yield higher fuel prices [25]. The portfolios of biofuel and biorefinery companies reveal that several prominent petroleum corporations invested in biofuel research. They manufactured both conventional and innovative biofuels. The latter includes bioethanol and alcohol-to-jet fuels. In many instances, petroleum refineries have been converted to biorefineries [26].

According to Guedes et al. [27] and Hull et al. [28] a challenge in implementing the technology is that biorefineries that convert algae can be effective only through integration with other industries, such as hydrothermal treatment. The latter should also be evaluated for its potential to transform algae and algae waste into liquid fuels.

Algae cultivation facilities require complex industrial settings to provide the climate algae require [29]. Microalgae can be cultivated with good productivity in highly controlled laboratory environments. The large-scale cultivation remains challenging, however. The latter can be conducted in photobioreactors, providing a closed, highly regulated environment for algae, which enhances nutritional and metabolic efficiency, resulting in higher biomass output per unit of substrate. Design criteria for photobioreactors must consider the reactor’s shape and structure, as well as concentration profiles and operating mode [30]. The proper choice of cultivation conditions enables greater growth rates [31]. For this, resources are required, including suitable land and water for crops. A recent study identified suitable sites in Karachi for microalgae cultivation using geographic information systems. Four categories were established: highly suitable, moderately suitable, less suitable, and unfavorable. The uncovered viable locations for algae cultivation may attract domestic and international interest, allowing for future developments in the field [32]. Crucial for the proper location of the cultivation site are the available infrastructure, transportation, and services [33]. All the above cause economic and sustainability drawbacks; however, producing bioethanol from macroalgae remains a technologically feasible alternative, owing to the low cost of macroalgae cultivation. Algae contain significant amounts of carbohydrates that can be efficiently converted into fuels, whereas their lignin levels remain low [34,35].

Bioprospecting can identify algae species with high lipid content that also show significant growth rates and considerable growth densities, yielding valuable co-products [36]. Therefore, they represent a low-cost means of commercial products. In real-world environments, there is a growing demand for genetic engineering and reproductive technologies, driven by the economic viability of these approaches. The industrial biotechnology sector has been slow to adopt genetically modified microalgae due to regulatory and scalability challenges. Over the past few years, significant progress has been made in micro-algal genetic engineering and strain improvement, particularly in biomass production, biomolecule production, and process optimization. Nonetheless, upon using genetically engineered microalgae in industrial and commercial applications, attention should be paid to regulatory and biosafety issues, as well as risk, hazard, and exposure assessments [37]. Bochenski et al. suggested implementing microalgae biorefineries in arid areas, where certain strains can grow at temperatures exceeding 28 °C and salinity levels above 30 ppt [38].

Any novel technology presents challenges, one of which is long-term sustainability [39]. Building appropriate industrial infrastructure and selecting suitable technologies are typical tasks in the production of algae-based biofuels. Scientists focus on biotechnological properties because the lipid yield of microalgae varies depending on the species and culture conditions.

Biorefineries should meet financial requirements by developing high-value products in addition to biofuels. This requires investigating innovative concepts and approaches [40]. A novel trend is the development of smart biorefineries that utilize artificial intelligence in algae production. It is expected that AI will enhance process efficiency and economic performance within a circular, sustainable framework [41].

2.2. Processes Involved in Obtaining Third-Generation Biofuels

According to the Royal Academy of Engineering, expanding commercially available processes could enable the long-term global production of third-generation biofuels by 2030 [42]. Although biomass represents a sustainable alternative to conventional biomass for biofuel production, Nashath and coworkers warn that microalgal biofuels are more costly than traditional biofuels due to their energy-intensive processing and demanding cultivation conditions. Nevertheless, strain selection is needed to identify appropriate species with suitable biochemical compositions [43]. Biomass can provide energy by biochemical conversion, chemical reactions, direct combustion, or thermochemical processes. Hybrid bioenergy systems can reduce overall costs by 15–37% and lower greenhouse gas emissions by 12–30%. Thermochemical technologies are highly efficient, biochemical methods are eco-friendly, while physicochemical methods are attractive owing to their simplicity. Each technique must address high capital costs, feedstock variability, and scalability issues [44]. Biochemical conversion processes, involving anaerobic digestion and fermentation, reached conversion efficiencies of 82–88%. Process optimization using artificial intelligence and machine learning could predict micro-algal growth characteristics, required nutrients, and the effects of environmental inputs. As a result, production costs decreased by 18%. Additionally, a 23% reduction in the carbon footprint was achieved. Overall, the environmental advantages were notable, resulting in a 78% reduction in net greenhouse gas emissions compared to fossil fuels [45]. Chlorella, Spirulina, Botryococcus, and Nannochloropsis demonstrated outstanding biomass production efficiency. They were also effective in bioenergy generation, carbon sequestration, nutrient recycling, and in algal-based wastewater remediation and bioethanol production [46].

3. Methodology—The Methodological Framework

The global importance of producing biofuels sustainably underscores the need to study third-generation biofuels. Therefore, the authors of this paper conducted a systematic literature review (SLR) following the guidelines for data systematization and presentation recommended by Snyder [47]. The results obtained by this methodological approach were evaluated by addressing a research question [48]. The aim was to determine the current state of knowledge regarding third-generation bioethanol derived from microalgae. Two categories were created in the review’s focus: (i) Infrastructure and industrial technology, and (ii) Processes for obtaining third-generation biofuels. The methodological design aligns with the phases of the 3G biofuel manufacturing process, specifically for bioethanol production. The database investigation included papers published in journals indexed in Scopus and Web of Science from 2019 to 2025. In this review, only a few authoritative policy documents dated before 2019 are cited.

3.1. Methodological Design and PRISMA Protocol

This study adopts a Systematic Literature Review (SLR) design, supported by a narrative synthesis to compare reported yields, productivities, and energy efficiencies. This paper conducted a systematic literature review (SLR) following the guidelines for data systematization and presentation recommended by Snyder [47]. The systematic review is organized into sequential phases, following the PRISMA 2020 (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines [49] for analytical and integrative documentary studies, along with the PRISMA checklist in Supplementary Materials. The main objective of this article is to analyze and describe the manufacturing processes for third-generation (3G), specifically bioethanol produced through biotechnological processes, within a sustainable management framework. The review aligns with two principal thematic axes: (i) industrial infrastructure and technology, and (ii) production processes. It also quantitatively extends the evidence from 2019 to 2025, while addressing identified gaps in scalability, efficiency, and sustainability.

Eligibility Criteria: The search strategies were structured according to the modified PICO framework (Population–Intervention–Comparator–Outcome–Study Type–Time), as shown in

Table 1. Search strategies in agreement with the modified PICO framework.

Element	Description
P (Population/Object of Study)	Industrial or experimental processes to produce third-generation bioethanol from micro-algal or macroalgal biomass.
I (Intervention)	Application of hydrolysis, fermentation, or biochemical conversion technologies for bioethanol production.
C (Comparator)	Alternative methods or variations within the same process (enzymatic vs. chemical, microalgae vs. macroalgae).
O (Outcome)	Bioethanol yield (g∙L−1, MJ∙kg−1), energy efficiency, GHG reduction, economic feasibility, and industrial scalability.
Included Study Designs	Experimental trials, laboratory studies, and simulation studies.
Languages	English and Spanish
Period	2019–2025

.

Information Sources: An exhaustive search was conducted across international academic and scientific databases, including Scopus, Web of Science, Springer, and Google Scholar, covering the period from 2019 to the present.

Search Strategy: A complete search strategy was reported for each database, including filters and limitations (2019–2025, title/abstract/keywords).

Table 2. The Boolean operators are used for searching the databases.

Search String
(“third-generation” OR “3G”) AND (bioethanol OR “bio-ethanol”) AND (alga* OR microalga* OR macroalga* OR seaweed*) AND (pretreat* OR pre-treat* OR hydrol* OR saccharif* OR ferment* OR “enzymatic hydrolysis” OR “acid hydrolysis” OR “simultaneous saccharification and fermentation” OR “hydrothermal” OR “hydrotropic”) AND (“yield OR productivity” OR “life cycle” OR scale*).

An * represents that the records were identified from the database.

summarizes the Boolean operators used.

Exclusion Criteria: Studies focused on non-ethanol biofuels (e.g., biodiesel) were excluded unless they included comparative or integrative data on bioethanol/ethanol. First- or second-generation (1G/2G) studies without 3G comparison, patents lacking verifiable data, and proceedings without full text were also excluded.

3.2. Reporting Guidelines

This systematic review was conducted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines [49], and the detailed PRISMA checklist (S1) was included in the Supplementary Materials. In addition, the PRISMA 2020 flow diagram for new systematic reviews, which included searches of databases and registers only, was used to document the study selection process, and a summary of each phase (identification, screening, eligibility, and inclusion) is presented below.

Phase 1: Identification

In this phase, full texts of potentially relevant articles were evaluated. Exclusion criteria were based on papers outside the main topic, such as non-ethanol biofuels (e.g., biodiesel) unless ethanol was included; theoretical works lacking techno-economic data; 1G/2G studies without comparison toward 3G; unverifiable patents; and incomplete conference papers.

Of the 850 initial records, 270 were removed (210 duplicates, 40 due to document type, and 20 for incomplete metadata), leaving 580 records for title and abstract screening.

Phase 2: Screening

Duplicate and independent screening was carried out at the title and abstract levels. Exclusion reasons were recorded for all full-text evaluations. Duplicates, documents outside eligible languages, or non-eligible document types (patents, abstracts without full text, editorials, errata) were eliminated. Three independent reviewers evaluated titles and abstracts, resolving discrepancies by consensus.

A total of 465 studies were excluded for not meeting inclusion criteria: 35 were duplicates of previously published data; 10 articles could not be retrieved due to lack of access; 47 did not correspond to 3G bioethanol or algal biomass; 112 focused exclusively on biodiesel or other fuels; 95 did not report experimental results or performance data; and 157 were outside the established time range.

Phase 3: Inclusion

The process concluded with 124 studies included for evidence analysis and synthesis (see Figure 1).

Data Extraction and Synthesis Phase: A standardized data extraction matrix was applied, recording main variables such as the type of biomass (microalgae/macroalgae), pretreatment method, type of hydrolysis (acidic, enzymatic, or combined), fermenting microorganism strains, yield (g∙L⁻¹ or % conversion), and country and year of study.

Data Synthesis: Given the heterogeneous nature of the experimental methods, a narrative synthesis was performed. Discrepancies among reviewers were resolved by consensus.

4. Infrastructure and Industrial Technology

The notable results for each category are discussed in detail in the following sections, which are impacted by physical parameters (light and temperature) and chemical factors (CO₂ concentration and possible nitrogen and phosphorus deficiencies).

4.1. Advancement in Algae Feedstock for Biofuel Production

The most significant domains of the chosen categories are infrastructure and industrial technology. Both reveal the considerable importance of costs, which require integrating biofuel production with other industries to provide complex infrastructure and economic viability for long-term use.

Biomass production is a complex technology that encompasses specific crops. Equally important is the adoption of sustainable production practices and doable land management strategies, precision agriculture, genetic engineering, and advanced processing technologies [50]. The need to integrate industrial infrastructure impacts the future of biorefineries and is also associated with more inclusive business models to attract investors.

This section discusses the technologies and advancements in 3G biofuels and bioethanol derived from microalgae and macroalgae. Algae (micro and macro) can be easily cultured on a large scale to harvest feedstocks suitable for biofuel and biogas production (Figure 2). Bioethanol is obtained by the alcoholic fermentation of algal biomass containing carbohydrates, starch, or cellulose [51].

Different parameters significantly affect the enrichment of lipids and carbohydrates in microalgae. Cultivation conditions include (i) physical parameters: temperature, light intensity, and pH, (ii) nutrient stress: macronutrient or micronutrient changes, and (iii) genetic modifications [52]. The photosynthetic pathway is a biochemical process that uses light energy to produce organic molecules (glucose, starch, lipids, and proteins). These compounds can serve as raw materials to furnish chemical energy that enables cell survival and to generate building blocks for cell division [53]. Cells synthesize these molecules with glycerate-3P [54], produced by the Calvin cycle that proceeds in chloroplasts, as depicted in Figure 3.

4.2. Harnessing Microalgae for Bioethanol Production

Microalgae are microscopic, photosynthetic organisms that are naturally found in freshwater and marine habitats. They can grow in various water bodies containing sufficient nitrogen and phosphorus, including freshwater, industrial wastewater, seawater, agricultural runoff, and municipal wastewater. This is a consequence of the adaptation capabilities of most microalgae, which can change the cultivation mode from photoautotrophic to heterotrophic, photoheterotrophic, or mixotrophic [55]. The use of wastewater for microalgae cultivation reduces biofuel production costs and, simultaneously, lowers nutrient concentrations to prevent eutrophication. Nevertheless, no wastewater can be as favorable as a synthetic commercial culture medium. Therefore, wastewater is analyzed for its physicochemical properties before use as a culture medium to maximize biomass yield. Meanwhile, wastewater contains a load of bacteria and other microorganisms that can potentially reduce the growth rates of microalgae. This unwanted phenomenon can be mitigated by utilizing screening technologies to identify suitable microalgal strains. Screening and selecting robust strains capable of thriving in unsterilized wastewater is a broad field of research aimed at reducing the cost of biofuel production.

High biomass yield is not the sole factor in enhancing biofuel production. Storing carbohydrates (starch and cellulose) is favorable for bioethanol production, see Figure 3. Several studies have aimed to increase the carbohydrate content in microalgal biomass. This review compiles the various approaches used to increase carbohydrate levels, regardless of their original purpose. In the future, this systematic review will help researchers select suitable strains and optimization strategies for producing carbohydrate-rich biomass as feedstock for bioethanol production. Carbohydrate (particularly starch) accumulation in microalgae is strongly influenced by environmental and cultivation parameters that trigger metabolic shifts away from protein synthesis and toward carbon storage [56].

Light is essential for microalgae growth since it enables photosynthesis. A high intensity of light favours the production of excess carbohydrates, and stored as an energy storage metabolite within cells [56]. Different microalgae strains can tolerate and utilise various light intensities to accumulate maximum carbohydrate content. This light intensity is known as the saturation flux of a particular strain. When light intensity exceeds the saturation flux, photoinhibition occurs in microalgal cells, inhibiting photosystem function and eventually reducing biomass production. Esteves and coworkers investigated the effect of significant light intensities on the growth of Chlorella vulgaris [57]. The high light intensity resulted in a better growth rate of 0.54 ± 0.02 day⁻¹ and the maximum productivity of 166 ± 13 mg∙L⁻¹∙day⁻¹, which triggered the accumulation of carbohydrates (30 ± 3% w/w) [57]. It was concluded that high irradiance during nutrient stress enhances carbohydrate accumulation. High light provides excess photosynthetic electrons, pushing carbon flux into starch pathways when nutrients for protein synthesis are limited. At the intensity of 600 µmol/m²/s combined with nitrogen starvation, the carbohydrate content of Scenedesmus obliquus increased to 51.8%, with nearly 80% of these carbohydrates being glucose [58]. This high glucose proportion makes S. obliquus a promising candidate for bioethanol production, with a reported bioethanol yield of 0.202 g/g biomass. Nutrient deprivation is one of the most effective triggers for carbohydrate accumulation. Under nitrate or phosphate starvation, carbon flux is redirected from protein synthesis toward starch formation. Calvin–Benson cycle intermediates (fructose-6-phosphate, glucose-6-phosphate) accumulate and are channelled into starch. Stress upregulates ADP-glucose pyrophosphorylase, the key enzyme converting glucose-1-phosphate into ADP-glucose for starch biosynthesis [59]. Parachlorella kessleri showed 50% higher carbohydrate under nitrogen stress [60], while Chlorella zofingiensis accumulates 66.9% carbohydrate with 66.7% just starch in nitrogen-limited conditions [61]. Şirin and Serdar reported experiments conducted under nitrogen-stress conditions, in which the carbohydrate content increased to 59% in Dunaliella tertiolecta [62]. The nitrogen starvation significantly increased the oligo- and polysaccharide content; in Dunaliella tertiolecta, it was determined to be 4.1 times higher for oligosaccharides and 3.6-fold higher for polysaccharides [62]. Markou et al. obtained 59.6% carbohydrate from Arthrospira platensis by reducing phosphorus levels by 98% in Zarrouk culture media [63]. Additionally, a study reported that phosphorus-depleted conditions combined with 2.0 g/L sodium acetate (CH₃COONa) as an organic carbon source resulted in 55% polysaccharide accumulation in Chlorella vulgaris [64]. Organic carbon supplies are also viable, including sodium acetate, glucose, and fructose. Adequate carbon availability reinforces carbon storage during stress. CO₂ enrichment increases Calvin cycle activity, generating more sugar phosphates for starch synthesis. Organic carbon (e.g., sodium acetate) further stimulates polysaccharide formation [64]. The carbon source for microalgal systems can be inorganic, such as CO₂ or NaHCO₃, with bicarbonate-based carbon sources proving more effective for microalgal cultivation [65]. The Net Energy Ratio (NER) of the bicarbonate-based cultivation system is 7.29, which is considerably higher than that of the CO₂-based bubbling approach, which has an NER of 0.85. [65]. Parachlorella kessleri showed maximal carbohydrate production under 5% CO₂ and a long photoperiod of 20 h ([66] Beigbeder and Lavoie, 2022), while Chlorella sp. AE10 exhibited a 42% increase in carbohydrate productivity under 10% CO₂ [67]. In the presence of D-xylose, L-arabinose, and KNO₃, Chlorella minutissima grown in tubular photobioreactors reached a carbohydrate accumulation of up to 66.4% [68]. Table 3 summarises various microalgal strains commonly optimized for carbohydrate and starch accumulation under the most widely applied stress conditions: nutrient limitation and light stress.

Salinity induces osmotic imbalance, leading to the synthesis of osmoprotective sugars. Microalgae produce soluble carbohydrates (glucose, sucrose, trehalose) to balance osmotic pressure. Salt stress also generates ROS (reactive oxygen species), which activates stress-response pathways promoting carbohydrate accumulation [69]. Wen et al. reported that Dunaliella spp. accumulated 50–60% carbohydrates under salinity stress [70]. Likewise, Scenedesmus obliquus increased carbohydrates by 30% under 50 mM NaCl [71], and Acutodesmus dimorphus reached 60.54% carbohydrates at 200 mM NaCl [72].

Rather than optimising operating parameters, some studies changed the mode of cultivation to enhance the accumulation of carbohydrates. Cultivation strategy significantly affects carbohydrate accumulation. Semi-continuous mode maintains cultures in an optimal growth zone with periodic nutrient limitation. Repeated cycles lead to the formation of polyploid cells with enlarged chloroplasts and enhanced starch storage [73]. In semi-continuous systems, cells maintain their photosynthetic capacity and avoid nutrient deficiency [73]. A mixed culture dominated by filamentous cyanobacteria, Geitlerinema sp. accumulated 57% carbohydrate when cultured in a semi-continuous stirred tank reactor using industrial wastewater [74]. However, the semi-continuous airlift bioreactor further increases the content of carbohydrate by 16% [74].

Based on the above research, it is evident that excess carbon and limited nitrogen in the medium promote carbohydrate accumulation in microalgae as a storage metabolite [75]. From an economic perspective, cultivating microalgae in industrial wastewater is more advantageous because it reduces media and water costs while simultaneously removing nutrients and treating wastewater, offering a more environmentally friendly alternative to chemical nutrient-removal processes.

In the field of applied microbiology, two-stage cultivation is a highly efficient method for targeted carbohydrate accumulation in microalgae. In general, stage 1 is used for biomass build-up under nutrient-replete conditions, while stage 2 is for carbohydrate induction via nutrient stress (typically N limitation). Chlorella sp. AE10 accumulated 77.6% [76], while Chlorella zofingiensis accumulated 66.90% of carbohydrate in the two-stage cultivation process [61]. Although these studies were not conducted using wastewater as the culture medium, it is expected that these Chlorella strains could also be used for wastewater treatment while accumulating carbohydrates for bioethanol production, as species within the Chlorella genus are generally robust and well-suited for growth in wastewater [77].

de Carvalho Silvello (2022) reported that, beyond cultivation strategies, genetic modification offers a promising avenue for increasing carbohydrate in microalgal biomass as a green innovative source of bioethanol, particularly through the AGPase-mediated starch synthesis route [78].

Among all stress factors, temperature is one of the most sensitive and critical parameters. It regulates the enzymatic activity of metabolic pathways, thereby directly influencing carbohydrate formation. Microalgae generally grow well between 18 °C and 35 °C, depending on the species [79]. Parachlorella kessleri produced a maximum carbohydrate content of 43% at 30 °C [80], while Tribonema sp. achieved the same maximum level (43%) at 25 °C [81]. Therefore, to reduce overall cultivation costs, it is practical to adopt seasonal cultivation strategies that align with optimal temperature conditions for carbohydrate accumulation.

Table 3. Studies on the effect of stress factors on carbohydrate accumulation in microalgae.

Microalgae Strain	Stress Factor	Effect on Carbohydrate/Starch Accumulation	Reference
Chlorella protothecoides Kruger (CCAP 211/8D)	Nutrient-limited conditions (both nitrogen and iron are limited)	Carbohydrate content 41% by dry mass	[81]
Chlorella vulgaris Beijerinck (CCAP 211/11B)	Nutrient-limited conditions (both nitrogen and iron are limited)	Carbohydrate content 55 wt.% by dry mass	[81]
Chlorella vulgaris (P12 41)	Nutrient-limited conditions (both nitrogen and iron are limited)	Starch content 41 wt.% by dry mass	[14]
Spirodela polyrhiza (ZH0196)	Nutrient-limited conditions	Starch content increased by 39.8 wt.% in 2 days	[82]
Spirodela polyrhiza	Nutrient starvation and abscisic acid (synergistic effect)	Starch content increased to 38.3 ± 1.9 wt.% (dry weight)	[83]
Spirulina	Presence of nitrates and phosphates in wastewater	Carbohydrate content up to 48.4 ± 2.9 wt.%	[84]
Scenedesmus obliquus	Nitrogen-limited conditions	Carbohydrate content 62.5 wt.% by dry mass	[85]
Chlorococcum humicola	Nutrient-limited conditions (both phosphorus and sulphur are limited)	Starch content 60 wt.% by dry mass	[86]
Chlorella vulgaris	Light stress, 140 μmol photons·m−2·s−1 with 7.5 g·L−1 NaCl/7.5 g·L−1 CaCl2	Carbohydrate content 52.71 wt.% by dry mass	[87]
Chlamydomonas sp.	Light stress, 750 μmol photons∙m−2∙s−1	Carbohydrate content 63 wt.% by dry mass	[81]

Table 3. Studies on the effect of stress factors on carbohydrate accumulation in microalgae.

Microalgae Strain	Stress Factor	Effect on Carbohydrate/Starch Accumulation	Reference
Chlorella protothecoides Kruger (CCAP 211/8D)	Nutrient-limited conditions (both nitrogen and iron are limited)	Carbohydrate content 41% by dry mass	[81]
Chlorella vulgaris Beijerinck (CCAP 211/11B)	Nutrient-limited conditions (both nitrogen and iron are limited)	Carbohydrate content 55 wt.% by dry mass	[81]
Chlorella vulgaris (P12 41)	Nutrient-limited conditions (both nitrogen and iron are limited)	Starch content 41 wt.% by dry mass	[14]
Spirodela polyrhiza (ZH0196)	Nutrient-limited conditions	Starch content increased by 39.8 wt.% in 2 days	[82]
Spirodela polyrhiza	Nutrient starvation and abscisic acid (synergistic effect)	Starch content increased to 38.3 ± 1.9 wt.% (dry weight)	[83]
Spirulina	Presence of nitrates and phosphates in wastewater	Carbohydrate content up to 48.4 ± 2.9 wt.%	[84]
Scenedesmus obliquus	Nitrogen-limited conditions	Carbohydrate content 62.5 wt.% by dry mass	[85]
Chlorococcum humicola	Nutrient-limited conditions (both phosphorus and sulphur are limited)	Starch content 60 wt.% by dry mass	[86]
Chlorella vulgaris	Light stress, 140 μmol photons·m−2·s−1 with 7.5 g·L−1 NaCl/7.5 g·L−1 CaCl2	Carbohydrate content 52.71 wt.% by dry mass	[87]
Chlamydomonas sp.	Light stress, 750 μmol photons∙m−2∙s−1	Carbohydrate content 63 wt.% by dry mass	[81]

4.3. Harnessing Macroalgae for Bioethanol Production

Like microalgae, macroalgae can grow in water of various purities, including wastewater [88]. Macroalgae are rich in carbohydrates (from 25 to 50 wt.%), but their lipid content is low (1–5 wt.%); hence, they represent a suitable feedstock for biofuel production, particularly for bioethanol manufacturing [52]. Macroalgae farming is more common in the food industry and agriculture, where they together produce almost 85–90% of the total macroalgae cultivated worldwide [89]. The use and cultivation of macroalgae for biofuel is a relatively new trend compared to the utilization of microalgae [90]; therefore, several aspects of macroalgae cultivation require further research [91].

Macroalgae are cultivated onshore and offshore using various methods. Offshore cultivation is cost-effective owing to lower maintenance and installation costs. Water and nutrients are obtained from seawater, and water flow is sufficient to provide dissolved CO₂. There are various solutions for installing macroalgae, including vertical, horizontal, and mixed-mode cultivation methods [92], which are schematically depicted in Figure 4. In vertical culturing, ropes are suspended vertically from supporting or floating raft ropes, which are connected using plastic or bamboo floaters to keep the entire cultivation frame near the surface. This setting provides optimum sunlight for maximum photosynthesis and biomass production [93]. Vertical culturing ropes are adjustable, allowing the spacing between vertical lines to be adjusted depending on cultivation intensity and the grow-out stage. The end of the vertical rope is attached to a weight, which keeps the rope straight downward and causes it to move in a pendulum-like motion. This type of cultivation is more common in deep-sea areas, where the water is low in turbidity. For Gracilaria vermiculophylla, the test panels were oriented upright, allowing rising air bubbles to trigger fluid motion across the panel surface. During cultivation at saturation light intensity in nutrient-replete medium, a growth rate of 8–9% per day was accomplished [94]. The only drawback of this approach is that the upper plant blocks the sunlight from the lower plant, which eventually decreases its growth rate.

In the horizontal cultivation method, supporting rafts are placed in parallel, and culturing ropes are positioned horizontally at the water surface, with two supporting rafts attached perpendicularly. This method is commonly used in shallow water on the seashore or in areas with intense sunlight. It is also used when water turbidity is high. In horizontal cultivation, the holdfast (root-like structure) receives more sunlight than the vegetative blade during weak water currents. This problem, however, is solved during the current intense period. A possible disadvantage is that long vegetative blades may become entangled, breaking and leading to biomass loss [94].

Mixed-mode cultivation combines vertical and horizontal cultivation. It appears that the horizontal cultivation method, with a weight placed between the cultivation ropes, creates a necklace-shaped structure that prevents entanglement of vegetative blades and does not block sunlight from the lower plant [95].

Macroalgal growth depends on the availability of nutrients present in seawater. Nutrients are more readily available near the seashore than offshore. This means that coastal waters show higher nutrient levels and algal blooms than offshore waters. Significant reductions in nutrient concentrations are required for achieving an appropriate environmental status [90]. According to Froehlich et al., a vast area, up to 48 million km² from the seashore, is suitable for macroalgae cultivation [96]. Though the nitrogen-to-phosphorus ratio represents a limiting factor for macroalgal growth, and this ratio varies nearshore [95], indicating that the nutrient availability in the area is sufficient to support significant levels of macroalgae cultivation [96].

The optimal strength of water currents enables the transfer of nutrients and CO₂, both of which favor macroalgal growth. Therefore, one should determine the water currents before installing any floating cultivation structures [97]. Grazers and epiphytes, more common in nearshore waters than offshore, affect macroalgae biomass production. Marsiglia and coworkers documented both positive and negative effects of several biotic interactions among these habitat formers and their associated symbionts [98]. Sea Urchin is the most common grazer of macroalgae, but it is not present offshore. Light intensity and temperature are factors that also affect the growth. Every macroalgae species has an optimal temperature range for maximum growth; hence, in each zone, only a specific species can be cultivated using offshore techniques. Offshore cultivation of microalgae is also considered for CO₂ removal. Alevizos and Barille calculated the ocean surface, appropriate for macroalgae offshore cultivation and sinking, which sums to 10.8·10⁶ km², while sinking-only areas are 3-fold larger (32.8·10⁶ km²) [99].

Light intensity is directly related to photosynthesis. Light availability depends on water turbidity, which is always lower in offshore waters and higher in nearshore waters. Intensity measurement is an essential factor that needs to be optimized to promote better macroalgae growth. Microalgae particles scatter light, increasing water turbidity and affecting biomass growth. Turbidity and reduced light penetration directly suppress increases in microalgal biomass [100]. Briefly, rather than trying to control cultivation parameters using offshore and nearshore techniques, it is more effective to leverage them. Optimizing cultivation parameters in onshore cultivation can achieve the maximum macroalgal biomass despite using seawater extensively.

Onshore macroalgae cultivation is like microalgal cultivation in photobioreactors and raceway ponds. Macroalgal growth in the onshore technique also depends on nutrients, light intensity, temperature, and CO₂ availability, as they are all required for microalgae cultivation in photobioreactors and raceway ponds, like offshore or nearshore macroalgal cultivation [91]. Macroalgae biomass is farmed using various techniques for biofuel production. Since lipid accumulation in macroalgae is lower than in microalgae, macroalgae are not appropriate for biodiesel production; nonetheless, three macroalgae in Indonesia—Gracillaria sp., Ulva sp., and Sargassum sp.—contained significant amounts of lipids, ranging from 7.25 to 8.40 wt.% [101].

The data demonstrates that macroalgae hold significant potential for biofuel production, with certain strains offering high carbohydrate content, ideal for 3G bioethanol. These findings are crucial for advancing sustainable bioethanol production using marine biomass.

Table 4. Bioethanol yields of different micro- and macroalgal feedstocks.

Feed Stock	Carbohydrate/Sugar Content	Hydrolysis Process	Ethanol Yield	Reference
Macroalgae
Ulva linza	111.91 mg/g dry biomass	3% acid treatment	0.12 g ethanol/g sugar	[102]
Ulva rigida	349.0 mg/g dry biomass	Enzymatic process: Amyloglucosidase and amylase enzymes	0.44 g ethanol/g sugar	[103]
Ulva lactuca	164.7 mg/g dry biomass	Thermal acid treatment followed by cellulase enzyme hydrolysis	0.41 g ethanol/g sugar	[104]
Ulva intestinalis	96.9 mg/g dry biomass	Steam explosion with cellulase enzyme hydrolysis	0.117 g ethanol/g sugar	[105]
Chaetomorpha linum	740 mg/g dry biomass	Wet oxidation method followed by cellulase hydrolysis	0.44 g ethanol/g glucan	[106]
Microalgae
Chlorella marina	198.38 mg/g dry biomass	3% acid treatment	0.232 g ethanol/g sugar	[102]
Arthrospira platensis	165.11 mg/g dry biomass	3% acid treatment	0.455 g ethanol/g sugar	[102]
Chlorella sorokiniana	622 mg/g dry biomass	H2SO4, amyloglucosidase, α-amylase	0.46 g ethanol/g sugar	[107]
Tetraselmis sp.	866 mg/g dry biomass	H2SO4, amyloglucosidase, α-amylase	0.42 g ethanol/g sugar	[107]
Skeletonema sp.	930 mg/g dry biomass	H2SO4, amyloglucosidase, α-amylase	0.43 g ethanol/g sugar	[107]
Mixed culture *	119.2 mg/g dry biomass	Trichoderma reesei and Aspergillus niger are used for enzymatic hydrolysis	0.46 g ethanol/g sugar	[108]
Chlorella sp. ABC−001	400 mg/g dry biomass	H2SO4 hydrolysis	0.43 g ethanol/g sugar	[109]

* The culture mainly contained Chlorella vulgaris, Scenedesmus obliquus, and Neochloris oleabundans.

summarizes various hydrolysis treatments applied to different micro- and macroalgal feedstocks, along with their reported carbohydrate content and bioethanol yields.

From the perspective of ethanol-yield efficiency, careful selection of algal strains is critical for bioethanol production. As shown in Table 4, high total carbohydrate content alone is not sufficient for choosing an optimal strain. Ismail et al. [102] demonstrated this by comparing micro- and macroalgal biomass; although A. platensis contained less total carbohydrate than C. marina, it produced a higher ethanol yield due to its greater proportion of fermentable sugars. Although macroalgae (seaweeds) generally contain complex sugars [106], they occur abundantly on beaches, making their carbohydrate-rich biomass a promising feedstock for bioethanol production.

5. Processes Involved in the Manufacturing of Third-Generation Biofuels

5.1. Technologies in Biofuel Production

The biochemical, physicochemical, thermal, and chemical processing was reported repeatedly in the search for cost-effective solutions. Microalgae represent a promising basis for biofuels. Current efforts are focused on improving cultivation methods, genetic engineering, and selecting suitable strains to enhance the productivity of lipids and carbohydrates, enabling efficient production of 3G bioethanol and thus reducing dependence on fossil fuels [110]. In addition, microalgae can retain CO₂ from combustion gases.

Lee et al. acknowledged that the high production costs of biofuels such as 3G bioethanol are not yet competitive with petroleum-derived fuels and other renewable energies in terms of thermochemical processes (gasification, liquefaction or pyrolysis) or biochemical conversions (anaerobic digestion, alcoholic fermentation or photobiological hydrogen production) [111].

Stages performed in microalgae biorefineries include the selection of suitable species, their cultivation and harvesting to extract lipids. An optimal biorefinery process pathway should integrate economic and environmental parameters with harvest scheduling under fluctuating climate conditions. An optimal biorefinery process pathway should balance the net operation margin and CO₂ emissions. Asterionella formosa achieved a high biomass yield, producing 2300 tons per year, with a short harvest interval of 17.5 days [112].

To produce 3G bioethanol, microalgae undergo metabolic changes from a photoautotrophic phase to a heterotrophic state. Essential is the accumulation of carbohydrates in starch, cellulose, hexoses, and pentoses, which eventually are converted into fermentable sugars to produce 3G bioethanol [113]

Table 4 shows the differences in sugar content among different microalgae and macroalgae. Other algae species are suitable for accumulating carbohydrates, including starch, cellulose, hexoses, and pentoses, which can be converted into fermentable sugars to produce 3G bioethanol.

5.2. Selection of Efficient Technologies for Third-Generation Bioethanol Production

Technological aspects in third-generation bioethanol production are crucial for ensuring efficiency and sustainability. Nonetheless, further development of algae-based biofuel technologies is needed, particularly in cultivation and harvesting.

Maliha and Abu Hijleh reported two algae cultivation methods: photoautotrophic and heterotrophic [114]. Photoautotrophic culture can be divided into open and closed systems. Open systems, such as ponds, lakes, and lagoons, are relatively easy to use and inexpensive because they rely on natural cultivation methods. One can cultivate algae in open ponds by harnessing atmospheric CO₂. The pond’s location is critical, as it determines how much sunlight the algae receive. Sunlight availability, water temperature, and evaporation are among the key factors affecting cultivation in open systems. Challenges associated with open-pond cultivation include unregulated temperatures, inadequate sunlight penetration, evaporation losses, and contamination.

Open pond cultivation can be conducted in any suitable open environment, making it a more economical option than photobioreactors. However, this system produces limited amounts of algae biomass. In contrast, closed systems, also known as photobioreactors, are strictly controlled, high-performance systems that maximize light availability and ensure proper mixing to meet specific biofuel requirements. The efficiency of closed systems stems from greater control over the production environment. These systems primarily use transparent materials, such as plastic or glass, to allow solar radiation to penetrate; they can take various forms, including flat panels, tubes, vertical columns, or bubble columns [115].

Several aspects should be considered when selecting the algae source. Additionally, one identified physical, chemical, and biological processes that facilitate enzymatic hydrolysis and fermentation, which are critical for biomass pretreatment. They can improve prospects in biorefinery applications. Khandelwal et al. highlighted the significance of bio-electrochemical systems, particularly microbial fuel cells, for algae cultivation and bioelectricity production [116,117]. Integrating algae cultivation with microbial fuel cell technology eliminates the need for external oxygen supply and aeration, making the process more sustainable.

Algae-supported microbial fuel cells represent a promising approach to renewable energy production. This technological process can provide a sustainable solution for the growing energy demand. Nevertheless, to optimize system performance, it is essential to understand the key factors affecting algae growth, including effluent composition, light intensity, and reactor configuration. They can improve algae biomass productivity and eliminate chemical oxygen demand in algae-supported microbial fuel cells.

The experience and advancement gained in 3G bioethanol production underscore the importance of economic, technical, and technological effects in this field. As a relevant example, Aparicio et al. demonstrated how Sargassum spp. biomass can be converted at high pressure; one can fractionate the biomass under different temperatures and residence time conditions, resulting in pre-treated solids enriched with glucan [118]. Subsequently, the authors applied high solid load enzymatic hydrolysis (13%, w/v) with an enzyme loading of 10 FPU∙(g glucan)⁻¹, resulting in a glucose yield of 43.01 g∙L⁻¹ and a conversion efficiency of 92.12%. Finally, the authors employed a simultaneous pre-saccharification and fermentation (PSSF) strategy to produce bioethanol. This operational procedure yielded 45.7 g glucose per liter during the pre-saccharification stage and 18.1 g∙(L of bioethanol)⁻¹, with a glucose-to-bioethanol conversion efficiency of 76.2%. These findings demonstrate how high-pressure technology can be effectively utilized to obtain glucan-enriched pre-treated solids, providing a viable strategy for bioethanol production.

During fermentation, microorganisms, such as yeast and bacteria, convert fermentable sugars into ethanol. Similarly, the separation and purification of bioethanol involve distillation, adsorption, and extraction processes along with other separation techniques. These processes also yield other alginate and lactic acid products. Sharma et al. emphasized that scaling up biofuel production to an industrial level is time-consuming due to numerous limitations imposed by currently available technologies and the associated cost increases [117].

Nanotechnology has gained importance in biofuel production, as highlighted by [91]. Owing to their large surface area, nanoparticles play a role in macroalgae biorefinery, enabling the modification of components within the algae biomass. An emerging approach is the immobilization of cellulase utilizing nanoparticles, which could minimize the consumption of hydrolytic enzymes. Additionally, hydrothermal liquefaction is noteworthy, as approximately 80–85% of the moisture content in macroalgae species is rapidly depleted during hydrothermal conversion for fuel generation.

6. Global Trends in the Production of Bioethanol from Algae

Algae bioethanol represents an exciting future industrial alternative that should be addressed within the framework of infrastructure, economic, environmental, and ecological viability to address the overall energy crisis and the search for sustainable fuels. One should prioritize technologies, research, design, and the acquisition of in-depth knowledge of the processes in algae bioethanol refineries. These are associated with microbiology, production routes, biochemistry, and cross-cutting social science research topics.

Manufacturing bioethanol from algae can render it a sustainable and economically viable alternative to fossil fuels. Nonetheless, several challenges remain, such as optimizing the manufacturing process, reducing production costs, and ensuring technology scalability. Bioethanol refineries need to consider factors related to economic investment, technological advancements, and environmental impact [118]. The industrial interest in promoting these technologies is strengthened by their ability to reduce greenhouse gas emissions substantially [119,120].

The future of 3G bioethanol production is promising, as technological advancements expand feedstock options and government policies support progress in this field. To produce microalgae-based 3G bioethanol, industrial managers should focus on developing efficient and sustainable production processes, as well as collaborating with stakeholders to achieve this goal. These challenges must be addressed for microalgae-based biofuels to become commercially viable, namely by reducing production costs, increasing process efficiency, and improving the scalability of production systems.

By overcoming the constraints above, the industrial potential of 3G bioethanol from microalgae becomes promising, and this technology is likely to play a crucial role in future sustainable bioethanol production. Effective management is essential to achieving this goal, as key factors must be optimized across cultivation, harvesting, extraction, conversion, and quality control. Both economic and financial features can aid the success and sustainability of a bioethanol production project.

Industrial management should also consider the high performance of microalgae, as they produce significantly more biomass per unit area than traditional raw materials used to produce feedstock. Additionally, algae have high growth rates, allowing them to double their biomass in just a few hours, thereby reducing land use and competition with food and biofuel crops.

Low capital investments and convenient operating costs represent additional benefits of 3G bioethanol production. Effective management of economic and financial factors will guarantee financially sustainable and environmentally friendly biofuel production; however, effective quality control will also be a fundamental component at each stage of bioethanol production, ensuring that the final product meets customer standards.

Although significant challenges remain, including increasing productivity and further cost reduction, the potential benefits of third-generation bioethanol production will make this an interesting area of research and investment in the future.

7. Price Comparison of Third-Generation Bioethanol from Microalgae vs. Macroalgae (Seaweeds)

Third-generation bioethanol production costs vary significantly between microalgae and macroalgae (seaweeds) due to differences in cultivation requirements, biomass composition, and processing pathways.

Microalgae-based 3G bioethanol is currently more expensive to produce because cultivation and harvesting remain the largest cost drivers [43]. Microalgae require controlled systems (e.g., photobioreactors or managed ponds), continuous nutrient supply, CO₂ delivery, mixing, and regulated environmental conditions. These factors increase capital and operational costs [121]. According to a recent assessment, it is reported that high production costs make microalgal bioethanol not yet competitive with fossil fuels or even other renewable fuels [122]. While microalgae offer high productivity and rapid biomass doubling, their processing, harvesting, dewatering, and conversion add high cost [43].

In contrast, macroalgae or seaweed-based bioethanol generally shows lower production costs, primarily because cultivation costs can be minimal or zero for naturally occurring beach-cast biomass. Macroalgae contain high moisture and complex carbohydrates, but the absence of costly cultivation infrastructure greatly reduces overall expenses [123]. As indicated in your examples (e.g., Sargassum hydrothermal conversion), pretreatment and hydrolysis technologies for macroalgae are increasingly efficient, making seaweed a cost-advantageous feedstock when large amounts of stranded biomass are available.

A recent cost assessment of bioethanol production from Chlorella vulgaris reported that bioethanol derived from defatted biomass costs approximately $0.73 L⁻¹, whereas biodiesel production costs about $2.30 L⁻¹ [124]. Given the lower production costs and simpler processing requirements of macroalgae, it is anticipated that bioethanol derived from seaweed biomass could be even cheaper than bioethanol produced from defatted C. vulgaris biomass.

8. Conclusions

Third-generation bioethanol production from algae faces major constraints, with multiple knowledge gaps limiting its technical and economic viability. Although process optimization has improved yields and reduced some operational costs, significant uncertainties remain in cultivation, harvesting, extraction, and biomass-to-ethanol conversion. Critical gaps include limited strain-specific metabolic models, inadequate real-time monitoring of biomass composition, and the absence of scalable, low-energy pretreatment systems for high-moisture algal biomass. The interaction between stress-induced carbohydrate accumulation and its fermentability also remains poorly understood.

The research reports covered in this study converge on cutting-edge approaches to optimize process parameters, thereby enhancing yields and reducing production costs while also considering energy and manufacturing efficiency. The challenges stem from alternative energy sources, primarily microalgae biomass, which capitalizes on their photosynthetic efficiency. The data extracted from these selected reports encompass the evaluation of algae strains suitable for biomass production and their application in biorefineries.

Effective management prospects are imperative for successfully producing microalgae-based third-generation bioethanol. Sustainable projects entail meticulous management of cultivation, harvesting, and extraction processes. Additionally, an efficient conversion process and rigorous quality control are necessary.

Although several challenges remain, including reducing production costs and improving process efficiency, the future of 3G biofuel production appears promising. Numerous research institutions and companies worldwide are dedicated to developing and scaling up third-generation biofuel production technologies, with increasing support from government policies and incentives. These undertakings are driven by the growing demand for sustainable energy sources and by concerns about climate change and energy security.

Regarding technological advancements, the production of 3G biofuels begins with selecting advanced genetic strains of algae to obtain variants with elevated lipid or carbohydrate content, accelerated growth rates, and enhanced resilience to environmental stress. Novel technologies are under development to convert algal biomass into biofuels, including hydrothermal liquefaction, pyrolysis, and gasification.

It is anticipated that advanced genetic engineering techniques will be deployed to modify bacteria and algae, facilitating the production of larger quantities of lipids or carbohydrates for subsequent conversion into biofuels. Additionally, the utilization of molecular biotechnology and advanced fermentation techniques is envisaged. These advancements aim to increase the efficiency, profitability, and sustainability of 3G biofuels.

Third-generation biofuels derived from microalgae have achieved a comprehensive understanding that encompasses diverse biological, chemical, technological, environmental, and ecological processes. Despite technological advancements and government support, significant gaps persist in terms of efficiency, profitability, and sustainability. Future research can yield further enhancements and breakthroughs in third-generation biofuel production.