Advancement of Abiotic Stresses for Microalgal Lipid Production and Its Bioprospecting into Sustainable Biofuels

Abstract

The world is currently facing global energy crises and escalating environmental pollution, which are caused by the extensive exploitation of conventional energy sources. The limited availability of conventional energy sources has opened the door to the search for alternative energy sources. In this regard, microalgae have emerged as a promising substitute for conventional energy sources due to their high photosynthetic rate, high carbohydrate and lipid content, efficient CO₂ fixation capacity, and ability to thrive in adverse environments. The research and development of microalgal-based biofuel as a clean and sustainable alternative energy source has been ongoing for many years, but it has not yet been widely adopted commercially. However, it is currently gaining greater attention due to the integrated biorefinery concept. This study provides an in-depth review of recent advances in microalgae cultivation techniques and explores methods for increasing lipid production by manipulating environmental factors. Furthermore, our discussions have covered high lipid content microalgal species, harvesting methods, biorefinery concepts, process optimizing software tools, and the accumulation of triglycerides in lipid droplets. The study additionally explores the influence of abiotic stresses on the response of biosynthetic genes involved in lipid synthesis and metabolism. In conclusion, algae-based biofuels offer a viable alternative to traditional fuels for meeting the growing demand for energy.

1. Introduction

The increasing concerns about energy security and climate change caused by traditional energy sources are driving the world to explore renewable and sustainable energy resources. The reduction in non-renewable fossil fuel resources and the escalating impact of global warming have led many countries to prioritize these issues [1,2,3]. To mitigate the future crisis of traditional fuels and their adverse consequences on the environment, the research community is continuously working to explore an alternative to fossil fuels that may be economical, non-toxic, renewable, and eco-friendly [4,5,6]. Among the many solutions to the global energy crisis, the use of algal biofuels as an alternative energy source is a potential strategy to meet our energy needs. Algal biofuels are renewable and viable energy sources that can contribute to the future global energy infrastructure. They offer several benefits, such as reduced dependence on foreign oil and low CO₂ emissions [2,3].

Although the production and utilization costs of fossil fuels severely affect the economies of several countries [7,8,9]. In this regard, the selection of potential and economically viable resource materials or substrates for biofuel production is one of the most crucial aspects. In the last few years, the identification of potential microalgal species has been carried out, which are considered suitable, economical, and effective for biofuel production [3,10].

Initially, first-generation biofuels obtained from edible plant biomass, including barley, beet, corn, potato, sugarcane, vegetable oils, and wheat were used as a replacement for fossil fuels and showed effectiveness in reducing CO₂ emissions in the atmosphere. However, the use of these first-generation biofuel sources raised concerns about potential food shortages and conflicts between food and fuel production. To address this issue, second-generation biofuels were developed using non-edible feedstocks, such as agricultural waste, wood residuals, and bioenergy crops. The carbon emissions associated with second-generation biofuels can be either neutral or negative. However, a drawback of these fuels is their dependence on the seasonal availability of raw materials. Algae have been categorized as third-generation biofuels, presenting a viable and advantageous alternative for biofuel production. They overcome the limitations of the previous generations and are also considered to be 10 times more efficient than second-generation biofuels [11,12]. Algal biomass cultivation can be achieved through three main methods: autotrophic, mixotrophic, and heterotrophic. Algae have a significantly higher growth rate than traditional fodder crops, and they contain approximately 30 times more oil content than conventional feedstocks. The lipid content of microalgae ranges between 20 and 60% of the total biomass, depending on the specific strain and the conditions under which they are cultivated [13,14,15]. The generation of algal biofuel involves the following steps: large-scale cultivation of algae, harvesting and biomass drying, lipid extraction, and the chemical conversion of extracted lipids into biofuel.

Currently, various renewable energy sources are being utilized. Biofuels derived from microalgal biomass are considered to be economically viable and eco-friendly, making them a promising choice for the next generation of biofuels [10]. Recent research conducted by [16], examined the potential of microalgal biomass as a zero-emission fuel, considering its distinctive phytochemical characteristics. The rate and quantity of metabolite formation in microalgae are affected by cultivation systems and growth conditions. Microalgae are grown in unregulated ponds and tanks in an open-air pond system with natural lighting [17]. Unregulated growth conditions in microalgae cultivation provide economic advantages and cost-effectiveness, but they are susceptible to contamination and exhibit variable yields [18]. In contrast, closed culture systems and bioreactor-based cultivation provide a controlled growth environment, ensuring optimal output [19].

Microalgae synthesize a diverse range of chemicals in response to environmental stress, in order to adapt to harsh environmental conditions [20,21,22]. Furthermore, many factors including CO₂ concentration, nitrogen and phosphorus starvation, light intensity, temperature, pH, heavy metals, and salinity could improve microalgal lipid content [23,24]. The physical characteristics of the medium, such as light, temperature, and nutrient supplementation, influence not only the strain’s metabolic machinery but also the composition and yield of microalgal lipids [25]. In contrast, choosing the right microalgal species, optimizing cultivation conditions, and factors affecting microalgal lipid content are the primary requirements for optimum biofuel production. The accumulation of lipids and lipid content can be changed by changing the growth conditions [26]. Therefore, researchers are focusing on abiotic factors to improve microalgal biomass and lipid content. However, there is still a need to develop new methodologies and techniques for the efficient production of biofuels from microalgal resources.

Microalgae store substantial amounts of neutral lipids, mainly triglycerides (TAGs), which are ideal for biodiesel production. Microalgal biorefineries not only have the potential to be a profitable approach for biofuel production, but they also have the ability to produce useful products of commercial value. In addition, microalgae-based biofuels address issues related to rising energy or fuel prices and contribute to CO₂ mitigation [27,28]. Microalgae, such as Chlorella vulgaris, Scenedesmus sp., Monoraphidium sp., Isochrysis galbana, Tetraselmis suecica, Nanochloropsis occulata, Botryococcus braunii, Dunaliella tricolecta, and Neochloris are widely distributed in the environment and used as a source of food, medicine, and biofuels [29]. The production of biofuels using microalgae faces several challenges, including strain selection, mass cultivation, harvesting, drying, extraction, and transfer processes [30,31]. The structure of this review consists of three main sections. Firstly, a comprehensive examination of microalgae species, culture methods, and harvesting systems is presented. Secondly, the focus has shifted to exploring the effect of diverse environmental stressors on the production of various types of biofuels derived from microalgae. Lastly, attention is given to the production of biobased energy products, lipid biosynthetic genes from microalgae, TAGs in lipid droplets (LDs), and software tools for microalgae biorefinery, along with the challenges and future research directions.

2. Microalgal Species and Biorefinery for Sustainable Biofuel Feedstock

Microalgae are photoautotrophs with the capability to survive in freshwater and marine ecosystems. Currently, it is estimated that approximately 200,000 to 800,000 microalgal species are present on the earth, of which only 50,000 have been identified and characterized [32]. The lipid content of microalgae is an important factor in the selection and screening of microalgae for biofuel production [33]. Microalgae consume CO₂ and fix it into carbohydrates, proteins, and lipids under different environmental conditions, playing an essential role in the production of a variety of renewable fuels [34,35].

Previous research has indicated that numerous algal species, including Chlorella vulgaris, Chlorella emersonii, Chlamydomonas reinhardtii, Nannochloropsis salina, Skeletonema sp., and Parachlorella kessleri, have significant lipid content [36,37,38,39,40]. However, some species, such as Chlorella pyrenoidosa, Chlorella zofingiensis, Chaetoceros muelleri, Coelastrella sp., and Chlorococcum pamirum, have relatively modest lipid content [41,42,43,44,45]. When considering the lipid content in various algal groups, a general trend can be observed: green algae > yellow-green algae > red algae > blue-green algae. Similarly, when examining lipid productivity, the trend follows: green algae > red algae [46]. Figure 1 shows different microalgal species with high lipid content that hold promise for potential biofuel production.

Microalgae are a group of microscopic organisms that have the potential for CO₂ sequestration and the ability to thrive in harsh environments [47,48]. However, due to their high photosynthetic nature, CO₂ mitigation capabilities, and excessive biomass generation, microalgae can be exploited for various applications in environmental management and bioenergy [18,20,49,50]. Microalgae are rich in carbohydrates, proteins, fatty acids, and other bioactive compounds, which make them valuable feedstocks for various industrial applications, including cosmetics, nutraceuticals, livestock feed, fertilizers, and biofuels [4,51]. Microalgae constitute both a large qualitative and quantitative market due to the variety of commercial products they produce. Microalgal products currently hold a market share of US$4.7 billion, according to market survey research, and are predicted to expand by 6.3% to US$6.4 billion by 2026 [52]. Once the necessary biomolecules have been isolated, the remaining biomass can be used as a raw material. This raw material can then be used to produce secondary products, such as 2–3 butanediol, bioethanol, and biobutanol [53,54].

To minimize energy investment and mitigate the risks linked to heat and solvents in extraction processes, alternative eco-friendly technologies have been developed. These technologies include the utilization of supercritical fluids, high pressure, microwave and ultrasound techniques, as well as electric fields [17]. The objective of these methods is to achieve a high yield of bioactive compounds. After extraction, the remaining algal biomass still contains carbon-rich biopolymers such as proteins, carbohydrates, and lipids. Microbial biomass is a good substrate for secondary fermentation because it contains sugars and nutrients. Numerous studies have investigated the conversion of microalgal biomass into bioenergy, including bioalcohol, biogas, and biohydrogen [55,56].

3. Microalgae Cultivation Systems

For an efficient industrial process, microalgae cultivation technology needs to be economically viable, environmentally friendly, and have a high yield [27]. Although photoautotrophic and heterotrophic growth conditions are the most common, some algal groups can also grow under mixotrophic conditions [57]. Several techniques for microalgal cultivation have been reported, including open, closed, and advanced systems. The choice of cultivation method for microalgae depends on the type of species, the nutrient source, and the end application of the biomass [21]. Other parameters, such as photobioreactor design, volume mixing, temperature, lighting, and CO₂ supplementation, also play a significant role in flue gas reduction from the environment and nutrient recovery from wastewater [20]. Microalgal cultivation systems can be categorized into four main groups, each with its own advantages and disadvantages (

Table 1. Various modes of microalgae cultivation with their advantages and disadvantages.

Cultivation Mode	Carbon Source	Energy Supply	Light Availability	Advantages	Disadvantages
Autotrophic	Inorganic carbon	Light	Obligatory	Low cost, low energy consumption, high pigments production.	Low growth rate and biomass, specific photobioreactor required.
Heterotrophic	Organic carbon	Organic	No requirement	High biomass productivity and lipid accumulation due to high growth rate, process of scaling up is simplified, organic substrates can be used to alter biomass composition.	Higher cost, easy to be contaminated by other microorganisms, only a few microalgal species that can grow in a heterotrophic environment, inability to synthesize metabolites triggered by light.
Mixotrophic	Inorganic and organic carbon	Light and organic carbon	No obligatory	Increased growth rate, biomass, density and lipid accumulation, extended phase of exponential growth, stopping the photoinhibition effect and reducing biomass loss due to respiration during the dark hours, switch between photoautotroph and heterotroph regimens at any time.	High cost, contamination problems, limited microalgae species will grow.
Algal turf scrubber	Organic and inorganic carbon	Light and organic carbon	Obligatory	Improved nutrient status, pollutant removal, high biomass productivity rate, easy harvesting and low maintaince, decreased the overall production cost	Requirement of sufficient space and infrastructure

).

3.1. Autotrophic Cultivation

Autotrophic cultivation is the preferred method for growing microalgae on an industrial scale because it is the most common and advantageous [58]. Microalgae use sunlight and CO₂ to produce organic molecules during autotrophic growth [59]. Autotrophic cultivation is often scaled up in open-air systems to improve lipid yield by using 2% CO₂ in the air [60,61]. In this situation, switching from a closed to an open system is more promising for reducing costs without sacrificing biomass productivity. Open systems have some limitations, such as the risk of contamination by other microalgae, fungi, bacteria, protozoa, and viruses, and the difficulty of maintaining consistent temperature, light periods, and pH [62,63]. Additionally, evaporation in open systems can lead to water loss, which requires constant monitoring and replenishment. This can lead to increased operational costs and may not be sustainable in water-limited regions. In contrast, closed-system photobioreactors (PBRs) have been developed to avoid the limitations of open-systems [64]. The high surface area to volume ratio of closed systems ensures stability and sustainability [65].

3.2. Heterotrophic Cultivation

Microalgal growth in heterotrophic cultivation depends on the availability and metabolism of organic carbon sources [66]. Under heterotrophic conditions, microalgae can accelerate cell density and growth, as well as decrease the costs of cultivation [67]. In this context, various alternative sources of carbon and nitrogen are used for microalgal growth within the heterotrophic system. A range of organic carbon sources, such as glucose, fructose, galactose, lactose, sucrose, and glycerol, are added as supplements to achieve optimal microalgal growth. However, some alternative waste products, which are economically cheap, have been used for microalgal growth. In a study, hydrolyzed corn steep liquor has been used instead of high-cost glucose as a source of carbon for optimum microalgal production [68]. The use of organic substrates and dark environments can increase the risk of contamination in microalgal cultivation [69]. The use of organic compounds in heterotrophic cultivation can increase the overall costs.

3.3. Mixotrophic Cultivation

Microalgae have the ability to use both external organic molecules and CO₂ as a source of carbon [69]. Under mixotrophic cultivation, microalgal species can grow in either heterotrophic or photoautotrophic conditions, or both, depending on the presence of organic substances and light intensity. Mixotrophic cultivation can produce higher yields of biomass than autotrophic and heterotrophic cultivation, especially when combined with a dark/light cycle [57]. Various reports have shown that algal strains, such as Chlorella sorokiniana, C. vulgaris, Chlamydomonas reinhardtii, Spirulina platensis, and Scenedesmus obliquus favor mixotrophic cultivation [61].

Mixotrophic cultivation has significant advantages, such as improving biomass productivity, increasing lipid content, and the ability to modify biomass composition [70]. However, there are some challenges that need to be resolved in order to achieve optimal production in mixotrophic cultivation. These include issues, such as culture contamination caused by bacterial or fungal growth, the presence of inhibitors such as hazardous metals, and lower photosynthetic efficiency under dark conditions. Microalgal cultivation could be more economically feasible if these factors were improved [71,72].

3.4. Algal Turf Scrubber and Hybride Systems

Algal turf scrubber (ATS) is an advanced cultivation system that uses a downward-sloped surface to grow an algal community [22]. Microalgae in the system grow efficiently by taking up organic and inorganic compounds and releasing dissolved oxygen. This cultivation system offers improved nutrient uptake, high biomass productivity, easy harvesting, and low maintenance, which all contribute to lower overall production costs [18]. However, this system has some drawbacks, such as the need for sufficient space, evaporation in open systems, and infrastructure, which limits its use [21]. A hybrid cultivation system, as the name suggests, is a combination of one or more open and closed systems [73]. This system is an advanced cultivation system with the integration of biomass harvesting for efficient resource recovery. The two-stage system was found to be effective in increasing lipid productivity [74]. The technique results in less contamination and increased productivity, but the system is complex and requires high maintenance and monitoring, which makes it a costly option [32]. In hybrid cultivation systems, each process is maximized to recover resources efficiently.

4. Microalgae Harvesting Techniques

Microalgae harvesting is the process of collecting microalgae from the liquid medium in which they are grown. The choice of microalgae harvesting method depends on a number of factors, including the properties of the microalgae, the desired properties of the end product, and the feasibility of recycling the growth medium. Microalgae cells can be harvested using biological, chemical, mechanical, or electrical methods. Microalgae harvesting is a time-consuming and challenging process that often requires the use of advanced chemical or mechanical techniques. Flocculation, filtration, flotation, sonication, centrifugation, and precipitation are some of the methods that are used to harvest microalgal biomass (

Table 2. A comparison of the principles, benefits, and drawbacks of various algal harvesting methods, adapted from [77].

Technique with Image	Principle	Advantages	Disadvantages
Flocculation	Aggregation of cells is achieved by enlarging their size through the addition of a flocculant, which can be in the form of chemicals (such as ferric chloride, ferric sulfate, and ammonium sulfate) or microbes (bacteria).	Fast and easy technique, used for large scale, less cell damage, applied to a wide variety of species, less energy requirements.	Chemicals may be expensive, high pH required, separating the coagulant from harvested biomass is difficult, limited culture medium recycling, increased microbial contamination.
Filtration	Large cells (size > 70 µm) can be filtered under pressure or suction whereas smaller cells (size < 30 µm require ultrafilters to be harvested.	High recovery efficiency, cost effective, no chemical required, low energy consumption, low shear stress.	Slow hence requires pressure or vacuum, not effective for small algae, membrane fouling/clogging and replacement increases operational and maintenance costs.
Flotation	Trapping algal cells by bubbling air.	Well-suited for large-scale applications, economically efficient with minimal space demands, short operation time.	Depends on bubble distribution into the suspension, needs surfactants.
Centrifugation	Sedimentation based on the velocity, cell size and density.	Fast and effective technique, high recovery efficiency (>90), applicable to all microalgae.	Expensive technique with high energy requirement, high operation and maintenance costs, risk of cell destruction.
Precipitation	Certain algae undergo self-precipitation, they settle at the bottom when circulation is halted.	No energy or chemicals are needed, it occurs naturally.	Species-specific, time periods vary depending on the species, not every species is self-precipitated.

). When choosing a harvesting method, it is important to consider the downstream processing requirements, as microalgal biomass will need to be processed further. Therefore, these processes must be carefully operated to avoid damaging or contaminating the microalgal biomass. It is also ideal if the chosen harvesting method permits the culture medium to be recycled [75]. Often, a combination of two or more harvesting methods is used to achieve greater separation efficiency and minimize costs [76].

5. Approaches to Stimulate Lipid Production through Abiotic Stresses

Abiotic stresses can play a vital role in stimulating the growth of microalgae and enhancing lipid production. The buildup of biomass and lipids in algal cells is influenced by various factors. This section specifically focuses on the effects of abiotic stresses, such as CO₂, nitrogen starvation, temperature variations, pH changes, salinity fluctuations, heavy metals, sulfur starvation, light intensity alterations, and the influence of nutrients. This study explores the impact of environmental stressors on lipid production in microalgae.

5.1. Effect of Carbon Dioxide

Microalgae can mitigate CO₂ emissions by CO₂ biofixing and can grow well in high CO₂ concentrations [23]. The growth rate and lipid content of microalgae can be affected by the concentration of carbon dioxide. Many studies have investigated the effects of CO₂ on the growth and fatty acid composition of microalgal strains. The mechanism involves the uptake of CO₂ by algae and converting the captured carbon into lipids. The carbon from microalgae is then converted into biodiesel through transesterification, as shown in Figure 2.

Increasing the CO₂ levels in the growth medium of microalgae leads to an increase in the production of cellular lipids and fatty acids [78]. A study found that increasing the CO₂ levels from 0.5% to 1% increased lipid production in C. vulgaris, but higher levels of CO₂ significantly decreased lipid content in other microalgae species [79]. Various organic carbon sources, such as glucose, fructose, sucrose, and glycerol can be used to cultivate Chlorella strains [80]. When the cellular growth requirements are met and excess organic carbon is present, it promotes lipid accumulation. In addition, the addition of CO₂ can increase the growth and lipid production of Chlorella sp., making them a promising candidate for industrial applications, especially due to their high polyunsaturated fatty acids (PUFAs) content [8]. The lipid content of C. vulgaris, Botryococcus braunii, and B. terribilis ranges from 2.5 to 20.0% under varying CO₂ concentrations. In C. vulgaris, CO₂ addition enhances the palmitic acid, while for Botryococcus sp., no such type of change occurs [81]. The lipid contents of Dunaliella salina increase nearly 20-fold with 0.01–12.0% CO₂ supplementation. Increasing the CO₂ concentration to 5% in Chlorella vulgaris cultures resulted in an increase in monounsaturated fatty acid (MUFAs) and PUFAs accumulation [82]. The lipid accumulation rate and lipid composition of microalgae can vary depending on the strain and cultivation mode.

Algae-based CO₂ sequestration stands out as a compelling carbon capture method when compared to other existing approaches. CO₂ sequestration, direct air capture (DAC), and carbon capture and storage (CCS) are three distinct approaches for mitigating carbon dioxide emissions, each with its unique characteristics and applications.

Table 3. Differences between CO₂ sequestration, direct air capture (DAC), and carbon capture and storage (CCS).

Parameters	CO2 Sequestration	Direct Air Capture (DAC)	Carbon Capture and Storage (CCS)
Source of CO2 capture	This method involves capturing carbon dioxide from various sources, including industrial processes, power plants, and natural systems like forests and oceans (algae). It encompasses a wide range of approaches, both natural (e.g., photosynthesis in algae) and engineered (e.g., mineralization).	DAC is designed to capture carbon dioxide directly from the ambient air. It targets atmospheric CO2 and is not dependent on specific point sources. DAC is ideal for offsetting emissions from sectors that are challenging to decarbonize, such as transportation.	CCS focuses on capturing CO2 emissions from specific industrial point sources, such as power plants and industrial facilities. It is primarily aimed at mitigating emissions from large, stationary sources.
Capture efficiency	The capture efficiency of CO2 sequestration methods can vary widely depending on the specific approach. For example, biological methods like algae cultivation can be highly efficient, while geological methods like enhanced oil recovery may capture a significant but variable portion of emissions.	DAC technologies are designed to be highly efficient at capturing CO2 from the atmosphere. They are engineered to achieve capture rates close to 100% of the CO2 passing through the system.	CCS technologies typically capture a substantial portion of CO2 emissions from point sources, but capture rates can vary depending on the technology and the type of industrial process.
Scalability	The scalability of CO2 sequestration methods can vary. Some approaches, like algae cultivation, may require extensive culture medium, while others, like mineralization, can be implemented on a smaller scale.	DAC systems can be scaled up or down relatively easily, making them adaptable to different emission scenarios. They can be distributed across diverse locations, depending on the need.	CCS facilities are typically large and centralized, making them less adaptable to different locations and requiring significant infrastructure investment.
Environmental impact	The environmental impact of CO2 sequestration methods can vary widely. Biological approaches may have positive environmental co-benefits, while some geological methods may pose risks related to underground storage.	DAC systems have a relatively low environmental footprint, but the energy requirements for DAC can impact the overall environmental assessment.	CCS can have environmental implications related to underground storage, potential leakage, and the energy required for capture and compression.
Energy requirements	Energy requirements vary significantly depending on the method, with some processes requiring minimal energy and others being more energy-intensive.	Generally energy-intensive due to the need for air circulation and chemical processes.	Energy is required for capturing, compressing, and transporting CO2 to storage sites, making it energy-intensive as well.
Carbon neutrality	May have a carbon-neutral or even carbon-negative impact.	Has the potential to achieve carbon neutrality but requires a sustainable source of energy to power the capture process.	Typically does not achieve carbon neutrality as it focuses on preventing emissions rather than removing carbon from the atmosphere.
Economic feasibility	Economic feasibility varies widely depending on the specific method, scale, and potential revenue streams from sequestered carbon.	Considered more costly compared to some other methods due to its energy-intensive nature but may become more economically viable with advancements in technology.	Economic feasibility depends on factors such as government policies, incentives, and infrastructure costs. It has been applied in various industries.
Technological maturity	Maturity varies by method, with some approaches well-established and others still in development.	Relatively newer technology with ongoing research and development efforts.	More mature technology with decades of operational experience in certain industries, such as enhanced oil recovery.
Primary application	Applied across various sectors, including algal cultivation, and industrial emissions reduction.	Often used to offset emissions from sectors that are challenging to decarbonize, such as transportation.	Primarily used in heavy industry and power generation to reduce emissions from stationary sources.

presents a comprehensive comparative analysis of three distinct carbon emission mitigation strategies.

5.2. Effect of Nitrogen Starvation

Nitrogen is a crucial macronutrient that directly contributes to the synthesis of proteins, carbohydrates and lipids. Microalgae can acquire nitrogen from sources such as nitrates, nitrites, urea, and ammonium [83]. Nitrogen is an essential nutrient that plays a critical role in the growth of microalgae. When algae experience nitrogen limitation, they downregulate the synthesis of many proteins that are involved in various cellular functions. The downregulation of protein synthesis in nitrogen-limited algae conserves energy and allows the algae to allocate more resources to lipid synthesis. For example, a study found that Scenedesmus quadricauda has a 2.27-fold increase in lipid synthesis when exposed to nitrogen stress [84]. Under nitrogen-starved conditions, the carbon flux within the cells is redirected towards lipogenesis instead of proteins and other nitrogen-containing compounds [85]. This metabolic shift increases fatty acids and TAG production, which are the primary components of algal lipids. In another study, it was observed that nitrogen depletion resulted in a 38.14% enhancement in the lipid content of Scenedesmus rubescens cells [86].

Nitrogen starvation triggers the upregulation of genes and enzymes involved in lipid biosynthesis. Crucial enzymes involved in lipid biosynthesis, such as acetyl−CoA carboxylase (ACC), fatty acid synthase (FAS), and diacylglycerol acyltransferase (DGAT), are often overexpressed during nitrogen deprivation [87,88]. These enzymes catalyze the conversion of carbon sources into fatty acids, which are then transformed into TAGs. As lipids accumulate in the algae, they are preserved in specialized organelles termed lipid droplets. The number and size of lipid droplets in algal cells increase during nitrogen starvation, leading to higher lipid content [89]. Different algal strains of the same species may respond differently to nitrogen starvation. The lipid accumulation efficiency of different algal species varies under nitrogen-depleted conditions. However, studies have shown that inducing lipid synthesis through nitrogen starvation can also lead to a decrease in biomass productivity [90,91]. In response to nitrogen depletion, cells degrade nitrogenous compounds to generate energy and carbon sources for the accumulation of neutral lipids as a response to stress conditions [92]. The correlation between nitrogen content in the culture medium and lipid synthesis may be attributed to the fact that lower nitrogen levels lead to a decrease in protein and carotenoid synthesis. The decrease in protein and carotenoid synthesis minimizes competition for carbon sources, which promotes lipid synthesis. The ability of algae to accumulate lipids under nitrogen-depleted conditions has important biotechnological implications, as algal lipids can be used to produce a variety of valuable products, including nutraceuticals, biofuels, and others. However, it is critical to carefully optimize the cultivation conditions and the duration of nitrogen starvation to achieve the desired lipid productivity without compromising the viability of the algae.

5.3. Effect of Light Intensity and Wavelength

Light is one of the most important environmental factors for microalgal growth. Previous studies have shown that high light intensity can significantly increase lipid accumulation in microalgae. The TAG content increased and the polar phospholipid composition decreased as the light intensity was increased in Cladophora sp. [93]. Nannochloropsis sp. cultivated under low light intensity (35 µmol m⁻² s⁻¹) had a lipid composition of 26% TAG and 40% galactolipid. A unicellular alga, Scenedesmus sp., accumulates a significant amount of lipid content when cultivated under light conditions of 250–400 µmol m⁻² s⁻¹ [94]. The total lipid content of Dunaliella salina, Isochrysis galbana, and Nannochloropsis oculata increases when the light intensity is increased to 150 µmol m⁻² s⁻¹ [95]. Microalgae grown under red light showed higher lipid accumulation than those grown under yellow or white light. Microalgae cultivated under green light had lower lipid accumulation than those grown under red light. For example, exposing a unicellular microalga Chlorella sp. to red light resulted in increased growth and lipid content [96]. In Acutodesmus obliquus, red light caused a lower degree of fatty acid desaturation than blue and green light [97,98]. The relative proportions of the 16:4, 18:3, and 18:4 fatty acids were up to 64% lower under a red light regime than under green and blue light. An additional study found that exposing T. obliquus to different frequencies of blue and red light increased lipid accumulation [99]. However, when they were alternately exposed to blue wavelength light, the lipid yield increased by 4-fold [100].

Another technique for improving microalgal growth and lipid accumulation is to use chemical dyes to change the light spectrum [101]. For example, organic dyes such as rhodamine 101 and 9,10-diphenylanthracene have been used to convert unused and/or harmful portions of incident sunlight into usable photons for microalgae cultivation, thereby increasing the productivity of biomass and lipids. The lipid yield of Chlorella vulgaris can be improved by cultivating it in a medium containing these chemical dyes [101]. Red paint produced the highest biomass output, while blue paint produced the maximum lipid content of 30% dry weight [102]. The lipid composition of microalgae is significantly affected by photoperiod. The effect of light intensity on the lipid composition of microalgae has been investigated in a variety of ways, and it has been found that PUFA levels decrease as light intensity increases [103]. Microalgae grown under high light intensity and long light periods had higher saturated fatty acids (SFAs) levels and lower MUFAs and PUFAs levels [104]. The lipid composition of Thalassiosira pseudonana grown under 12:12 h light/dark periods showed higher SFA and MUFA levels and lower PUFA levels [105].

5.4. Effect of Temperature

Temperature changes influence not only lipid synthesis but also lipid content in the microalgae. In most microalgae, a decrease in temperature resulted in an increase in polar lipid content, while an increase in temperature resulted in an accumulation of non-polar lipids [106]. Exposing Acutodesmus dimorphus to temperature stress at 35 °C led to a 22.7% increase in lipid yield and a 59.9% increase in neutral lipid accumulation [107]. A decrease in temperature from 30 to 25 °C in Chlorella vulgaris has been shown to increase lipid content by 2.5-fold, without any change in growth rate [108]. Low temperatures simultaneously stimulate lipid content and reduce growth in Chlorella sorokiniana [109].

Although most studies focus on total lipid content, few have investigated the impact of temperature on specific lipid classes. Microalgae cultivated at low temperatures have a high content of PUFAs, which are important for survival in harsh conditions. When the temperature was decreased from 25 to 10 °C, Isochrysis galbana showed a significant increase in PUFA content [110]. Phaeodactylum tricornutum also showed a similar increase in PUFA content when the temperature was decreased from 25 to 10 °C [111]. Lower temperatures prompt microalgae to produce lipids with a higher concentration of unsaturated fatty acids (UFAs). These UFAs help algal strains maintain membrane fluidity in cold conditions [109].

5.5. Effect of Salinity

In response to salt stress, microalgae typically undergo biochemical changes that regulate lipid production. However, the salinity tolerance of different microalgae strains varies. Dunaliella sp. is an example of a microalga that can withstand high salt concentrations. Dunaliella salina is a well-studied microalga because it can use salt stress to increase both biomass productivity and lipid yield. Dunaliella tertiolecta exhibited an increase in both total lipid content and a notable percentage of TAGs when the amount of NaCl was increased [112]. Scenedesmus sp., when subjected to salinity stress of 400 mM during a biphasic cultivation process, resulted in a lipid content of 34.77% [113]. The lipid content of various microalgae, including Scenedesmus sp., Chlorella vulgaris, and Chlamydomonas mexicana, increases under salt stress conditions [22,113,114]. In most microalgae, the lipid content peaks at a certain salt concentration, and then declines significantly beyond this concentration. In Chlorella minutissima, salt stress affects both biomass production and lipid content [115]. Nitzschia laevis had its best growth and lipid production at a specific salinity level. Under salinity stress, the alterations in lipid content and composition serve to decrease membrane fluidity and permeability [116]. This adaptive response aids microalgae in reproduction and enables them to adapt to the new environment.

5.6. Effect of pH

The pH affects the photosynthetic process, the solubility of inorganic nutrients, the rate of lipid accumulation, and the activity of enzymes in the cell. For microalgal growth, a certain pH range is required, which is limited and strain-specific. Microalgae are able to accumulate higher lipid content within a pH range of 7 to 9.5. Chlorella sp. showed an increase in lipid content up to 23% at pH 8, and Tetraselmis suecica showed a similar trend at pH 7.5. A study of the impact of various pH values (6, 7, 8, 9, and 10) on the biomass and lipid content of Nannochloropsis salina found that N. salina exhibited optimal growth rates and lipid content at pH 8 and 9 [117].

The relationship between CO₂ and pH for microalgal cultivation is intricately linked through a process known as carbon dioxide dissolution or carbonic acid equilibrium. pH is important for microalgal growth because it affects the solubility of CO₂ in the culture medium. As the pH increases, the solubility of CO₂ decreases. This means that there is less CO₂ available for microalgae to use. The optimal pH range for microalgal growth varies depending on the species of microalgae. However, most microalgae grow best in a pH range of 6.5 to 8.5 [27]. At pH levels below 6.5, the solubility of CO₂ is too low, and at pH levels above 8.5, the toxicity of CO₂ increases. When CO₂ dissolves in water, it forms carbonic acid (H₂CO₃). This reaction can be represented as follows:

CO₂ + H₂O ↔ H₂CO₃

The relationship between CO₂ and pH is complex. As microalgae grow, they take up CO₂ from the medium, which causes the pH to increase. This can be counteracted by injecting CO₂ into the culture, or by adding a buffer to the medium. The amount of CO₂ that needs to be added or the amount of buffer that needs to be added depends on the pH of the medium, the species of microalgae, and the growth rate of the culture. As CO₂ dissolves in the culture medium where microalgae are cultivated, it decreases the pH of the medium. This means that as more CO₂ is added to the culture, the pH of the medium becomes more acidic due to the formation of carbonic acid. Conversely, when CO₂ is removed from the medium (for instance, through microalgal photosynthesis), the pH tends to increase, becoming more alkaline. This pH fluctuation is essential to monitor and control in microalgal cultivation systems because microalgae have specific pH preferences for optimal growth. The pH of the culture medium affects the availability of CO₂ as a carbon source for photosynthesis, and it also influences the solubility of other nutrients essential for microalgal growth. Therefore, maintaining an appropriate pH level in microalgal cultivation systems is crucial for ensuring that there is a sufficient and stable supply of CO₂ for photosynthesis, which in turn supports healthy and efficient microalgal growth.

The lipid profile of microalgae can be affected by changes in the culture medium pH. Alkaline pH stress tends to induce the synthesis of saturated lipid components in many microalgae. For example, Chlorella sp. shows a decrease in the levels of glycolipids and polar lipids, but an increase in the accumulation of TAGs. Additionally, high pH values lead to a rise in both saturated and polyunsaturated lipid content, while the content of monounsaturated lipids decreases. Nannochloropsis sp. had its optimal lipid content at a pH of 8. There were no significant changes observed in the synthesis of saturated and mono/polyunsaturated lipids when the pH was either decreased or increased [118]. Pinguiococcus pyrenoidosus has the highest proportion of PUFAs at a pH of 7, accounting for 38.75% of the total fatty acids [119].

5.7. Effect of Metal

The lipid content of microalgae is affected by the presence of trace metals, and the amount of lipids produced depends on the concentration of these metals in the growth medium. Heavy metals, including copper, zinc, and cadmium has been shown to increase lipid yield in some microalgae. Iron stands out as particularly effective in enhancing photosynthetic enzyme activities. For example, the overall lipid content of Chlorella vulgaris increases by approximately 3 to 7 times when it is exposed to FeCl₃ [120]. Similarly, the lipid content of Chlorella sorokiniana is highest when exposed to an iron concentration of 104 mol/L [121]. At this concentration, the lipid content was 2.8-fold higher than that of samples without iron. A copper concentration of 4 mg/L resulted in elevated lipid content in three microalgae species: C. vulgaris (0.2 mg/g), C. protothecoides (0.16 mg/g), and Chlorella pyrenoidosa (0.11 mg/g) [122]. A separate study found that subjecting Chlorella protothecoides to copper stress at a level of 31.4 mg/L resulted in a lipid content of 5.78 g/L [123]. Interestingly, it has been found that increasing the concentration of a metal can lead to the production of higher amounts of saturated fatty acids in the treated cells [124].

5.8. Effect of Sulfur Starvation

Algae require sulfur in the form of sulfate as an essential nutrient for their growth. Sulfur limitation in the growth medium can induce lipid accumulation in algae. In response to sulfur limitation, algae may alter their metabolic pathways to prioritize lipid synthesis as a way to cope with the stress. For example, under sulfur starvation, Chromochloris zofngiensis and Scenedesmus acuminatus exhibit high lipid accumulation [125,126]. Sulfur stress can also change the fatty acid profile of algal lipids. This could lead to changes in the proportion of saturated to unsaturated fatty acids and the length of fatty acid chains [126]. Sulfur stress can alter the regulation of key enzymes involved in lipid biosynthesis pathways. For example, it is known that the enzymes acetyl-CoA carboxylase and fatty acid desaturases are regulated by sulfur availability [127]. Changes in their activity can affect the rate of lipid synthesis in algae. It is important to note that the effects of sulfur stress on algal lipid synthesis can vary depending on the algae species, growth conditions, and other environmental factors. Therefore, there are currently many comprehensive studies underway in this field to understand and utilize the potential of sulfur stress for the sustainable production of algal lipid-based biodiesel.

5.9. Effect of Phytohormones

To stimulate lipid accumulation in microalgae, it is important to use both traditional abiotic stress conditions and innovative techniques. This is necessary because there is a reciprocal relationship between biomass and lipid accumulation under abiotic stress [128]. The effects of phytohormones on microalgal metabolism, especially in relation to lipid production, are still not fully clear. However, it has been noted that auxins, a type of phytohormone, can promote the growth of Scenedesmus sp. This growth regulator has been found to increase the content of TAGs and MUFAs, while simultaneously reducing the content of PUFAs [129]. The combination of indole-3-butyric acid (IBA) at a level of 10 mg/L and 6-benzylaminopurine (BAP) at a level of 5 mg/L exhibited a synergistic effect. The synergistic effect led to a 2.34 g/L increase in biomass production and a 42.43% increase in lipid content [130]. Similar findings were reported in Chlorella sp., where the application of IBA promoted maximum growth and lipid yield [131].

Fluvic acid, an additional phytohormone, has been used to enhance lipid yield in microalgae. This effect was achieved by modulating gene expression and the actions of principal enzymes such as phosphoenolpyruvate carboxylase and acetyl-CoA carboxylase [132]. The application of salicylic acid (SA) at a concentration of 10 ppm resulted in a substantial increase in lipid, reaching 475 mg/L in the early stationary growth phase. The application of salicylic acid (SA) was found to be crucial for the formation of omega-3 fatty acids, specifically eicosapentaenoic acid (EPA, C20:5) [133]. Higher concentrations of methyl jasmonate (MeJA) were found to promote the formation of MUFAs, especially oleic acid (C18:1). Most phytohormones have demonstrated the ability to enhance biomass production, and some have also shown promise in improving lipid yield and modifying lipid composition. There is a pressing need to conduct extensive large-scale experiments to ascertain the feasibility and cost-effectiveness of using phytohormones in industrial-scale cultivations, which are crucial for biofuel production and involve more diverse culture conditions.

6. Microalgal Biorefinery for Biofuels Production

A microalgal biorefinery is a specialized facility designed to harness the full potential of microalgae for the production of biofuels and various other valuable products. Microalgal biomass can be processed into an extensive array of products, including proteins, carbohydrates, lipids, pigments, polyunsaturated fatty acids, antioxidants, nutraceuticals, vitamins, biofertilizers, animal feed, biosurfactants, and bioenergy products (Figure 3). A microalgal biorefinery, in combination with other processes, has the potential to solve the current bioeconomic problem by producing multiple high-value products [134]. Microalgae are an efficient feedstock for generating various types of biofuels, depending on the chosen generation route (Figure 3). Microbial biomass conversion to biofuels is an alternative approach to the increasing demand for fossil fuels. Microalgal biorefineries hold promise as a sustainable and versatile solution for biofuel production, offering the potential to address energy needs while promoting environmental sustainability and economic diversification [135,136].

The zero-waste principle represents a fundamental shift in the paradigm of microalgal biorefinery, emphasizing the efficient utilization of all components within microalgal biomass to minimize waste generation [137,138]. In traditional microalgal cultivation and processing, there is often a focus on extracting a single high-value product, such as lipids for biodiesel production, while the remaining biomass and by-products are discarded. However, the zero-waste approach recognizes the intrinsic value of every constituent within microalgae and seeks to extract and utilize multiple valuable products from a single biomass source. The zero-waste principle endeavors to achieve the highest degree of resource efficiency by extracting, separating, and valorizing all components of microalgae. This entails not only the extraction of lipids but also proteins, carbohydrates, pigments, and other bioactive compounds [139]. For instance, after lipid extraction, the residual biomass can be processed to recover proteins suitable for food and feed applications, pigments for cosmetics, or carbohydrates for bioethanol production [140,141]. This comprehensive utilization optimizes resource efficiency, minimizes waste, and reduces the environmental footprint of microalgal biorefineries. Moreover, it helps diversify revenue streams by generating multiple valuable products from the same starting biomass, thereby enhancing economic viability.

Industry examples showcase the practical implementation of the zero-waste principle in microalgal biorefineries. One such instance is the production of omega-3 fatty acids and astaxanthin from microalgae [142,143]. Omega-3 fatty acids are highly valued for their health benefits and are used in dietary supplements and functional foods. Meanwhile, astaxanthin, a potent antioxidant and pigment, finds applications in nutraceuticals, cosmetics, and aquaculture feeds [144]. By cultivating microalgae for these high-value compounds, the zero-waste approach ensures that the residual biomass, which still contains valuable proteins, carbohydrates, and pigments, is put to use, reducing waste and enhancing the economic feasibility of the entire process.

Embracing the zero-waste principle in microalgal biorefinery aligns with broader sustainability objectives and the transition towards a circular bioeconomy. It reduces the environmental impact associated with waste disposal and provides an environmentally responsible alternative to resource-intensive practices. Furthermore, by efficiently utilizing microalgal biomass, the zero-waste approach contributes to the development of a sustainable and circular bioeconomy, where resources are conserved, reused, and repurposed [143]. This not only helps mitigate environmental pressures but also fosters economic resilience and innovation in the microalgal industry.

Further, microalgae biorefinery has emerged as a promising approach for sustainable and diversified bioproduct production, driven by the concept of multi-product valorization. Traditionally, the focus in microalgal cultivation has primarily been on a single high-value product, such as biofuels or nutraceuticals. However, the realization of the full potential of microalgae lies in their ability to yield multiple valuable products simultaneously. One compelling example of multi-product production in the microalgal industry is the cultivation of Haematococcus pluvialis. This microalga is renowned for its ability to accumulate astaxanthin, a potent antioxidant and red pigment used in nutraceuticals and cosmetics [145]. While astaxanthin is a high-value product, the residual biomass still contains valuable components, including proteins and carbohydrates. In response, some companies have developed integrated biorefinery processes to simultaneously extract astaxanthin, proteins, and carbohydrates from H. pluvialis, thus diversifying their product portfolios and reducing waste.

Another compelling instance involves the production of value-added nutraceuticals from microalgae. Companies have successfully cultivated microalgae such as Chlorella sp. and Spirulina sp. to produce not only lipids for biofuels but also high-protein biomass suitable for dietary supplements [146,147]. The versatility of microalgae enables the simultaneous production of lipids for biofuel and proteins for nutraceuticals, underscoring the potential for multi-product production in microalgal biorefineries. This approach enhances economic viability by tapping into multiple markets and revenue streams.

Additionally, some microalgal species, such as Nannochloropsis and Schizochytrium, offer the unique advantage of co-producing biodiesel and omega-3 fatty acids. While Nannochloropsis is known for its lipid-rich biomass suitable for biodiesel production, it also produces valuable long-chain omega-3 fatty acids such as eicosapentaenoic acid (EPA) [148]. Similarly, Schizochytrium produces docosahexaenoic acid (DHA), another omega-3 fatty acid, alongside lipids [149]. The co-production of biodiesel and omega-3 fatty acids exemplifies the potential for microalgae to serve multiple industries, including biofuels and nutraceuticals. Embracing the concept of multi-product production in microalgae biorefineries not only enhance economic feasibility but also aligns with the broader goals of sustainability, resource efficiency, and circular bioeconomy development. Here are some of the biofuels that are commonly produced using biorefineries.

6.1. Biodiesel

Biodiesel is an alkyl ester derived from fatty acids produced through the transesterification of lipids. Microalgal-derived biodisels are a sustainable alternative to petroleum-based biodeisels. Microalgal-based biodiesel can be generated using two methods: direct transesterification reaction with a heterogeneous catalyst, or in-situ transesterification of the lipids present in microalgae [21]. In addition, the various physio-chemical properties of different microalgae make them a useful variant of microalgal-derived biodiesel. It is biodegradable, nontoxic, oxidatively stable, and exhibits good performance in engines [150,151]. Microalgae reported for biodiesel production, including Chlorella vulgaris, Neochloris sp., Tribonema sp., Porphyridium sp., Chlorella pyrenoidosa, Scenedesmus obliquus, Desmodesmus sp., Nannochloropsis sp., Monoraphidium sp., Isochrysis, Phaeodactylum sp., Schizochytrium sp., Scenedesmus dimorphus, and Synechocystis sp. [152,153,154,155]. However, the productivity of microalgal-derived biodiesel may be enhanced by using the modern concept of metabolic engineering, which involves altering the metabolic pathways of microalgal species.

6.2. Bio-Oil

Bio-oil, a dark brown liquid, is generated by converting biomass through pyrolysis and hydrothermal liquefaction methods. Microalgae are favored over land-based oil-producing crops for biofuel production because they have a high level of polyunsaturated fatty acids. Pyrolysis is used for biomass with low moisture content, while hydrothermal liquefaction can be applied to any type of biomass, regardless of its moisture content [156]. Pyrolysis and hydrothermal liquefaction are thermochemical conversion processes that involve the depolymerization of organic substances under anaerobic conditions [157]. The reactive and unstable nature of these organic compounds leads to their reorganization into oily substances that exhibit diverse molecular weights. Liquefaction is a costly thermochemical conversion technique that decomposes moist microalgal biomass into high-quality liquid fuel. In the process of direct liquefaction, fast pyrolysis is used to transform liquid tars and oils into gases. In indirect liquefaction, catalysts are used to transform non-condensable gases into liquid products through pyrolysis or gasification processes [158]. Studies have indicated that the pyrolysis of microalgae, specifically Chlorella sp., has the potential to be an alternative energy source [159]. According to [160], bio-oil can be directly blended into turbines without requiring any modifications and can be used for transportation purposes.

6.3. Biobutanol

Carbohydrate-based microalgae can be a source for producing biobutanol, an alternative fuel that can replace bioethanol [161]. The remaining green residue from microalgal oil extraction can be effectively used for biobutanol production. Biobutanol is conventionally generated from microalgae through the fermentation process of acetone-butanol-ethanol, which involves Clostridium anaerobic bacteria. This fermentation process produces byproducts, such as ethanol, gases, acetone, and organic acids [162]. The production of byproducts, particularly organic acids, during the biobutanol fermentation process, reduces the pH of the medium, leading to lower efficiency in the process. Biobutanol offers advantages, such as high water solubility, energy content, volatility, and molecular similarity to gasoline, making it a viable alternative to bioethanol and biomethanol. Biobutanol is also widely used as a solvent in various industrial processes [163]. Chlorella vulgaris, Chlorella reinhardtii, Tetraselmis subcordiformis, and Scenedesmus obliquus are all considered for biobutanol production [164]. In a regulated fermentation procedure, the highest yield of bio-butanol was achieved [165]. According to reports, treating C. vulgaris with 2% sulfuric acid resulted in the production of 3.37 g/L of butanol through acetone-butanol-ethanol (ABE) fermentation [166]. A study observed that the addition of butyrate during ABE fermentation resulted in an increase in biobutanol production [167]. This was attributed to enhanced metabolic processes, specifically the transformation of acetoacetyl Co-A to butyl Co-A instead of acetoacetate [167]. Continued research and development in this field could contribute to a more sustainable and diversified energy landscape.

6.4. Bioethanol

Bioethanol is the predominant liquid biofuel, and it is produced through the process of alcoholic fermentation using carbohydrates as a feedstock [168]. The conventional production method of bioethanol is energy-intensive and costly because it requires pretreatments, expensive enzymes, and purification steps [169]. Microalgae are used for ethanol production through hydrolysis and fermentation processes. Microalgae-based feedstocks are more efficient for carbohydrate conversion to ethanol than other feedstocks due to their low lignin content [165,170]. Novel methods for bioethanol production, such as dark fermentation and the use of genetically modified strains in photofermentation, are also being implemented [171,172]. Algal species, including Chlorococcum humicola, Chlorella vulgaris, Scenedesmus bijugatus, Synechocystis sp., and Synechococcus elongatus, have been reported to be suitable candidates for bioethanol production [173,174,175]. In a study, Chlorella vulgaris containing approximately 37% starch content was found to produce around 65% ethanol [176]. Genetically modified Chlamydomonas perigranulata can self-ferment carbohydrates to produce bioethanol [177]. The anaerobic fermentation of microalgal biomass for bioethanol production is increasingly being seen as a simpler process than other fermentation methods.

6.5. Biogas

The high concentration of diverse molecules in microalgal biomass can be converted into biogas through anaerobic digestion (AD). Additionally, the AD technique can be used to recycle nutrients from wastewater, and the reclaimed water from the AD process can be used to cultivate microalgae [178,179]. Methanogenesis is the most common process for biogas production, and it is often carried out in co-digestion systems with sewage sludge (SS) or other co-substrates [180]. Researchers investigated the effects of co-digesting microalgae biomass and primary sludge (PS) on biogas production and microcontaminant removal. The co-digestion process resulted in a 65% increase in methane (CH₄) productivity and up to 90% micro-contaminant removal efficiency. The methane yield in co-digestion is highly dependent on the operational conditions. Therefore, it is essential to consider and optimize these conditions to improve methane production.

There is a critical need for innovative and environmentally friendly technologies to upgrade biogas. Several conventional biogas upgrading technologies exist, such as chemical adsorption, membrane separation, and pressure swing adsorption, but they are all costly [181]. Currently, research is being conducted on microalgal-based biogas upgrading, with the aim of overcoming the significant drawbacks of conventional biogas methods [182]. Biogas upgrading using microalgae can achieve an efficacy of around 97%, which is influenced by factors such as reactor design, operating parameters, and the specific microalgal strains chosen for the upgrading process [181]. Studies have shown that microalgal-based biogas upgrading is very effective in removing CO₂ and H₂S, and the upgraded biogas can have a methane purity of 95% [183]. The removal of CO₂ from biogas using photosynthetic microalgal biogas upgrading is more efficient during the light phase than the dark phase [184,185]. The integration of microalgae with wastewater treatment processes can improve the efficiency of nutrient removal and lead to the production of purified methane [186].

The use of microalgae with elevated protein levels as feedstock for AD can lead to an imbalance and even failure of the AD process. Protein breakdown releases ammonia as an intermediate product. Excessive ammonia in an AD system can inhibit the activity of methanogenic bacteria, which can lead to reduced biogas production [187]. Additionally, the degradation of proteins can result in the formation of volatile fatty acids, causing acidification of the AD reactor [188]. Low pH conditions can negatively affect the AD process, leading to inconsistent results and reduced efficiency. High protein content can disrupt the AD process and reduce its efficiency and reliability. To mitigate these issues, it is crucial to carefully manage the feedstock composition in AD systems that utilize microalgae with high protein content. Co-digestion of microalgae with other substrates that have a balanced carbon-to-nitrogen ratio, such as agricultural residues or wastewater sludge, is one approach to improve the overall performance and stability of the AD process [189]. Pre-treatment techniques, such as hydrothermal processing or enzymatic hydrolysis, can be used to break down complex proteins into simpler compounds that are more easily degraded by microbes during AD. By acknowledging this limitation and employing appropriate strategies, the capacity of microalgae to serve as a valuable feedstock for anaerobic digestion can be harnessed effectively, contributing to sustainable energy solutions.

6.6. Biohydrogen

Biohydrogen is a renewable and sustainable energy source that has the potential to replace conventional fuels [190,191]. Biohydrogen is a more environmentally friendly fuel than fossil fuels because it has a high energy yield and produces water as a byproduct [192]. The algal hydrogen metabolic system relies on two important enzymes, nitrogenase and hydrogenase [193]. The efficiency of microalgae in producing hydrogen is affected by several factors. The most important parameters for hydrogen production are oxygen levels, nutrient media composition, and salinity [194]. Biohydrogen production from microalgae can be carried out using indirect or direct photolysis processes [195]. In the direct photolysis process, the hydrogenase enzyme is used to split water into hydrogen ions and electrons. The electrons then move to the Fe-Fe hydrogenase enzyme, which produces biohydrogen. The presence of oxygen hampers the functionality of the enzyme’s binding site, resulting in a reduction in the biohydrogen yield [190]. In indirect photolysis, oxygen is reduced to form biomolecules, which are then broken down to produce electrons. Algal species, such as Chlamydomonas reinhardtii, Chlamydomonas moewusii, Micractinium reisseri YSW05, and Scenedesmus obliquus are used for biohydrogen production [196,197,198]. Genetic modifications can be used to enhance the productivity of biohydrogen.

6.7. Bioelectricity

Microalgae have a higher potential for bioenergy generation than other microorganisms and plants. Bioenergy products, such as bio-oil and biohydrogen are typically used to generate bioelectricity [199]. Microalgae-based microbial fuel cells (MFCs) have significant applications in wastewater treatment and bioelectricity generation. Additionally, this technique plays a role in carbon sequestration, biohydrogen production, and desalination [200,201]. MFCs are bioelectrochemical systems that consist of an anode and a cathode, where various active microorganisms are used to generate bioelectricity [202]. Oxidation and reduction reactions are essential for bioelectricity generation in MFCs [203]. The photosynthesis pathway is used in microalgae-based MFCs to convert CO₂ into organic biomass and release O₂ as a byproduct. During this process, the released O₂ accepts electrons. The electrons flow through the metabolic processes and generate a current [204]. The following algae species are capable of producing bioelectricity: Chlamydomonas sp., Oscillatoria sp., Scenedesmus sp., Botryococcus braunii, Phormidium sp., Chlorella sp., Golenkinia sp., Chlorococcum, Synechococcus sp., and Spirulina sp. [205,206,207,208,209,210]. To implement and commercialize bioelectricity on a large scale, advanced and integrated approaches are needed.

The production of biofuels from microalgae is influenced by a number of crucial parameters, such as species of microalgae, growth medium, light intensity, temperature, pH, and salinity. Different microalgae species have different lipid, carbohydrate, and protein content. The choice of microalgae species is therefore important for the production of a particular type of biofuel. For example, species with high lipid content are suitable for the production of biodiesel, while species with high carbohydrate content are suitable for the production of bioethanol. In addition to these crucial parameters, other factors such as the mixing rate, the aeration rate, and the CO₂ concentration can also affect the production of biofuels from microalgae. The effect of different parameters on the lipid content of microalgae species for biofuel production is shown in

Table 4. The impact of crucial parameters on diverse microalgal species for the production of various types of biofuels.

Parametric Study	Algae Species	Biofuels	Production	Ref.
Effect of reactive oxygen, nitrogen species, time and depletion of cations	Chlorella sp.	Bioethanol	0.43 g/g sugars	[211]
Effect of acid concentration, temperature and time	C. vulgaris	Bioethanol	0.07 g/g microalgae	[212]
Effect of culture duration and fermentation time	Nannochloropsis oculata and Tetraselmis suecica	Bioethanol	7.26%	[213]
Effect of moisture contents of wet algae, temperature, pressure and hydrogenation on process integration	Acutodesmus obliquus	Biohydrogen	13,944 kg/h	[214]
Catalytic activity and effect of ZnCl2, CuB loading, NaBH4 concentration and temperature	C. Vulgaris	Biohydrogen	17,833 mL/min g catalyst	[215]
Organic carbon sources and lighting regime	Parachlorella kessleri	Biohydrogen	2.2 mmol/L	[216]
Effect of temperature, pH, light and glucose supplementation	Anabaena sp.	Biohydrogen	57.6%	[217]
Temperature 55 °C	Porphyridium cruentum	Biogass	179 mL CH4/g VS	[218]
Temperature 35 °C	C. vulgaris	Biogass	168.9 mL CH4/g COD	[219]
Temperature 37 °C	Haematococcus pluvialis	Biogass	91.9 mL CH4/g VS	[220]
Temperature 37 °C	Chlorella pyrenoidosa	Biogass	147 mL CH4/g VS	[221]
Temperature 37 °C	Scenedesmus obliquus	Biogass	0.16 m3 CH4/kg VS	[222]
Temperature 53 °C	Nannochloropsis limnetica	Biogass	0.41 m3 CH4/kg VS	[223]
Temperature 35–55 °C	Chlorococum sp.	Biodiesel	0.010–0.015 g FAME/g DW biomass	[224]
Light (180 µmol photons m−2 s−1 ) on a 14:10 (light: dark) photoperiod with salinity (NaCl 18 g/L)	Chaetoceros gracilis	Biodiesel	0.36 g FAME/g DW biomass	[225]
Light (180 µmol photons m−2 s−1 ) on a 14:10 (light: dark) photoperiod with salinity (NaCl 18 g/L)	Tetraselmis suecica	Biodiesel	0.18 g FAME/g DW biomass	[225]
Light (180 µmol photons m−2 s−1 ) on a 14:10 (light: dark) photoperiod with salinity (NaCl 18 g/L)	Chlorella sorokiniana	Biodiesel	0.18 g FAME/g DW biomass	[225]
Red light intensity 350 µE m−2 s−1	Dunaliella tertiolecta	Biodiesel	0.22 g FAME/g DW biomass	[226]
Temperature 20 °C	Nannochloropsis oculata	Biodiesel	0.07–0.24 g lipids/g DW biomass	[108]
Temperature 25 °C	C. vulgaris	Biodiesel	0.06–0.14 g lipids/g DW biomass	[108]
Nutrients limitations (0.1 mM Sulfur, 7.0 mM Nitrogen and 0.43 mM phosphorus)	Chlamydomonas reinhardtii	Biodiesel	60%	[227]
Light intensity (51 µmol photons m−2 s−1) with pH 7.8	Botryococcus braunii	Biodiesel	85.7%	[228]
CO2 (390 ppm)	C. vulgaris	Bio-oil	46.60%	[229]
Increased alkanity	Chlorella sorokiniana	Bio-oil	62%	[230]
Hydrothermal treatment	Desmodesmus sp.	Bio-oil	49.40%	[231]
Effect of different metals (Co, Ni, and Fe)	Spirulina	Bio-oil	43.60%	[232]
Catalytic hydrotreatment	Nannochloropsis gaditana	Bio-oil	24.60%	[233]
Kitchen wastewater	Scenedesmus obliquus	Bio-oil	55.59%	[234]

.

7. Abiotic Stress Induced Genes Responsible for Lipid Production

Biosynthetic genes involved in lipid production respond to various abiotic stresses. Microalgae can be triggered to produce more lipids by stress, which upregulates the expression of genes involved in lipid biosynthesis. The enzymes involved in fatty acid synthesis, lipid droplet formation, and lipid accumulation pathways are encoded by these genes. The specific response of these genes to abiotic stresses varies depending on the microalgal species and the severity of the stress. Optimizing lipid production in microalgae under challenging cultivation conditions is important for understanding the gene response to abiotic stresses. The schematic overview of the principal enzymes linked to enhanced triacylglycerol synthesis under stress conditions in the lipid synthetic pathways is provided in Figure 4.

A deep understanding of the mechanisms that control lipid production in microalgae is essential for the development of new technologies for algal biorefinery. Researchers have investigated the reprogramming of biosynthetic pathways for different biomolecules to favor lipid accumulation [236]. Cultivating microalgae under stressed conditions has been found to activate regulatory mechanisms in the algae, which results in increased synthesis of neutral lipids, especially TAGs. This response serves as a defense mechanism and aids in cell survival [237]. Numerous research studies have reported the regulation of genes related to abiotic stress and the enzymes responsible for TAG production. Pyruvate dehydrogenase (PDH) is a key enzyme that is involved in the pathways that produce lipids and contribute to the accumulation of lipids in microalgae [238]. Similarly, an increase in the activity of ACCase has been observed, which facilitates the utilization of malonyl-CoA for the excessive production of lipids. Fatty acid synthase (FAS) is also an enzyme that acts as a rate-limiting step in the synthesis of lipids. Studies have shown that the overexpression of genes related to FAS complexes, such as 3-ketoacyl-ACP synthase (KAS-III), can result in increased levels of palmitic acid. Additionally, up-regulation of KAS-I has been found to significantly enhance lipid synthesis. It has been suggested that the coordinated regulation of multiple genes involved in lipid synthesis may be more effective than targeting a single gene [239]. Overall, abiotic stress induction and gene regulation are feasible approaches to promote lipid synthesis

8. Cell Membrane Lipid and Triglycerides Accumulated in Lipid Droplets (LDs) in the Cells by Abiotic Stress for Biodiesel Production

Algae lipids can be broadly categorized into two main groups: storage lipids and membrane lipids. Storage lipids serve as reserves of energy and carbon, while membrane lipids play an essential role as the fundamental components of both photosynthetic and non-photosynthetic membranes. TAGs are the primary storage lipids in algae, while sterol esters play a minor role. The lipid composition of membranes is more complex and heterogeneous, and it varies between different cell compartments, cell types, and even different organisms [235]. Cellular membranes are essential for maintaining cell integrity and functionality. Lipids are a major constituent of cellular membranes, and their composition can have a significant impact on membrane fluidity, permeability, and stability. Abiotic stressors can disrupt membrane integrity by altering the composition of lipids in the membrane. Microalgae are able to remodel their membrane lipids in response to environmental challenges, which is an efficient way for them to adapt to these challenges. The modification of membrane lipid structure is a dynamic process that is controlled by a variety of factors, including membrane repair, lipid metabolism, fatty acid trafficking, cellular signaling, and homeostasis mechanisms [240,241]. Increased levels of DAG pools and acyl-CoA molecules in the cell, which are derived from the dynamic interplay of membrane lipids, desaturases, lipases, and acyltransferases, are used in the synthesis of TAGs. Consequently, this can lead to the accumulation of lipid droplets within the cell. Galactolipids, which are the main lipids in the membrane and respond significantly to abiotic stresses, play a role in controlling stress responses and TAG metabolism. Monogalactosyldiacylglycerol (MGDG), a type of galactolipid, is found in high concentrations in the membranes of algae. It is essential for the proper functioning of the thylakoid membrane [242]. In Chlamydomonas, a specific lipase called plastid galactoglycerolipid degradation-1 (PGD-1) has been found to be essential for redirecting the flow of fatty acids from MGDG to TAG during nitrogen starvation. This process enhances the stability of thylakoid membranes in response to nutrient stress [243]. Artificially controlling MGDG levels could induce membrane lipid remodeling, which could lead to the accumulation of TAG and improved stress resistance in microalgae.

Microalgal lipids, such as galactolipids, phospholipids, and TAG, are widely used in biodiesel production. Neutral lipids are the preferred feedstock for microalgal biodiesel production because they do not contain sulfur and phosphorus [244]. The presence of phosphorus in phospholipids can interfere with the transesterification reaction, which can lead to a lower biodiesel yield [245]. The fatty acid composition is a key factor in microalgal biodiesel production, as it can affect the fluidity of biodiesel at low temperatures and its oxidative stability [246]. Maintaining a low level of long-chain saturated fatty acids is advantageous for preserving fluidity at low temperatures in biodiesel. A greater amount of SFAs and MUFAs is beneficial for ensuring oxidative stability in the biodiesel.

Lipid droplets are spherical organelles that store neutral lipids, mainly TAGs, which are major precursors for biodiesel production. The accumulation of TAGs in lipid droplets within cells under abiotic stress conditions is a promising development for biofuel production. Studies have shown that LDs are dynamic structures that play essential roles in cellular activities. These findings have revealed the multifunctionality of LDs within cells [247]. Abiotic stress can cause changes in nutrient availability and energy demands in cells, which can lead to alterations in TAG accumulation. Numerous microalgae have shown an enhanced capacity to accumulate TAGs in lipid droplets in response to stresses such as nutrient deprivation, temperature fluctuations, and changes in light conditions [248,249]. After 11 days of nitrogen limitation, the microalgae Nannochloropsis can accumulate TAGs up to 37% (w/w) of their biomass [250]. Chlorella sorokiniana HS1 can grow and hyperaccumulate LDs under hypersaline conditions [251]. This oxidative stress leads to the phenomenon of liquid-liquid phase separation (LLPS), where TAGs separate from the cytosol and coalesce to form LDs [252]. Lipid droplets play a significant role in maintaining cellular redox balance. Investigating the specific effects of different stressors on triglyceride accumulation can help identify stress-resilient organisms and optimize their use as biodiesel feedstock sources.

9. Software Tools for Microalgae Biorefinery

Nowadays, creating more efficient processes is a key challenge for greener production. This objective necessitates a combination of economic, technical, and environmental factors, which are frequently addressed using simulation techniques. This technique is vital for biorefineries, as they offer eco-friendly fuels and chemicals, aligning with sustainable development goals. However, simulating such systems using raw mathematical formulas and data can be complex and time-intensive. Software tools alleviate this complexity by providing organized databases and calculations, streamlining prospective algae modeling. Advanced software tools, such as AlgaGrow, Design Expert, Aspen Plus, SuperPro Designer, BioSTEAM, Unisim, IPSEpro, COPABI, STELLA, AIMMS, COMSOL, DAYCENT, LabVIEW, and others are available for algal growth and biorefinery systems [253,254]. These tools, chosen based on feed, process, and product conditions, enable more efficient simulation. Appropriate software solutions are essential for resolving intricate process challenges, optimizing processes, selecting methods, and improving efficiency and productivity in biorefineries. The distinct stages of biorefineries and their corresponding software are shown in Figure 5.

AIMMS software is a powerful tool that can be used to model and optimize complex processes, such as integrated algae biorefineries. The concept of integration involves utilizing the wastewater and CO₂ emissions from biorefinery facility processing wheat straw as inputs for the microalgae biorefinery. The resulting residual materials from the algae process are then returned to the wheat straw biorefinery to generate additional valuable compounds [255]. The outcomes of their study indicated that the integrated approach offers economic benefits compared to an independent algae biorefinery, primarily due to potential cost savings of up to 80% in biodiesel production.

In the study conducted by [256], they employed both MATLAB and Aspen Plus software tools to develop a multiobjective optimization framework. This framework was designed to address the process planning of integrated biorefineries in the presence of uncertainties. They applied this optimization methodology to a combined multiproduct biorefinery facility that manufactured improved biofuels (ethanol) and bio-based chemicals (succinic acid). Through this, they showcased the effectiveness of their proposed approach.

Further, in a study conducted by [257], they investigated the production of biodiesel through the transesterification of microalgae oil. This process was carried out using methanol and ethanol solvents under both supercritical and non-catalytic conditions. For the purpose of refining the operational parameters of the procedure, a combination of Response Surface Methodology (RSM) and Central Composite Design (CCD) was utilized. The researchers employed Design Expert software, version 8.05 to evaluate the experimental data and forecast optimal conditions. The outcomes indicated that the model effectively forecasted the experimental data with satisfactory precision. In their study, the optimal biodiesel yield values achieved using methanol and ethanol solvents were approximately 91% and 88%, respectively.

Moreover, in a study conducted by [258], they examined the generation of bioethanol from Chlamydomonas reinhardtii using a biorefinery methodology. They improved the biomass and carbohydrate productivity by fine-tuning the physicochemical factors. They utilized a de-pigmented and de-fatted algal biomass as a substrate to evaluate the potential of bioethanol fermentation. Enhancements in the biomass and carbohydrate levels of C. reinhardtii were achieved by adjusting various physicochemical parameters. Multiple attributes of the media, including initial pH, cultivation temperature, and concentrations of acetate and ammonium chloride, were optimized using Minitab18 software. This optimization process involved altering one variable at a time and subsequently examining how the optimized variables interacted. Notably, the combined adjustment of these physicochemical factors, along with CO₂ sequestration, was found to positively impact the augmentation of biomass and metabolite concentrations in C. reinhardtii. It is important to choose the software that aligns best with specific research goals, simulation needs, and level of expertise.

10. Challenges of Microalgal Biofuel Production

Microalgae show great promise and are considered a viable option for sustainable biofuel production in the long run. However, there are several research challenges associated with microalgal-based biofuel production systems, which have raised concerns about their feasibility. One major challenge lies in selecting suitable microalgal strains for optimal biofuel production. Obtaining suitable microalgal strains for biofuel production is often a time-consuming and labor-intensive process that involves collecting, isolating, purifying, and identifying the desired strains. It is essential to customize the growth media and culture conditions to promote optimal microalgal proliferation. Species identification typically involves a combination of morphological and genetic analyses. To optimize in vitro experiments, it is essential to consider the natural conditions at the sampling site. Once a microalgal strain is isolated, it is cultivated in advanced systems to achieve high biomass and lipid production. This cultivation process involves utilizing high-tech methods and equipment [259]. A potential approach is to cultivate the microorganisms in their native environments using sufficient inoculum. Moreover, microalgae that demonstrate favorable performance in laboratory conditions may not thrive in their natural habitats, necessitating the development of bioengineered microalgal strains. While the production of biofuels from microalgae can be costly and resource-intensive in terms of water, nitrogen, and CO₂ requirements, they offer significant environmental benefits, as mentioned earlier. The ample production of biomass and biofuels necessitates substantial investments in equipment, energy, water, and nutrients.

The limited photon conversion efficiency is a notable limitation in microalgal biofuel production systems. Microalgae employ extensive light-harvesting complex (LHC) systems to maximize photon capture in their natural environment. However, as a survival mechanism, a smaller fraction of captured photons are released as heat via a mechanism known as non-photochemical quenching (NPQ). The NPQ phenomenon is primarily witnessed at the upper layer of densely populated microalgal cultures, resulting in a self-shading impact that restricts light penetration to the lower layer. As a result, both the photon conversion efficiency and the self-shading effect contribute to a decrease in photon conversion efficiency. To address this issue, potential solutions include modifying the structure of the LHC and designing photobioreactors in a way that optimizes light distribution throughout the culture. To attain high photon conversion efficiency, it is essential to use photobioreactors that allow for improved light penetration and distribution. The amount of light that reaches the algae in a photobioreactor is affected by the surface area-to-volume (A/V) ratio of the reactor. Maximizing the A/V ratio is critical in ensuring optimal light availability to the microalgae, thereby enhancing the efficiency of photon conversion [260]. Significant advancements in process success and economic viability are necessary before widespread adoption of this technology can occur.

Furthermore, it is essential to conduct a thorough examination of biofuels from a meta-economic standpoint, considering the secondary expenses linked to their extraction and utilization. Initial investigations have already uncovered various hidden expenses linked to the use of biofuels, revealing that the actual cost of fuel extends beyond the price paid at the pump. Although the initial evaluation is important, it is vital to acknowledge that the actual undisclosed expenses associated with the utilization of biofuels are significantly greater.

Therefore, conducting in-depth economic analyses is essential to determine the accurate competitive cost of biofuels. Additionally, due to the multitude of uncertainties surrounding the design particulars of a practical microalgal biofuel facility, conducting a detailed cost assessment is presently challenging. Consequently, a comprehensive cost analysis remains unattainable at the moment. Furthermore, extensive research is imperative before the microalgal biofuel production method can be effectively commercialized. At the laboratory stage, certain operational and technological challenges may not be evident, but they can arise when scaling up to a larger production level. Therefore, it is essential to conduct practical pilot-scale research to effectively transform these methods into viable and practical technologies. It is worth noting that while microalgal biofuels show great potential, there are still several technical and economic obstacles that need to be tackled before they can be commercially viable on a large scale. Continued research and development are focused on enhancing the efficiency and cost-effectiveness of processes involved in producing biofuels from microalgae.

11. Future Perspective

Microalgal systems are rapidly emerging as highly promising platforms for the production of renewable biofuels. However, further research is needed to enhance microalgal biofuel production, as current biomass and lipid yields are not sufficient for making biofuels commercially viable as an energy source. Hence, there is a need to investigate different approaches to enhance the capacity of microalgae to produce biofuels. A deep understanding of the complex metabolic pathways in microalgae, which are influenced by environmental factors, is crucial to meet the demand for biofuels. Currently, the main challenge lies in enhancing the efficiency of biofuel production by genetically modifying existing microalgal strains using genetic engineering and synthetic biology approaches. Synthetic biology advancements offer opportunities to engineer microalgal cells and enable them to produce specific value-added products, such as biobutanol, bioethanol, biodiesel, biohydrogen, biogas, and electricity. The progress in synthetic biology and microbial genetic engineering, coupled with the acceptance and utilization of biofuel energy, will determine the microalgae’s potential as a significant future source of energy.

12. Conclusions

The urgent need to replace fossil fuels has become increasingly prominent in recent years. Biofuels derived from microalgae are considered a promising solution as they are renewable and environmentally friendly. However, the current lipid production from microalgae falls short of meeting the current fuel demand. Consequently, researchers have directed their attention toward enhancing lipid yield through modifications in cultivation conditions. Temperature, light intensity, light spectrum, photoperiod, CO₂ concentration, pH, and phytohormones are crucial parameters in the cultivation of microalgae, as they significantly influence their growth and lipid production. This review has extensively explored methods to enhance both biomass productivity and lipid content in microalgae. Additionally, it has conducted a comprehensive analysis of the influence of environmental stress factors on cultivation conditions, particularly focusing on microalgal lipid yield and fatty acid composition. However, future research should concentrate on integrating the manipulation of multiple abiotic stresses to simultaneously improve biomass and lipid production in microalgae. Furthermore, there is a need for further investigation and innovative approaches to sustainability analysis and quality control in order to facilitate large-scale production and commercialization of biofuels derived from algae.