New strategy for the biosynthesis of alternative feed protein: Single‐cell protein production from straw‐based biomass

With rapid growth of global population, meeting the increasing demand for food has become a significant challenge. This challenge is further compounded by limited arable land and the necessity to address the nutritional needs of both humans and animals. However, the utilization of straw biomass, which is readily available as an agricultural by‐product, presents a sustainable solution to this problem. Microbial fermentation has emerged as a highly effective method for converting non‐food biomass into protein, particularly known as single‐cell protein (SCP). Compared to traditional protein sources, SCP production through microbial fermentation is rapid and efficient, and requires minimal land resources. This review provides a comprehensive review of the research advancements in SCP from agricultural biomass, including pretreatment methods, microbial strains, and fermentation processes involved in the bioconversion of straw biomass. Due to the complexity of straw‐based biomass (SBB), it is essential to customize industrial strains and optimize the fermentation process to achieve the highest protein yield and productivity. Additionally, improving the compatibility between tailored processes and cost‐effective industrial strains can lead to the production of protein substitutes that are not only highly nutritious but also economically viable. Hence, the application of SCP derived from SBB presents a dual solution by reducing the need for managing agricultural residues and providing a sustainable source of protein. However, the production of SCP from SBB also has some limitations, such as protein‐synthesis efficiency, production cost, and difficulty to scale‐up the production process. In the future, there is great potential for significant advancements in the targeted conversion of SBB into protein by customizing high‐performance microbial strains. Several sensor and machine learning technologies will predict and monitor real‐time dynamic changes in the fermentation process of SBB, offering an opportunity to improve the production of sustainable SCP in an environmentally friendly and precise manner.


| INTRODUCTION
The world's population is continuously increasing, and it is predicted to reach 9.3 billion by 2050 (Bratosin et al., 2021;Reihani & Khosravi-Darani, 2019).As a result, the global demand for animal proteins and derivatives may reach 1.25 billion tons annual (Ritala et al., 2017).Soybean meal, which is a by-product of soybean oil extraction, is commonly used as a source of vegetable protein in highquality animal diets.As a consequence, 85% of the world's soybeans are made into soybean cakes or soybean oil, and 97% of the resulting soybean dregs are used in feed (Ren et al., 2019).Additionally, due to the low efficiency of converting plant protein into animal products, as well as a possible conflict with human food production due to the conversion of 685 million hectares of farmland to livestock feed production, blindly expanding the scale of meat production will not be sustainable (Boland et al., 2013).Therefore, it is essential to find a protein source that is rich in amino acids, comes from a healthy source, and does not involve moral problems (Kim et al., 2019).
Straw-based biomass (SBB) is an inedible by-product of crops that is produced in vast quantities worldwide.However, most straw resources are burned or discarded, causing resource waste and environmental pollution, in addition to fires and other problems (Man & Wiktorsson, 2001).Although some straw resources are used as feed for ruminants after silage, ammonification, and other treatments, its low crude protein content (<15%) is far from the crude protein content of traditional feed protein sources such as soybean meal (43%-48%) and fishmeal (50%-70%; Sufyan et al., 2022).Moreover, straw protein feed has more crude fiber, resulting in poor palatability and low digestibility, which become the main factors limiting the preparation of microbial protein from straw for feeds (Zhu et al., 2017).By applying lignocellulose degrading strains that produce complex cellulase, SCP feedstuff can be produced, which is a way of utilizing straw resources (Eibl et al., 2021).The development of crop straw can make full use of resources and play a positive role in alleviating the above problems.
Since the early 1950s, there have been studies into new, unconventional protein sources that can be used to replace traditional animal and plant proteins.As a result, a novel method of protein production known as single cell protein (SCP) has been developed.SCP primarily involves the use of bacteria, fungi, yeast, and algae in the production of protein resources.This innovative approach has opened up new possibilities for sustainable protein production and has garnered significant attention in recent years (Bud, 2001;Suman et al., 2015).SCP refers to the biomass or protein extract derived from a single or mixed culture of microorganisms.It has the potential to serve as a valuable source of animal feed or human food, either as a standalone product or as a supplement (Onyeaka et al., 2022).With its rich nutritional composition, SCP offers a sustainable and efficient alternative to traditional protein sources.It can be utilized to address the growing demand for protein while reducing the reliance on conventional agriculture and livestock production.The versatility of SCP makes it a promising solution for meeting the nutritional needs of both animals and humans.As a matter of fact, the fungus Paecilomyces variotii was approved as a food source in Finland as early as 1983.This particular fungus boasts an impressive protein content of 55% (Tegge, 1983).It has a variety of advantages as a substitute for animal and plant protein, and since the production of SCP is neither affected by season nor climate, it can be produced throughout the year (Nangul & Bhatia, 2013).In addition, SCP production offers the advantage of requiring minimal land area while still achieving a high protein content with low fat.This characteristic makes SCP an excellent alternative to traditional feed protein supplements.
Compared to conventional methods of obtaining protein, such as animal farming or large-scale soy cultivation, SCP production is much more space-efficient.This efficient land utilization allows for the sustainable production of significant amounts of protein without exacerbating land scarcity or deforestation (Rischer et al., 2020).By utilizing SCP as a feed protein supplement, the environmental impact can be reduced while still meeting the nutritional needs of livestock and other animals.Microbial protein offers a balanced nutritional profile, characterized by essential amino acids, vitamins, and trace elements.Notably, its nutritional value surpasses that of soybean protein, and it does not pose the risk of sensitization associated with plant protein (Aidoo et al., 2023).The production process of SCP from SBB involves several key steps.First, fresh or stored straw undergoes necessary mechanical processing and sterilization.Additionally, appropriate pretreatment methods are employed to enhance the sugar utilization ratio of straw.Second, one or more microorganisms are cultured and fermented to produce SCP.Third, the resulting SCP is separated and concentrated, and in some lignocellulosic materials, microbial fermentation, microbial strains, pretreatment, single-cell protein, straw-based biomass cases, drying may be required.At last, the SCP undergoes final processing and packaging to create finished products (Amara & El-Baky, 2023;Banks et al., 2022).These steps ensure the production of high-quality SCP that can be utilized as a valuable protein source in various applications.Recent interest in SCP has focused on increasing the protein content of the final product, while a number of low-cost straw substrates for the production of short-chain starch are emerging on a commercial scale, with increasing numbers of protein-rich products being used in food or feed (Ritala et al., 2017).
In this review, we summarize the research progress and problems of SCP production from SBB.First, the necessary pretreatment of straw can improve the complex structure of straw and increase the utilization efficiency of microorganisms.Second, the SCP and other fermentation products can improve the nutritional composition of feeds while alleviating the shortage of traditional protein sources.Solving the problem of animal feed is intimately linked to solving the food security problem of billions of people around the world.At the same time, it provides new ideas and directions for straw bioconversion, reducing carbon emissions and realizing a low-carbon economy.
2 | PRETREATMENT OF STRAW RESOURCES 2.1 | Structure and chemical composition of SBB SBB represents a typical example of lignocellulosic biomass (LCB).To ensure the rational and efficient utilization of lignocellulose, it is crucial to gain a deep understanding of the intricate structure and inherent resistance of LCB.By comprehending the complex composition and recalcitrant nature of LCB, we can develop innovative strategies and technologies to unlock its full potential and transform it into valuable products.This knowledge plays a vital role in driving the advancement of sustainable and eco-friendly solutions for utilizing LCB effectively (Zhao et al., 2012).Generally speaking, LCB mainly consists of cellulose, hemicellulose, and lignin, the proportions of which vary in straw from different sources, as shown in Table 1.The variations in biomass composition can be attributed to a multitude of factors, including genetic traits, geographic location, environmental conditions, and crop harvest season (Keshav et al., 2021).These differences in composition have a significant impact on the overall characteristics and potential applications of LCB.By comprehending the intricate interplay between these influencing factors, we can develop tailored approaches to effectively harness the diverse potential of LCB.Considering the unique characteristics and challenges associated with different straw resources, researchers need to specify targeted strategies to maximize the efficiency and effectiveness of processing (Mankar et al., 2021).Cellulose, the primary structural component of lignocellulose, consists of a linear chain of repeating βd-glucose units (Mussatto & Dragone, 2016).These glucose units are connected by β-1,4 glycosidic bonds, which contribute to the rigidity and stability of the LCB.Hemicelluloses, on the other hand, are composed of a mixture of five-and six-carbon sugars that are interconnected by β-1,4 glycosidic bonds (Lynd et al., 2002).Acting as a molecular binder, hemicellulose forms a complex network of covalent bonds between cellulose and lignin.Lignin, is a complex biopolymer with a three-dimensional reticular structure that is formed by the interconnection of three phenyl propane units through ether and carbon-carbon bonds (Ashokkumar et al., 2022).This unique reticular system of lignin acts as a scaffold, surrounding and reinforcing cellulose and hemicellulose.Ultimately, the recalcitrant nature of LCB stems from this intricate architecture, which hinders its efficient utilization.Pretreatment plays a crucial role in the production of microbial protein from straw by enhancing its accessibility for enzymes that convert cellulose and hemicellulose into fermentable sugars.During pretreatment, the straw biomass undergoes several beneficial transformations, which result in increased specific surface area and porosity, structural modifications and removal of lignin, depolymerization of hemicellulose and reduction of cellulose crystallinity (Meenakshisundaram et al., 2021).Previous studies have demonstrated that untreated straw biomass can only release approximately 20% of the sugars, whereas with pretreatment, the recovery of fermentable sugars can reach 80%-83% (Singhvi et al., 2014).It is important to note that the pretreatment process should also consider creating an optimal environment for subsequent microbial growth and enzyme activity by avoiding extreme pH conditions or the presence of residual inhibitors that may hinder cell growth (Hendriks & Zeeman, 2009).In industrial production, the feasibility of a pretreatment method is determined not only by its effectiveness but also by its energy consumption and cost considerations.Additionally, the environmental friendliness of the chemical reagents or methods employed in the pretreatment is also a key factor to be taken into account.Pretreatment methods are generally categorized as physical, chemical, or physicochemical.

| Traditional physical methods
Physical pretreatment methods are among the most straightforward and effective ways to break down the structure of straw material.Commonly used physical pretreatment techniques and tools include milling, extrusion, microwave, and ultrasonic methods.During mechanical milling, lignocellulose is disintegrated into fragments with an average size of 0.2 mm, helping to decrease the degree of crystallinity while improving the accessibility for subsequent microbial or enzymatic reactions (Sitotaw et al., 2021).Extrusion, also known as mechanical grinding, another straw pretreatment method similar to mechanical milling, destroys the internal structure of LCB by mechanical force (Wang et al., 2020).
Microwave pretreatment is rapid and shortens the reaction time due to the dual effects of temperature and molecular rotation.This method is suitable for increasing the surface area and improving the hydrolysis efficiency (Huang et al., 2016;Khan et al., 2021).The increase in temperature and pressure caused by the thermal effect disrupts lignin and cell wall components.Conversely, thermal and non-thermal forces are generated through the relaxation and polarization of dielectric substances under the influence of an electromagnetic field.The rearrangement of polar molecules results in the disruption of hydrogen bonds, decreasing cellulose crystallinity, thus promoting hydrolysis (Kumar et al., 2020).It is worth mentioning that the use of microwave heating presents numerous advantages in pretreatment, including short process duration, prompt and homogeneous heating, low activation energy of reactions, high yield of products, limited formation of by-products, cost-effectiveness, environmental friendliness, and energy efficiency (Li et al., 2016).
Ultrasound pretreatment utilizes the phenomenon of cavitation, whereby countless microscopic vapor bubbles are generated.The rapid implosion of these bubbles leads to the generation of extremely high temperatures (1700-4700°C) and pressures (1800 bar) within microseconds.This process generates shockwaves that exert mechanical forces capable of breaking larger molecules into smaller fragments, resulting in the reduction of particle size (Li et al., 2018).High-power ultrasound treatment, when applied for a longer duration, can reduce the degree of polymerization and crystallinity of cellulose while increasing the pore volume (Song et al., 2013;Wang et al., 2008).In one study, sequential multimodal ultrasound and microwave pretreatments with a natural ternary deep eutectic solvent (NATDES) were used to deconstruct corn straw, resulting in delignification rates of 37.86% and 52.36% (Yan et al., 2021).
Physical pretreatment methods are simple, easy to implement, and environmentally friendly.However, they require large-scale equipment in specialized industrial sites, which increases costs and energy consumption.Moreover, physical pretreatment methods alone have limitations in degrading LCB and are often used in conjunction with other pretreatment methods in actual production processes.

| Chemical methods
Chemical pretreatment is one of the most extensively studied approaches, with better degradation effects and higher efficiency compared to physical pretreatment methods (Abraham et al., 2020).Dilute acid (DA) pretreatment as a simple and feasible method that is widely used in the industrial pretreatment of biomass.Generally, higher concentrations (10%) of acid are more effective in treating straw, although high concentrations can severely corrode the equipment used (Xu et al., 2023).Consequently, a DA with a concentration of less than 5% is often used in combination with high temperature and pressure conditions to achieve effective straw treatment.DA pretreatment has a significant degradation effect on hemicellulose.After DA pretreatment, corn straw was degraded using cellulase, resulting in xylose yields of 70% and 77% (Schell et al., 2003).In the pretreatment of corn cobs with 1.75% (w/w) dilute phosphoric acid, optimal treatment conditions were found to encompass a temperature of 140°C, a duration of 10 min, and a liquid-to-solid ratio of 15:1.Under these conditions, the highest glucose titer reached 11.46 g/L (Satimanont et al., 2012).However, it is important to note that the degradation of carbohydrates by acid pretreatment produces biological inhibitors such as organic acids, phenols and aromatic aldehydes (Klinke et al., 2004;Zabed et al., 2023).This can lead to loss of polysaccharides, resulting in high detoxification costs in performing downstream microbial single-cell protein production.
In comparison to DA pretreatment, alkali pretreatment is better suited for the degradation of lignin in straw materials.For instance, pretreatment of sugarcane tops with a 4% sodium hydroxide solution can reduce the lignin content by 90%.The resulting changes of physical and chemical structure can increase the yield of reducing sugars by up to seven times during enzymatic saccharification (Sindhu et al., 2014).The combination of urea and sodium hydroxide has also been shown to be effective in degrading corn stalk lignocellulose.The glucose yield obtained using NaOH/urea (6 and 12 wt%, respectively) at 80°C for 20 min was approximately 0.54 g/g corn stover (Shao et al., 2020).Alkaline treatment is effective in degrading lignin in SBB and to some extent hemicellulose as well as cellulose.Alkaline conditions are usually provided by chemicals such as lime, sulfite, ammonia, and sodium hydroxide.The most frequently used alkaline chemical is sodium hydroxide, which effectively degrades lignocellulosic materials to produce fermentable sugars (Akus-Szylberg et al., 2021;Shao et al., 2020).However, similar to other pretreatment methods, the production of biological inhibitors and high costs of post-treatment must be considered in the largescale application of alkali pretreatment.Pretreatment of SBB with inorganic salts, such as FeCl 3 and CuCl 2 , has been shown to improve the hydrolysis rate and yield of cellulose or hemicellulose (Loow et al., 2015).Among all the inorganic salts, FeCl 3 has proven to be the most effective inorganic salt for SBB pretreatment, serving as an alternative to acid.When corn straw was pretreated by FeCl 3 at 160°C, the yield of enzymatic hydrolysis increased from 22.8% to 98%.This process effectively removed almost all hemicellulose and disrupted ether bonds as well as some ester bonds between lignin and carbohydrates, without affecting delignification (Liu et al., 2009).It is worth noting that the inorganic salt treatment of SBB may result in the production of a small amounts of fermentation inhibitors due to pentose dehydration.For example, when organic solvents were used with FeCl 3 , lower concentrations of furfural (0.011 g/L) and HMF (0.148 g/L) were produced (Kim et al., 2010).Thus, inorganic salt pretreatment produces significantly less inhibitors than acid pretreatment.

| Physicochemical methods
Hydrothermal pretreatment refers to the use of highpressure hot water for 30 min or longer treatment.Compared with other methods, hydrothermal pretreatment has lower cost and less impact on the environment (Ma et al., 2014).The effect of hydrothermal treatment mainly lies in the solubilization of hemicellulose, and the key to this process lies in the control of temperature and time, as excessive treatment may lead to the degradation of products such as glucose and sucrose (Santo et al., 2018).Steam explosion is a special method of hydrothermal pretreatment, which is widely used in industrial production.It dissolves hemicellulose and alters the cell wall structure of SBB to make it more accessible (Singh et al., 2015).This process involves pressurizing SBB in steam for a certain period of time and then rapidly decompressing it, resulting in an explosive reaction that breaks down the lignocellulosic structure.The high temperature and pressure (180-240°C) disassemble the 3D lignin fraction and catalyze the depolymerization of cellulose and lignin by decomposing some hemicelluloses into glyoxalate and acetate (Singhvi et al., 2014).After the corn straw was treated at 2.5Mpa for 200 s, the contents of cellulose, hemicellulose, and lignin decreased by 8.47%, 50.45% and 36.65%,respectively (Chang et al., 2012).However, during steam explosion, some hemicellulose is hydrolyzed, resulting in dry matter loss and the production of organic acid inhibitors, such as glucuronic acid (Zhang et al., 2022).The lower energy cost compared to traditional physical methods and the reduced recovery cost compared to traditional chemical methods due to the lack of chemical reagents added to the process.This makes steam explosion an ideal pretreatment process for large-scale industrial production.
To avoid higher temperatures and inhibitors during steam explosion and improve enzymatic hydrolysis, the ammonia fiber explosion (AFEX) method was developed.AFEX pretreatment includes the treatment of SBB with anhydrous liquid ammonia (1:1 w/w) at moderate reaction temperatures (60-170°C) and high pressures (15-30 bar) for short periods (5-60 min; Uppugundla et al., 2014).The AFEX method has several benefits, including cellulose de-crystallization, increased surface area, and partial degradation of hemicellulose as well as lignin.This is reflected in an increase of cell wall porosity in corn straw treated by AFEX, particularly in the compound middle lamella and the S1 secondary walls, as visualized by transmission electron microscopy (Chundawat et al., 2011).Additionally, the residual nitrogenous compounds present in the NH 3 -treated substrate serve as an important source of nitrogen for subsequent microbial fermentation processes.This process makes it possible to reduce or even eliminate the need to supplement additional nitrogen sources during subsequent microbial fermentation (Chundawat et al., 2010).

DERIVED FROM SBB
The pretreated SBB can undergo enzymatic and microbial biomass conversion, leading to the breakdown of its biomass into various pentose and hexose sugars, such as glucose, galactose, xylose, and arabinose (Dutta et al., 2023).Biotransformation using sugars produced via the enzymatic digestion of SBB has gained significant attention in recent years, particularly in the production of SCP, bioenergy, and other products.
Bioethanol production from agricultural biomass has emerged as a promising option with the potential to stimulate economic growth and contribute to environmental improvement, given its relatively low greenhouse gas emissions during combustion (Chandel et al., 2007;Sun & Cheng, 2002).However, the high cost still limits the application of cellulosic ethanol.In terms of energy equivalents, the cost of cellulosic ethanol is estimated to be two to three times that of petroleum fuel (Mathew et al., 2016).The pretreatment of SBB, detoxification of the enzymatic hydrolysate, and nutritional supplements are all high-cost components in the process.On the other hand, the biobutanol industry has the potential to generate various valueadded by-products, including fibers, solvents, coatings, and plastics, among others.These diverse product offerings have the capacity to contribute to economic growth in the industry (Bellido et al., 2014).However, the toxicity of butanol to downstream microorganisms remains a significant problem, while inefficient butanol recovery significantly increases production costs.Additionally, straw biomass can be specifically treated and transformed to produce a variety of organic chemicals, which have a wide range of applications (Liao et al., 2020).The demand for lactic acid has grown significantly in recent years due to the potential widespread use of polylactic acid (PLA) as a biodegradable plastic (Lu et al., 2010).Lactic acid contains a chiral carbon atom, resulting in either D-PLA or L-PLA, with the former offering better material performance than L-PLA but at a higher production cost.Lactic acid is produced using fungal and bacterial strains such as Rhizobium and Lactobacillus strains.Bagasse, wheat straw, and peanut meal can be used as fermentation substrates (Assavasirijinda et al., 2016;Cizeikiene et al., 2018).Lactic acid production depends on aeration, inoculum, moisture content, pH and type of fermentation.
Microorganisms can be used to produce high-quality SCP from SBB under simple culture conditions.This process also produces functional substances such as flavors, enzymes and fats, solving the problem of protein feed shortage while efficiently utilizing straw biomass (Suman et al., 2015).SCP has been commercialized in the food industry since the 1980s and began to be sold in the UK in 1985.Now it is sold under the brand name Quorn™ (Monde Nissin Corporation, Philippines) in most countries, mainly in the form of burgers, slices, and nuggets (Finnigan et al., 2017).Quorn™ is made starting from a culture of the aerobic ascomycete Fusarium venenatum.Although SCP has been successfully commercialized in the UK for decades, many researchers are still investigating optimal fermentation conditions, various potential substrates and a wide range of microbial species due to worldwide protein scarcity.To evaluate the nutritional value of SCP, several aspects should be taken into consideration, including the composition of nutrients, amino acid profile, vitamin content, nucleic acid content, as well as potential allergies and gastrointestinal effects (Bajpai, 2017a).In addition, conducting long-term feeding trials is necessary to assess any potential toxicological and carcinogenic effects.

| Microorganisms producing single-cell proteins
The current utilization of microorganisms for SCP production is limited, with more microbes approved for use in animal feed than for human consumption.SCP animal feeds using bacteria, yeast, algae, and fungi are being developed and produced around the world (Voutilainen et al., 2021).The selection of strains for fermenting SBB to produce SCP is crucial as it directly impacts the quality and quantity of the products.Several key criteria should be considered when selecting a suitable strain for SCP production.First, the microorganisms should be easy to cultivate and thrive under industrial fermentation conditions, enabling successful utilization of straw biomass for SCP production.Second, they should exhibit resistance to changes in environmental conditions during the fermentation process to ensure stable SCP production throughout.Third, the chosen microorganisms should possess a high protein content (45%-85%) and have a short life cycle, allowing for the accumulation of SCP in large quantities within a short period of time.Fourth, the microorganisms should be amenable to genetic modification, enabling easy adaptation to genetic and metabolic engineering techniques.
Additionally, the central metabolism of the microorganisms should remain stable, maintaining optimal biochemical and physiological properties during fermentation.Most importantly, the metabolite species of the microorganisms must comply with relevant indicators, avoiding the production of harmful by-products and ensuring that the components of the final product are directly usable by humans or animals.Table 2 lists some of the strains that have been developed for SCP production, including bacteria, yeasts, filamentous fungi and co-culture (Figure 1).

| Bacteria
Bacteria possess several advantageous characteristics for SCP production, including fast growth rates and short fermentation times.Bacterial populations can often double in as little as 2 hours, providing them with a significant advantage as SCP-producing strains.Numerous bacterial species have been harnessed for the degradation of LCB, including Bacillus sp.(Wongputtisin et al., 2014), Cellulomonas sp.(Sun & Cheng, 2002), Clostridium sp.(Lin et al., 2011), Rhodococcus jostii (Jiao et al., 2015), and Sphingobium sp.(Khan et al., 2021).However, it should be realized that bacteria cannot degrade lignin as effectively as fungi, although bacteria are able to quickly decompose cellulose and hemicellulose in straw (Kato et al., 2004).Bacterial SCP with high protein content (50%-80%) is widely produced, but it is worth noting that bacteria have equally high nucleic acid content, which makes them unsuitable for animal feed or human food (Anupama & Ravindra, 2000).This means that bacterial SCP must be properly pre-treated to remove excess nucleic acid before it becomes a commodity.Bacteria can use a wide range of carbon sources, from conventional sugars and starches to petrochemical products such as methanol and ethanol.Therefore, the substrates for bacterial SCP production are usually industrial as well as agricultural wastewater, most commonly used to ferment Cellulomonas and Alcaligenes (Bhalla et al., 2007).Bacteria have the highest nucleic acid content of any microorganism.Moreover, the high rate of RNA breakdown produces purine compounds that increase plasma levels of uric acid, leading to gout and kidney stones (Sadler, 1990).SCP with a high nucleic acid content is only recommended for feeding short-lived animals.

| Yeasts
Yeasts are probably the most widely accepted and used microorganism for SCP production.Among them, the most popular species are Candida (Rajoka et al., 2006), Pichia (Rachamontree et al., 2015), Kluyveromyces (Yadav et al., 2014), and Saccharomyces (Hezarjaribi et al., 2016).During World War I, the Germans added a strain of Candida to their sausages and soups as a protein supplement.Currently, the yeasts are the most widely studied of all microorganisms for the production of SCP (Pessoa et al., 1996).Yeasts generally possesses a higher protein content than bacteria with a superior amino acid profile.Moreover, their lower nucleic acid content makes them more acceptable as a source of microbial protein (Obaeda, 2021).Candida is a single-celled microorganism with great potential, and this microbial biomass is rich in essential amino acids, especially lysine.Candida cells have the ability to collect and absorb more elements from the medium than necessary, forming biologically valuable conjugates such as selenium and magnesium complexes (Kieliszek et al., 2017).This characteristic allows for the customization of Candida biomass to produce SCP feed with a rich nutritional composition.Candida intermedia can use hexoses, pentoses and cellobiose to accumulate biomass.When Miscanthus straw and corn pulp were used as carbon and nitrogen sources to produce SCP, the productivity reached 0.23 g/L/h, while xylose was converted into xylitol (Wu et al., 2018).However, many yeast-based protein supplements are deficient in sulfated amino acids, particularly methionine, which restricts their extensive use as the sole source of protein.

| Fungi
Fungi have been widely used in SCP production, with Fusarium being commercially available as the most common SCP product.Fungal SCPs typically contain 30%-45% protein, which is usually high in threonine and lysine, but often low in methionine (Nasseri et al., 2011).Another SCP product has been produced by the PEKILO process from the filamentous fungus P. variotii grown on sugars in sulfite waste liquor or wood hydrolysates.The product is mainly used in animal feed, but a series of studies have assessed its potential as a food additive (Koivurinta et al., 1979).As the natural lignin degraders, white rot fungi are used in the microbial degradation of SBB due to their excellent ligninolytic ability.The finding that the white-rot fungus Phanerochaete chrysosporium could efficiently bridge the lignin barrier has attracted particular attention (Li, He, et al., 2020;Li, Wang, et al., 2020).In addition, Cyathus stercoreus and Pycnoporus sanguineus also offered high sugar yields in the microbial degradation of corn stover (Saha et al., 2016).Gloephyllum trabeum, a brown rot fungus, has the ability to reduce the hemicellulose content and crystallinity of lignocellulose.It is considered a potential biological pretreatment agent for  (Gao et al., 2012;Monrroy et al., 2011).However, the long growth and degradation cycles hinder their use in SCP production, as fermentations lasting weeks or even months are not conducive to SCP production.Shorter degradation cycles or synergy with other strains is something that should be considered when using white-rot fungi.Cellulase is the essential complex enzyme that directly determines the efficiency of lignocellulosic sugar conversion of straw biomass.Trichoderma viride is recognized as a high cellulase producing strain, but Chaetomium cellulolyticum also breaks down cellulose while growing much faster and producing 80% more protein than Trichoderma (Bhalla et al., 2007).Thus, C. cellulolyticum is more suitable for SCP production while Trichoderma is more suitable for extracellular cellulase production.Aspergillus niger is also frequently used for SCP production, whereby applying corn stover as the substrate can increase the cellulase production of A. niger (Ghori et al., 2011).Paynor et al. utilized a variety of bamboo endophytic fungi for the production of SCP using corn cob as substrate.Among them, Cladosporium cladosporioides and Fusarium sp. 2 achieved the highest crude protein production of 13.64 and 11.54%, respectively (Paynor, 2016).However, some fungi may produce mycotoxins with allergenic or carcinogenic potential, and their effects on human health may be significant.In particular, about 50 species of Fusarium are producers of mycotoxins (fumonisins), which may cause damage to the central nervous system (Alamgir, 2018).To avoid this hazard, adequate toxicological evaluation should be carried out before being recommending a strain for SCP production.
3.1.4| Co-cultures   In nature, various species of microorganisms coexist and interact with each other, many of which are most effective when they are in association with other species.This has led to an increased interest in using co-cultures for the production of SCP (Shi et al., 2020).It is widely recognized in the scientific community that the efficient utilization of lignocellulosic raw materials requires a synergistic combination of enzymes.In this regard, it has been observed that the metabolic byproducts of one species can serve as substrates for another species, or these species may exhibit complementary catabolism by acting on different substrates (Nyyssölä et al., 2022).For example, in a study on SCP production from rice straw, a mixed fermentation using multiple strains of Neurospora crassa, Candida utilis, and P. chrysosporium resulted in the highest true protein yield of 8.89% (Jia et al., 2019).However, it is important to note that negative interactions can also occur during mixed fermentation, such as the production of toxic metabolites or the creation of an antagonistic The process of producing SCP from straw.which makes process control more complex (Li, Wang, et al., 2020).Nevertheless, the co-culture of different strains offers a broader lignocellulolytic enzyme spectrum and richer nutritional structure, which can make straw-based SCP more competitive relative to traditional protein sources.
3.2 | Fermentation technology of straw-based SCP 3.2.1 | Submerged fermentation Submerged fermentation offers several advantages, including a short fermentation time, high efficiency, suitability for industrial production, and easy control of conditions (Nasseri et al., 2011).In this process, the cellulose and hemicellulose present in the straw, which are difficult to utilize, need to be degraded into simple sugars and separated from the solid substrate to serve as a carbon source during submerged fermentation.The success of SCP production in this process heavily relies on the composition of the culture medium and environmental conditions (Reihani & Khosravi-Darani, 2019).Numerous studies have been conducted to investigate the factors that influence SCP production, and these findings have contributed significantly to our understanding of the process (Gibbs et al., 2000).In the utilization of SBB, additional carbon sources are often not required due to the abundant cellulose and hemicellulose content.Instead, a percentage of additional nitrogen sources for protein synthesis is usually required to maintain microbial growth.The common nitrogen sources used in SCP production include urea, ammonium salts, nitrate, as well as organic nitrogen found in different waste products.According to different substrates and fermentation methods, the selection of an optimal nitrogen source is an indispensable part of production.Ammonium sulfate ([NH 4 ] 2 SO 4 ) is widely used as an inorganic nitrogen source in SCP production processes utilize microorganisms such as Saccharomyces cerevisiae and Candida sp.(Adoki, 2008;Hezarjaribi et al., 2016).However, urea was reported to have the best effect in the fermentation of corncob hydrolysate, while corn pulp resulted in a higher rate of biomass accumulation in the submerged fermentation of Miscanthus straw (Wu et al., 2018).In an optimized experiment using F. venenatum to ferment date palm waste, (NH 4 )H 2 PO 4 was found to yield higher biomass formation and protein production compared to peptone as a nitrogen source (Reihani & Khosravi-Darani, 2018).Corn steep liquor (CSL), a low-cost by-product of the starch industry, is also popular as an organic nitrogen source in SCP production.In the process of using Candida utilis to ferment defatted rice polishings to produce SCP, 5% CSL was found to be the optimal nitrogen source, resulting in the highest specific growth rate coefficient (0.31 h −1 ), cell quality benefit (0.65 g/g), and cell quality productivity (1.24 g/L h; Rajoka et al., 2006).Furthermore, a consortium of Knallgas bacteria, specialized in gaseous nitrogen assimilation, has been cultivated for the production of SCP, offering an alternative strategy for nitrogen utilization in SCP production (Hu et al., 2020).
Temperature is the most crucial factor influencing the yield and productivity in the process of SCP production (Yunus et al., 2015).For fungi such as Candida intermedia or Aspergillus niger, the common optimal fermentation temperature is 30°C, resulting in the highest protein yield (Said et al., 2023;Wu et al., 2018).However, bacteria such as Bacillus subtilis are often used for SCP production at 37°C (Gomashe et al., 2014;Wongputtisin et al., 2014).Because of the growth morphology of filamentous fungi, the use of simpler reactors, such as airlift fermentation, has been widely used in the culture of filamentous fungi as an alternative to traditional stirred-tank reactors (Finnigan et al., 2017).The chemostat principle has been effectively implemented in industrial fermentation, based on the harvesting of the product while continuously adding fresh media to the production process (Cooney, 1986).Implementing this method maximizes the production cycle of SCP and is a cost-effective option.Submerged fermentation utilizes highly precise control of conditions for pure cultures of microorganisms to obtain high purity SCP under optimal conditions.The addition of inducers allows the microbes to produce unique nutrients, which facilitates the duction of tailored feeds with different nutrient profiles.
3.2.2| Solid-state fermentation Solid-state fermentation refers to a fermentation process carried out under the condition of almost no free water, but the substrate must contain enough water to maintain the metabolism and growth of microorganisms (Pandey, 2003).During microbial solid-state fermentation, an enzyme system is produced that can break down plant proteins, into fragments, small peptides, or free amino acids, which are more readily bioavailable than the macromolecular plant proteins they were derived from (Sadh et al., 2018).Microbial solid-state fermentation can degrade antinutrient factors (ANF) and produce some major nutrients in feed, providing probiotics and their metabolites (Kiarie et al., 2011).Microbial solid-state fermentation can significantly improve the nutritional value of feed, resulting in greater palatability and enhancing the immunity of animals.
Because the whole product can often be directly in animal feed, solid-state fermentation is considered to be more suitable for low-tech applications of SCP and does not require additional waste treatment at the end of the process (Abdul Manan & Webb, 2017).A comparison of the characteristics of solid-state fermentation and submerged fermentation is presented in Table 3.Additionally, the fermentation modes used for some SCP products derived from SBB are listed in Table 2.
In solid-state fermentation, water plays a crucial role in substrate utilization by cells as it helps to expand the substrates and facilitate their utilization by microorganisms (Said et al., 2019).However, a high water content (>75%) may affect the mass transfer of oxygen and prevent the growth of aerobic microorganisms due to the decrease of matrix porosity.By contrast, when the moisture is too low, the normal metabolism of microorganisms cannot be maintained, thus slowing down or even stopping growth (Rayhane et al., 2019).Typically, a water content of 60%-70% is the most favorable for microbial growth on a solid substrate, and this water content is closer to the natural level at which the vast majority of aerobic microorganisms can carry out their normal metabolism (Bajpai, 2017b).The efficient degradation and utilization of lignin have been a significant factor limiting the utilization of SBB.Due to the complex molecular structure of lignin, which contains a variety of aromatic structures, it requires the cooperation of a variety of enzymes to achieve optimal degradation (Iimura et al., 2021;Jiménez-Barrera et al., 2018).Due to their unique growing environment, filamentous fungi are abundant in lignocellulose-degrading enzymes, including cellulase, xylanase, laccase, and peroxidase (Chilakamarry et al., 2022).However, a single strain usually cannot express several or all species with high enzyme activity.Several studies have shown that the efficiency of lignin degradation by composite colonies is superior to that of single strains.For example, solid-state fermentation (SSF) of the composite colony using Lenzites betulina and Trametes versicolor increased both laccase and peroxidase activities by 40%, and the lignin degradation under synergistic action (50%) was higher than that of single strains (26.6% and 37.2%; Cui et al., 2021).Moreover, single-cell protein (SCP) produced through solid-state fermentation effectively reduces antinutritional chemicals in the raw material, such as glucan, xylan, phytates, tannins, and polyphenols, due to the complex enzyme system of the microorganisms involved (Du et al., 2022;Shi et al., 2020).
While solid-state fermentation offers extensive technological possibilities based on simple investments, it also presents certain challenges.These include high labor intensity, susceptibility to contamination and impurities, as well as difficulties in accurately determining and automating process parameters.A new type of bioreactor needs to be developed in the future to make the process more controllable while retaining the benefits of solidstate fermentation.

| Nutritional benefits of SCP
As previously mentioned, compared with soy (38.60%), fish (17.80%), meat (21.20%) and whole milk (3.28%), higher production efficiency and lower land occupation make microbial SCP an attractive substitute (Xu et al., 2023).For SCP to be effective, it needs to meet the nutritional requirements of animal feed and potentially even human food, including the desired protein content, a balanced amino acid composition, and protein digestibility (Linder, 2019).In addition to considering its protein content, the nutritional value of SCP is contingent upon its biochemical composition, encompassing amino acids, nucleic acids, minerals, enzymes, and vitamins.Nevertheless, SCP still has considerable advantages in terms of economics and space utilization compared to alternative plant-and animal-based protein sources (Bogdahn, 2015).Unlike conventional protein sources, which can be influenced by weather conditions, SCP The fermentation conditions are easy to control More complex post-processing steps The growth rate of microorganisms is higher under the optimal conditions SBB needs to be saccharified before it can be used as a substrate can maintain consistent yields and quality throughout year, with different SBBs being used as substrates based on the season.The use of different microorganisms and substrates results in varying amino acid ratios in SCP products (Aidoo et al., 2023).Scientists can add more valuable amino acids to proteins through genetic engineering to make them more nutritious than traditional proteins.The most abundant vitamins present in SCP are riboflavin, thiamine, pyridoxine, niacin, folic acid, pantothenic acid, biotin, and cobalamine.Among them, the contents of cobalamine and retinol are particularly high in bacteria and algae (Anupama & Ravindra, 2000).
In SCP, approximately 70%-80% of the total nitrogen is present in the form of amino acids, with the rest in nucleic acids, and this high nucleic acid content of the biomass structure is characteristic of all fast growing organisms (Mondal et al., 2012).Excessive intake of nucleic acids will increase the level of serum uric acid, which can cause of kidney stone formation (Suman et al., 2015).To address this issue, various methods have been proposed to reduce nucleic acid levels in SCP production.For instance, in the production of Quorn™ mycoprotein, the fermentation broth is heated after reaching the desired solids concentration, which halts the growth of the mycelium.This process helps to decrease the amount of RNA in the SCP due to the action of natural nucleases present in the mycelium (Whittaker et al., 2020).It is worth mentioning that the presence of uricase enzyme in most animals mitigates concerns regarding uric acid in feed applications.Furthermore, some recent studies have shown that the addition of exogenous hydrolytic enzymes (e.g., phytase) to feeds can improve the digestion of protein feeds (Rezaei et al., 2007;Yadav & Sah, 2005).In recent years, biotechnology and large-scale fermentation process have made remarkable progress, so that microorganisms can not only produce SCP but also synthesize specific hydrolytic proteases and other nutritional factors.The expression of abundant hydrolytic enzyme systems reduces the content of large proteins in SCP, which can increase digestibility when used as a feed product.

| CONCLUSIONS AND OUTLOOK
Utilizing agricultural biomass for the production of SCP offers several advantages, including improving the nutritional value of non-food biomass, reducing pressure on farmland, and efficiently providing high-quality protein sources.However, there are several challenges that need to be addressed in the current production of SCP from agricultural straw.At the current stage, there are still major issues of small production scale and high production costs.One of the main issues is the small production scale, which limits the availability and accessibility of straw-derived protein.Additionally, the production cost of straw-based SCP is relatively high, making it less economically viable.Furthermore, the nutritional profile of straw-based SCP is often not rich enough, which may affect its overall value as a protein source.
To overcome these challenges, there is an urgent need for the development of equipment and technologies specifically designed for straw-based SCP production.This includes improving the pretreatment and storage processes of raw materials to optimize the production efficiency.In the future, advancements in sensor and machine learning technologies will enable the prediction and real-time monitoring of dynamic changes in the fermentation process of SBB.This development offers a significant opportunity to enhance the production of sustainable single-cell protein (SCP) in an environmentally friendly and precise manner.In addition, future research should also focus on screening and transforming high-performance and highly-robust strains, as well as increasing the richness of metabolites in straw protein.Due to the complexity of SBB, machine learning approach predict and screening tailor-made lignocellulolytic enzyme cocktails and microbial strains based on biomass structure features.This method eliminated the need for expert-level prior knowledge of reaction mechanisms and experimental datasets, making the paring strains-substrate-process interactions more efficient and accessible.
Further, the addition of more mineral nutrients beneficial to humans and animals can enhance the nutritional profile of straw protein.It is foreseeable that the targeted transformation of strains and the development of corresponding fermentation processes will greatly promote the development and application of microbial protein.As research progresses, a wide range of biomass sources (including but not limited to straw-based materials), such as food residues and one-carbon (C1) molecules, can be utilized to produce single-cell protein (SCP) products.These SCP products have the potential to replace conventional feeds like soybean meal, fishmeal, and other mainstream feedstuffs.This sustainable approach contributes to addressing the global food crisis in an environment friendly manner.However, the success and widespread adoption of microbial straw protein in the market will depend on favorable legislation, public acceptance, and competitive costs.Given the current environmental impact of agricultural production, there is promising potential to explore the use of agricultural straw for microbial fermentation as a substitute for protein feed.

AUTHOR CONTRIBUTIONS
Zherui Zhang: Formal analysis; writing -original draft.Xiaoyi Chen: Writing -review and editing.Le Gao: analysis; project administration; writing -original draft; -review and editing.

T A B L E 1
The composition of common straw-based biomass resources.
T A B L E 2 Advantages and disadvantages of different fermentation methods.
T A B L E 3