Bacillus licheniformis is widely used for γ-PGA production; particularly, the B. licheniformis 9945a (NCIM 2324) strain is well known for γ-PGA production. Multiple studies have optimized γ-PGA production to retrieve maximum yield. Bajaj et al. (2009) adopted solid-state fermentation to enhance B. licheniformis NCIM 2324-based γ-PGA production. They followed the ‘one factor at a time’ method to examine the effects of moisture content, solid substrates, pH, nitrogen and carbon sources, TCA cycle intermediates, and amino acids on γ-PGA production. The optimized media produced a maximum γ-PGA yield of 98.64 mg/g dry solids with solid fermentation. Response surface methodology was further applied to optimize the nutrient concentrations in the medium, which were experimentally tested. A significantly higher γ-PGA yield (26.12 g/L) was noted with the optimized medium [glycerol (62.4 g/L); ammonium sulphate (8.0 g/L); citric acid (15.2 g/L); and L-glutamic acid (20 g/L)] as compared to the basal medium (5.27 g/L). The produced γ-PGA had a molecular mass of 2.16105 Da. Based on these findings, the authors developed a more efficient system and obtained a γ-PGA production of 35.75 g/L using the B. licheniformis NCIM 2324 strain. This is the highest reported production of γ-PGA with a B. licheniformis strain in the submerged fermentation process. This was achieved by supplementing B. licheniformis NCIM 2324 medium with L-glutamine (0.07 g/L) and α-ketoglutaric acid (1.46 g/L), which served as metabolic precursors for γ-PGA production. γ-PGA yield was considerably high (35.75 g/L) in the metabolic precursor-supplemented medium in comparison to the medium without these precursors (26.12 g/L). Thus, precursors facilitated better utilization of L-glutamic acid by the studied strain. Mabrouk et al. (2012) followed the Plackett–Burman design to optimize the medium [glucose (50 g/L); K ₂ HPO₄ (6.4 g/L); NH₄Cl (3 g/L); MgSO₄.7H₂O, (0.8 g/L); yeast extract (2 g/L); NaCl (0.8 g/L); FeSO₄.4H₂O (0.006 g/L); CaCl₂.2H₂O (0.00084 g/L); trace element solution (0.1 mL), and culture volume (25 mL)] and achieved a γ-PGA yield of 28.2 g/L using an exogenous glutamate-independent strain B. licheniformis A13. Medium volume and yeast extract mainly affected the γ-PGA production. The Plackett–Burman experimental design has also been applied to assess the culture requirements of B. licheniformis SAB26 (Soliman et al., 2005). γ-PGA production was evaluated against fifteen variables mainly including (NH₄)₂SO₄, K₂HPO₄, KH₂PO₄, and casein hydrolysate. The use of an L-glutamic acid nitrogen source alleviated the γ-PGA production of B. licheniformis SAB-26 and thus it was classified as a glutamate-independent γ-PGA producing strain. Ogunleye et al. (2015) noted a three times higher γ-PGA yield (33.5 g/L) on an optimized medium as compared to a basal medium.

Bovarnick (1942) discovered that B. subtilis fermentation leads to γ-PGA secretion into the medium. This discovery attracted the researchers to further investigate B. subtilis-based γ-PGA production. B. subtilis strains have been more thoroughly studied for γ-PGA production than B. licheniformis strains. Scoffone et al. (2013) recently evaluated the γ-PGA production by knocking out two γ-PGA-degrading enzyme genes (ggt and pgdS) in B. subtilis 168 (laboratory strain). They studied the effects of double (deletion of both genes) and single (one gene at a time) mutations on γ-PGA yield. Single mutations did not significantly improve the γ-PGA production whereas double mutation led to a twofold increase (40 g/L) in γ-PGA yield compared to the WT strain. However, the weight average molecular mass and number average molecular mass of produced γ-PGA were comparatively lower in double-mutant strains than in single-mutant and WT strains. The highest molecular mass (36,106 Da) was noted in the pgdS mutant strain, which could be attributed to decreased endo-degradation activities. Huang et al. (2011) have reported cost-effective high-yield and large-scale γ-PGA production with B. subtilis ZJU-7 (B. subtilis CGMCC1250). They stated that 30 g/L L-glutamate, 40 g/L yeast extract, 20 g/L initial glucose, and a glucose concentration range of 3–10 g/L significantly enhanced the γ-PGA production (1.4 to 3.2-fold) following a fed-batch approach in comparison to batch fermentation. Overall, γ-PGA concentration and productivity remained at 101.1 g/L and 2.19 g/L, respectively. Jian et al. (2005) optimized the solid-state fermentation of B. subtilis CCTCC202048 for γ-PGA production. They achieved a maximum γ-PGA yield of 83.61 g/L (kg dry solids) with a mixed substrate (11:9 w/w) of soybean cake powder and wheat bran along with glutamate (40.14 kg/L), NH₄NO₃ (20.05 kg/L), citric acid (18.50 kg/L), and mineral salts (FeCl₃.6H₂O, MgSO₄.7H₂O, MnSO₄.H₂O, and CaCl₂.2H₂O). Low production costs and high yield favor solid-state fermentation for large-scale γ-PGA production. Shih et al. (2005) used the B. subtilis C1 strain to synthesize a novel glycerol-γ-PGA derivative in an L-glutamate-lacking medium. B. subtilis C1 depended on glycerol and citric acid for γ-PGA production and the absence of any of these compounds significantly impacted the yield. The conjugate’s molecular mass (16,107 Da) was found to be higher than the super-high-molecular mass of γ-PGA reported by Park et al. (2005), which could be attributed to the glycerol in the medium. The conjugate presented an γ-PGA to glycerol ratio of 10:1 and had a higher D-glutamic acid units (97%) concentration as compared to L-glutamic acid units. Interestingly, the enantiomeric composition of glycerol-γ-PGA conjugate remained unaffected against Mn²⁺. Contrarily, Wu et al. (2006) have established the effects of Mn²⁺ on the enantiomeric and stereochemical composition of B. subtilis NX-2 produced γ-PGA. The D-glutamate proportion was noted to rise (18 to 77%) in response to variations in Mn²⁺ concentrations (0–0.09 g/L). Mn²⁺ affected γ-PGA stereochemical properties by modifying glutamate racemase activity (Ogunleye et al., 2015).

Cao et al. (2013) cloned γ-PGA synthase genes (racE and pgsBCA) from a non-L-glutamate-dependent γ-PGA producer B amyloliquefaciens LL3 and an L-glutamate-dependent γ-PGA producer B. licheniformis NK03 in E. coli JM109 and evaluated γ-PGA yield. pgsC and pgsB genes of both strains shared high similarities of 93.96 and 93.13% whereas racE and pgsA genes presented 84.5 and 78.53% similarities, respectively. The engineered strains (4) yielded γ-PGA in both L-glutamate and glucose media after 24 h of culturing. Irrespective of the harboring vector, PgsBCA of B. amyloliquefaciens LL3 exhibited better catalytic efficiency than B. licheniformis NK-03, and B. amyloliquefaciens LL3-pgsBCA yielded a higher γ-PGA quantity than B. licheniformis NK-03-pgsBCA. B. amyloliquefaciens LL3-derived RacE and PgsBCA and B. licheniformis NK-03-derived RacE and PgsBCA displayed significantly enhanced D-isomer content and productivity of γ-PGA in comparison to B. amyloliquefaciens LL3-derived PgsBCA and B. licheniformis NK-03-derived PgsBCA. It depicts that racE incorporation enhanced the γ-PGA productivity and D-isomer content. B. subtilis and Corynebacterium glutamicum were co-cultured using a mixed carbon source (sucrose and glucose) to avoid the addition of exogenous L-glutamic acid, which reduced the production cost and fermentation time, and γ-PGA had an average molecular mass of 1.246106 Da (Jian et al., 2005).

Bacillus anthracis-based production of pure γ-PGA D-enantiomer has been established (Zwartouw and Smith, 1956). However, the production mechanism of B. anthracis differs from other Bacillus species as its γ-PGA remains bound to peptidoglycan and is not secreted into the medium. Thus, it complicates the purification and recovery process, which involves cell autolysis and autoclaving. Moreover, B. anthracis toxicity also restricts industrial scale γ-PGA production. The anchored γ-PGA is associated with a non-immunogenic B. anthracis capsule, which is linked to the lethal toxin (Ezzell et al., 2009). Therefore, the γ-PGA-anchoring capping gene should be targeted to avoid B. anthracis toxicity (Candela and Fouet, 2006). Bacillus thuringiensis vs. Monterrey strain BGSC 4AJ1 is also known to produce γ-PGA capsules like B. anthracis (Cachat et al., 2008). B. anthracis (Ames) and B. thuringiensis sv. Monterrey strain BGSC 4AJ1 share four alleles (gmk-1, tpi-1, pta-1, and pur-1) while differing in three other alleles (pycA-52, glpF-57, and ilvd-52) by three, two, and two nucleotides, respectively. γ -D-PGA-producing plasmid (pAJ1-1) genes share similarities with B. anthracis. The discovery of γ-PGA capsule in the B. thuringiensis strain indicates its potential pathogenicity under specific conditions. Poli et al. (2015) isolated Bacillus horneckiae strain APA from a shallow hydrothermal vent of Panarea Island, Italy. They characterized it as an extracellular poly-γ-glutamic acid (γ-PGA)-producing strain and studied its immunomodulatory and antiviral effects against the HSV-2 virus (Herpes simplex virus type 2). Tarui et al. (2005) adopted the Agrobacterium infection technique to introduce indispensable pgsBCA complex into tobacco leaves. γ-PGA production was noted only in plant tissue with all three pgsBCA genes, which yielded 600 mg γ-PGA/g leaf material (Ogunleye et al., 2015).

The cell wall of Archaebacterium Natronococcus occultus is known to contain L-glutamate (Niemetz et al., 1997). Natrialba aegyptiaca strain 40 T is extremely halophilic and was the first archaebacterium to produce extracellular poly-γ-D-(glutamate) (PGA) (Hezayen et al., 2000, 2001). Then, archaebacterial strain 56 T was also characterized to produce exopolymer containing PGA (65% w/w), carbohydrates (15% w/w), and unidentified material (20% w/w) (Hezayen et al., 2002).

Spizizen (1958) devised techniques to develop recombinant Bacillus strains for the commercial production of the B. subtilis enzyme. The transformation proved a significant milestone in molecular biology. Engineered plasmids are obtained either from natural plasmids or plasmids of closely related organisms, which are inducted into a host strain through electroporation, competent cell transformation, and protoplast fusion. Plasmids can be inherited, replicated, or transcribed into host strains. Recombination occurs over several generations during the fermentation process of Bacillus species. The deletion of plasmid sequences could lead to problematic replication and segregation steps in modified plasmids. Thus, the development of mutant strains lacking relevant recombination enzymes dramatically enhanced the stability of recombinant strains. Native promoters (α-amylase gene) directing the production of extracellular proteins (20 g/L) are frequently employed in industrial microbiology. Direct DNA insertion into chromosomes by designing constructs flanked with homologous sequences to chromosomal genes produce more stable clones. The knowledge of Bacillus species nucleotide sequence offers the possibility of novel modifications to produce strains with desired traits. Gene cloning via the encoding of novel plasmid enzymes into antibiotic-resistant B. subtilis hosts helps to maintain plasmid. The induction of a dal-bearing plasmid into a dal-mutant is performed into a dal-mutant, which is unable to produce D, L-alanine racemase. D, L-racemase is necessary for the production of the cell’s D-alanine component. The genetic constructs retain selective pressure on plasmid maintenance in these hosts. Chromosomal integration of novel enzyme-expressing genes could also generate high-yielding B. subtilis strains. The amplification of genes could facilitate the development of highly efficient enzyme-producing strains for large-scale fermentation (Gupta et al., 2002).

B. licheniformis strains are crucial for commercial applications as their enzyme-yielding efficiency is higher than B. subtilis. A high enzyme production in these strains needs a combination of high-performance expression systems from traditional high-yielding strains and multiple copies of the target gene. The ideal strains should have a genetically stable and chromosomally integrated gene with higher expression levels and product secretion. B. licheniformis and B. subtilis have successfully fulfilled these criteria (Markcus et al., 2004).

γ-PGA can be a homopolymer (poly -γ-D- glutamic acid, poly-γ-L-glutamic acid) or a heteropolymer (poly-γ-DL-glutamic acid). The growing peptide chain either incorporates L-glutamic acid monomers from the medium or is synthesized by enzymes (2-oxoglutarate aminotransferase or glutamate dehydrogenase) (Ashiuchi et al., 2001). However, racemase activity is necessary for D-glutamic acid synthesis, which catalyzes D-glutamic acid formation from L-glutamic acid and the process is referred to as racemization (Ashiuchi et al., 1998; Luo et al., 2016). B. subtilis contains two glutamate racemase homologous genes (yrpC and racE (glr)) (Ogunleye et al., 2015). These genes do not directly participate in γ-PGA biosynthesis but are essential for the strain’s growth in nutrient-rich (racE) and minimal medium (yrpC) (Wang et al., 2016). Contrarily, glr plays a key role in γ-PGA synthesis and forms D-glutamic acid from L-glutamic acid. The overexpression of the glr gene could enhance the D-glutamate enantiomeric ratio in B. licheniformis (Jiang et al., 2011).

B. anthracis contains plasmid-encoded γ-PGA biosynthesis genes whereas such genes are chromosomally inherited in some Bacillus species (Ashiuchi and Misono, 2002; Ogunleye et al., 2015). The release or anchoring of synthesized γ-PGA depends on the gene function. Cap genes are crucial in B. anthracis for capsule formation where γ-PGA is attached to the cell surface. Contrarily, B. licheniformis or B. subtilis release γ-PGA outside the cells under the influence of pgs genes. B. anthracis cap genes (cap B, C, A, E) are homologous to B. licheniformis and B. subtilis cap genes (pgs B, C, A, E) (Figure 3). The cluster of cap genes plays a key role in γ-PGA production; however, equivalent importance of all the four pgs genes remains questionable. The investigations have concluded more importance of the pgsC and pgsB genes whereas the pgsE gene has been reported as non-essential (Wu et al., 2008; Luo et al., 2016).

Polymerization is an ATP-dependent process where substrate-dependent hydrolysis of ATP leads to the transfer of phosphoryl group to a terminal carboxyl group of elongated γ-PGA. Then, glutamic acid’s amino group conducts a nucleophilic attack on the phosphorylated carboxyl group to form an amide linkage and continues to γ-PGA polymerization at the synthase complex (PgsBCA) active site. PgsC and PgsB collectively form most parts of complex’s catalytic site whereas PgsA contributes to the elongated chain removal from the active site that allows the addition of next monomer and might also participate in γ-PGA transportation. PgsBCA activity is known to depend on Mg²⁺. Shorter phospholipid-containing less compact cell membranes could facilitate extracellular γ-PGA transportation (Ashiuchi et al., 2001; Urushibata et al., 2002; Ashiuchi et al., 2004; Candela and Fouet, 2006; Buescher and Margaritis, 2007; Wang et al., 2017).

Troy (1973) was the first to report a depolymerase enzyme that could break down PGA to glutamic acid monomer in the late stationary phase of Bacillus licheniformis. This discovery urged several researchers to further investigate this hydrolase enzyme (Kunioka and Goto, 1994; Gross, 1998; King et al., 2000). γ-PGA-synthesizing Bacillus strains contain two γ-PGA breaking enzymes (exo- and endo-glutamyl peptidase). Increased depolymerization time results in reduced dispersity as endo-glutamyl peptidase, secreted by B. licheniformis and B. subtilis in the medium, breaking high molecular weight γ-PGA into fragments (1,000 Da to 20 kDa) (Tsao, 2004). B. subtilis is known to contain endo-glutamyl peptidase encoding genes, which have the same orientation as the pgsBCA operon. Their protein contains a cleavage site (30A-E-A32) proximal to the N- and a hydrophobic cluster (10F-L-L-V-A-V-I-I-CF-L-V-P-I-M24) (Sebastián et al., 2013).

Some Bacillus species secrete peptidase enzymes (GGT (gamma-glutamyltranspeptidases)) under stress conditions or starvation for γ-PGA hydrolysis and utilize released glutamic acid as a nitrogen and carbon source. Gamma-glutamyl-hydrolase encoding pgdS gene is present downstream of the pgsBCA operon, which could degrade γ-PGA into two glutamate residues. The capD gene belongs to the family GGT, which performs dual functions such as γ-PGA anchorage and depolymerization via cleavage and relocates it to water or an acceptor molecule leading to hydrolysis or transpeptidation. Several studies have reported γ-PGA depolymerase presence and activity in B. subtilis NX-2, which carries out γ-PGA depolymerization in the batch culture (Ashiuchi et al., 2003b; Kimura et al., 2004; Candela and Fouet, 2006; Wu et al., 2008; Yao et al., 2009; Morelli et al., 2014; Ogunleye et al., 2015). The extracellular activity of this endo-hydrolase has been reported in the culture, which is encoded by the gene ywtD (pgsS). During a study, the ywtD gene was cloned and expressed in E. coli to obtain purified YwtD protein. The enzyme remained active within a pH range of 5–8 and a temperature range of 30–40°C. A reduced γ-PGA molecular mass (1,000 to 20 kDa) was noted at optimal temperature (30°C) and pH (5) and dispersity was alleviated in response to depolymerization time. Extracellular enzyme activity was also observed during the late stationary phase. The process presented a mild technique for controlled molecular mass reduction, which could be employed as a better alternative to chemical and physical degradation methods (Ogunleye et al., 2015).

The ComP–ComA signal transduction system is known to regulate γ-PGA yield in B. subtilis (natto) (Tran et al., 2000). Stanley and Lazazzera (2005) have reported an additional two-part system (DegS–DegU, DegQ, and SwrA) as unusual γ-PGA synthesis regulators. The transcriptional effects of ComPA, DegSU, and DegQ occur in response to phase variation signals, quorum sensing, and osmolarity, whereas SwrA activity is considered post-transcriptional. Multiple studies have investigated the DegU and SwrA relationship and revealed that phosphorylated DegU (DegU-P) and SwrA are necessary to completely activate the pgs operon leading to γ-PGA production. The effect of genes on γ-PGA production and pgs transcription remains negligible (Stanley and Lazazzera, 2005; Osera et al., 2009). Contrarily, Ohsawa et al. (2009) demonstrated direct activation of pgs expression at a high DegU-P level rather than SwrA and high degQ levels. However, SwrA was still crucial for γ-PGA synthesis under certain conditions. Do et al. (2011) knocked out the degQ gene in B. subtilis (natto) to identify its role in γ-PGA production. They further isolated suppressor mutants, which could produce γ-PGA in the absence of the degQ gene. The results were compared with a domestic B. subtilis 168 strain, which cannot produce γ-PGA due to lower biosynthetic pgs transcription though is genetically more competent than the wild B. subtilis (natto) strain. degQ alteration could significantly restrict the γ-PGA production in B. subtilis (natto) and downregulate the synthesis of degradation enzymes (Stanley and Lazazzera, 2005; Osera et al., 2009; Do et al., 2011). Thus, the degQ gene of B. subtilis (natto) is necessary for γ-PGA production (Do et al., 2011).

Media components and culture conditions are important factors for microbial γ-PGA production as they can impact its characteristics and yield. Nutritional requirements of γ-PGA production vary with L-glutamic acid-independent and -dependent Bacillus strains (Kunioka and Goto, 1994; Gross, 1998). Leonard et al. (1958) formulated the Medium E that has been extensively used to culture bacteria (Gross, 1998). Medium E contains high carbon content and mainly comprises L-glutamic acid (20 g/L), glycerol (80 g/L), citric acid (12 g/L), NH₄Cl, 7 0.0; K₂HP0₄ (0.5 g/L), MgS0₄.7H₂0 (0.5 g/L), CaCI₂.2H₂0 (0.15 g/L), FeCI₃.6H₂0 (0.04 g/L), and MnS0₄.H₂0 (0.104 g/L). Different media have been explored for enhanced γ-PGA production. L-glutamic acid-producing strains can even produce γ-PGA in L-glutamic acid-lacking media. Different types of fermentation media (wastes and synthetic) have been tested for γ-PGA production (Ju et al., 2014). Optimized medium composition efficiently promotes cell growth and facilitates high accumulation of precursors for γ-PGA production (Gao C. et al., 2016). Engineered strains that can metabolize cheaper substrates offer a cost-effective option for γ-PGA synthesis. Carbon substrates pose direct or indirect impacts on synthetic enzyme activity and γ-PGA production. Glutamic acid, sucrose, glucose, glycerol, and citric acid are common carbon sources with varying metabolic pathways, which influence Bacillus species-based γ-PGA production (Moraes et al., 2012; Tork et al., 2015). Carbon source selection mainly depends on bacterial strain for better PGA production.

L-glutamic acid performs a diverse role in PGA-producing strains. Strain and medium components determine the required L-glutamic acid amount for PGA production. Significant interactions of L-glutamic acid with medium components require its optimization, which can directly impact PGA production cost. The L-glutamic acid to PGA conversion rate determines its effective concentration in the medium. Most studies have recommended an L-glutamic acid concentration of 20–30 g/L for PGA production (Cromwick et al., 1996; Du et al., 2005; Kongklom et al., 2015).

PGA production is supported by glycerol and glucose in most glutamic acid-independent strains. Ko and Gross (1998) reported that B. licheniformis ATCC 9945A converts glucose to acetyl-CoA and TCA cycle intermediates which form L-glutamic acid to synthesize PGA. Troy (1973) stated glycerol-based stimulation of polyglutamyl synthetase, which catalyzes glutamic acid polymerization to PGA. Wu et al. (2010a) revealed that glycerol not only stimulates PGA production but also reduces its molecular weight in B. subtilis NX-2 culture.

Glycerol improves cell permeability more than glucose to facilitate the production and release of γ-PGA (Shih et al., 2001; Du et al., 2005). Moreover, the presence of glycerol in the fermentation medium decreases γ-PGA chain length leading to reduced broth viscosity and enhanced substrate uptake for better cell growth and γ-PGA synthesis. Glycerol can efficiently regulate the molecular weight of γ-PGA (Wu et al., 2010).

Citric acid serves as an effective precursor for PGA production (Jain et al., 2005; Du et al., 2005). Kongklom et al. (2015) have reported the promoter activity of glutamic acid and citric acid for the production of γ-PGA. Goto and Kunioka (1992) revealed that B. subtilis IFO 3335-based production strongly depends on the citric acid presence in the fermentation medium. During a study, maltose, xylose, and fructose positively affected cell growth but remained unable to impact γ-PGA production. Zhang H. et al. (2012) noted a maximum γ-PGA yield of 28.3 g/L in the presence of citric acid as compared to other organic acids (oxalic acid, succinic acid, and malic acid).

The addition of metabolic precursors as carbon sources for γ-PGA production might achieve higher yields through enhanced enzyme activity (Nair et al., 2023). Bajaj and Singhal (2009) used B. licheniformis NCIM 2324 to screen different TCA cycle intermediates (pyruvic acid, a-ketoglutaric acid, succinic acid, or malic acid) for PGA production and noted a rise in yield (26.12 to 34.98 g/L) after the addition of α-ketoglutaric acid (10 mM) to the medium. Da Silva et al. (2014) reported that α-ketoglutaric acid and L-glutamine added culture medium could enhance the γ-PGA yield of B. subtilis BL53 by 20%. Kunioka (1995) demonstrated a higher PGA yield without using other byproducts in an L-glutamine/citric acid medium. Bajaj and Singhal (2009) investigated the impacts of glutamic acid family amino acids (L-proline, L-glutamine, L-ornithine, and L-arginine) and amino acids associated with the PGA biosynthetic pathway (L-aspartic acid and L-alanine) on PGA production and achieved maximum production with 0.5 mM L-glutamine. PGA yield and molecular weight (570 kDa) were further enhanced with the addition of α-ketoglutaric acid and glutamine combination to the medium (Lim et al., 2012).

Biomass materials or by-products can also be converted into high-value γ-PGA. Agro-industrial wastes (rapeseed meal, cane molasses, soybean residue, corncobs, crude glycerol and its hydrolysate, rice straw, and monosodium glutamate) have been investigated for γ-PGA production (Zhang D. et al., 2012; Tork et al., 2015; Zhang et al., 2019). Moreover, carbonaceous substances could replace common carbon sources including dairy products, algae, animal feathers, and chicken manure (Chen et al., 2005; Scheel et al., 2019). Zhang et al. (2019) noted a high γ-PGA yield (65 g/L) in an optimized culture medium supplemented with chicken manure, glutamic acid extracts, and soybean residue. Nitrogenous compounds can also impact the γ-PGA production. NH₄⁺ is known to form γ-PGA monomers with α-ketoglutarate via transamination reaction. The presence of free amino groups is necessary during the γ-PGA synthetic pathway. Glutamate serves as a common nitrogen source for most B. subtilis species. Other nitrogenous substances can also replace traditional nitrogen sources. Birrer et al. (1994) optimized the culture medium with fishmeal waste as the nitrogen source and achieved a rise in γ-PGA production up to 25 g/L.

Inorganic salts (MnSO₄ and CaCl₂) can significantly affect the stereochemical composition and yield of PGA. CaCl₂ addition to the medium can reduce culture broth’s viscosity and increase extracellular glutamic acid consumption (11.4%) resulting in a higher yield of PGA than controls (Shih and Van, 2001; Huang J. et al., 2011). CaCl₂-based enhanced enzymatic activities of ODHC (2-oxoglutarate dehydrogenase complex), GDH (glutamate dehydrogenase), and ICDH (isocitrate dehydrogenase) have been reported in the PGA biosynthesis pathway, which directed 2-oxoglutarate for higher glutamic acid production (Bajaj and Singhal, 2009; Huang J. et al., 2011). Metal ion cofactors help in carbon source utilization. Mn₂⁺ could considerably improve glutamate racemase activity leading to higher yield (9.25 to 28.42 g/L) of γ-PGA (Wu et al., 2006; Kongklom et al., 2015). Sodium salts in the culture medium can regulate γ-PGA yield and molecular weight in extremophilic bacteria and halotolerant strains (Wei et al., 2010). In addition to phosphorylation-based derivatives of γ-PGA, sulfonation or esterification can be directly achieved by adding phosphoric acid to the growth media. γ-PGA derivatives are more potent than native γ-PGA with diverse applications (Ashiuchi, 2010). Thus, exogenous culture additives are effective in γ-PGA production.

Fermentation conditions can be modified for higher γ-PGA productivity, concentration, and yield (Table 1). Suitable culture conditions (temperature, pH, inoculation amount, and oxygen content) can efficiently enhance γ-PGA yield (Bajaj and Singhal, 2009). pH is an important γ-PGA biosynthesis factor as glutamate-involving reactions exhibit pH sensitivity (Luo et al., 2016). pH control promotes glutamate utilization for better γ-PGA yield. Cromwick et al. (1996) stated the dramatic impact of pH on PGA production and found that pH reduction (7.4 to 6.5) caused a four-fold rise in PGA yield with B. Iicheniformis (Bajaj and Singhal, 2009). Bacillus species are mostly aerobic and mesophilic, which flourish in the optimum temperature range and affect the synthesis of γ-PGA (Zeng et al., 2014). However, thermoacidophiles can survive at high temperatures by maintaining thermal stability. Cromwick et al. (1996) investigated the role of agitation and aeration in carbon usage, PGA production, and biomass production. Higher oxygen supply to B. licheniformis ATCC 9945A could double its dry cell weights. Citric acid and L-glutamic acid depleted more quickly at high aeration and increased PGA yield (23 g/L) compared to the yield (6.3 g/L) at a low oxygen supply. A high aeration rate led to a higher rate of molecular weight reduction with time in comparison to a low aeration rate. This can be associated with depletion of the carbon source and higher activity of PGA depolymerases at a high aeration rate (Birrer et al., 1994).

To obtain high γ-PGA production, the oxygen supply can be maintained by placing culture in the fermenter or adding an oxygen-carrying agent (Da Silva et al., 2014). The inoculation amount depends on culture conditions, culture composition, and bacteria (Meissner et al., 2015). An inappropriate inoculum concentration results in nutrient competition or cell growth inhibition, which ultimately leads to reduced γ-PGA production. Studies have focused on the integration and optimization of culture conditions to reduce production cost and fermentation time for sustainable γ-PGA yield and productivity. Several techniques such as continuous culture, fed-batch, batch, and symbiotic fermentation (co-cultured strains or mixed strains) have been studied for better γ-PGA productivity (Ashiuchi, 2013; Hirasawa and Shimizu, 2016; Jose et al., 2018). The yield of γ-PGA was increased to 2.19 g/L in a fermenter using a fed-batch glucose supply (Huang B. et al., 2011).

Intracellular glutamic acid combined with a membranous mechanism synthesizes extracellular PGA. Therefore, intracellular substrate amount is a crucial factor for γ-PGA biosynthesis, and its higher level could more efficiently produce this polymer (Wu et al., 2008). Cellular stimulation for γ-PGA secretion also alleviates the stress linked to the accumulation of intracellular γ-PGA and substrate leading to enhanced γ-PGA formation. Improved cell membrane permeability increases extracellular substrate consumption and PGA secretion. The addition of glycerol, Dimethyl sulfoxide (DMSO), or Tween 80 addition to the medium enhances extracellular substrates’ uptake and γ-PGA secretion (Du et al., 2005; Wu et al., 2008). During a study, DMSO and Tween 80 supplemented medium improved the extracellular glutamate uptake (3.88 mmol/g DCW/h and 3.06 to 3.92). Wu et al. (2010) have revealed that DMSO and Tween 80 could increase carbon sources’ utilization and affect cell membrane permeability to regulate substrate availability.

Most native γ-PGA producers are associated with Bacillus species; particularly, B. licheniformis and B. subtilis are widely applied in industrial applications (Bajaj et al., 2009). Fermented soybean products and certain environments are the main sources of γ-PGA-producing strains. However, the lower rate of substrate conversion and higher production cost of glutamate-dependent strains emphasize the utility of glutamate-independent strains. Genetic manipulation is an established method for the regulation of γ-PGA synthesis (Cao et al., 2018). Genetic manipulation facilitates the development of genetically engineered strains containing construction plasmid and knockout genes. Genetic manipulation in Bacillus species can indirectly or directly inhibit or promote enzyme expressions to influence γ-PGA biosynthesis and breakdown.

Genetic manipulation in the γ-PGA synthesis pathway of native strains is a common technique. Zhang et al. (2015) reported that the individual deletion of gudB (glutamate dehydrogenase) or rocR (transcriptional regulating) genes in B. amyloliquefaciens LL3 efficiently increased γ-PGA yield. The deletion of gudB, fadR, aspB, lysC, pckA, rocG, and proAB genes in B. amyloliquefaciens strain NK-A6 partially blocks the downstream metabolic pathways. B. amyloliquefaciens NK-A7 can be genetically manipulated by inserting a strong PC2up promoter to enhance the NADPH level. B. amyloliquefaciens NK-A11 is genetically manipulated by deleting itu and srf operons. These engineered bacteria have been reported to, respectively, produce 4.84 g/L, 7.53 g/L, and 6.46 g/L γ-PGA, which are higher than original B. amyloliquefaciens LL3 strains (Gao et al., 2019). A gene cluster (ywsC-ywtAB or PgsBCA) integrated with a regulating SwrA gene enhances intracellular SwrA and DegU-P levels and γ-PGA production. Similarly, chromosomes embedded with PgsBCA via a strong promoter can significantly improve γ-PGA yield. The unavailability of external nutrients could lead to γ-PGA hydrolysis into nitrogen or carbon sources by its producing strains (Cao et al., 2018). γ-PGA hydrolase repression through genetic engineering could mitigate the γ-PGA production loss and alter its molecular weight (Scoffone et al., 2013; Mitsunaga et al., 2016). γ-PGA hydrolase expression has been manipulated in various studies by adopting the gene knockout technique. Kimura et al. (2004) have reported higher γ-PGA molecular weight and comparable yield of the ggt-deleted mutant B. subtilis NAFM5 with wild strains. The deletion of the cwlO gene in B. amyloliquefaciens and B. subtilis is known to promote γ-PGA yield with higher molecular weight compared to the wildtype strain, whereas deletion of pgdS gene only caused a minor increase in molecular weight (Mitsui et al., 2011; Zhao et al., 2013). However, a combined deletion of the ggt, pgdS, and cwlO genes in B. amyloliquefaciens and B. subtilis strains significantly increased γ-PGA yield (93%) in comparison to wildtype strains (Ohsawa et al., 2009; Tian et al., 2014). Feng et al. (2015) reported a minor improvement in the B. amyloliquefaciens NK-E10 strain’s γ-PGA production capability after the deletion of the luxS gene. Furthermore, improved carbon flux conversion to γ-PGA production, elimination of undesired precursor or by-products, and inhibition of drain energy pathways can enhance γ-PGA purity and production (Nair et al., 2023). Extra ATP can positively affect carbon metabolism for improved γ-PGA production (Peng et al., 2016). Polysaccharides are major γ-PGA contaminants and by-products, which utilize a considerable amount of metabolic energy and carbon sources (Cao et al., 2018). Genetic mutation can knock out lipopolysaccharide and exopolysaccharide synthesizing genes to achieve pure γ-PGA (Feng et al., 2015; Tian et al., 2019). The removal of lipopolysaccharides from the B. amyloliquefaciens NK-E5 culturing medium increased γ-PGA purity to 95% compared to lipopolysaccharides containing medium (Feng et al., 2015). Acetate and lactate negatively impact the bacterial cell growth. Thus, mutants with knocked-out acetate and lactate synthesis genes yielded pure γ-PGA. However, the elimination of glutamate-consuming pathways remained unable to promote γ-PGA yield because the gene manipulation led to a metabolic imbalance with related cellular pathways (Feng et al., 2015; Cao et al., 2018). The synthesis of energy-consuming substances (antibiotics) coincides with γ-PGA production. The repression of such substances via gene knockout could improve γ-PGA production compared to native strains (Gao et al., 2019).

Heterologous or homologous hosts’ recombinant expression is also an efficient technique for enhancing γ-PGA production. Corynebacterium glutamicum and Escherichia coli-based genetic manipulation is referred to as heterologous expression whereas homologous expression is recombined by Bacillus. The introduction of xylose-induced plasmid pWH1520 with pgsBCA operon into B. subtilis MA41 disrupts the native pgsBCA gene and facilitates the successful expression of γ-PGA synthetase (Ashiuchi et al., 2006). Energy-saving NADPH-GDH pathway-containing plasmid in B. amyloliquefaciens NK-1 could elevate the γ-PGA production by 9%. γ-PGA yield is mainly influenced by genetic expression patterns such as inducible and constitutive expressions (Scoffone et al., 2013; Chen et al., 2018). Inducible expression is capable of rapidly accumulating γ-PGA. During a study, simultaneous cloning of racE of B. amyloliquefaciens LL3 and pgsBCA operon of B. licheniformis NK-03 was carried out into an induced plasmid followed by co-expression in E. coli JM109 for the microbial production of γ-PGA. Jiang et al. (2006) recombined Geobacillus toebii gene’s (D-AAT (D-amino acid aminotransferase)) constitutive promoter (PHCE) and expressed it in E. coli/ pCOpgs for yielding γ-PGA (3.7 g/L) in an optimized medium. Native L-glutamate-yielding C. glutamicum could also serve as a suitable host for γ-PGA’s recombinant production. Cao et al. (2018) revealed that heterologous expression of PgsBCA operon in C. glutamicum yielded 0.7 g/L of γ-PGA even in a glutamate-deficient medium.

One of the major hurdles for scaling up high purity γ-PGA manufacturing is the downstream process. Several downstream procedures are critical for recovering and characterizing γ-PGA produced in the fermentation medium (Nair et al., 2023). Extracellular γ-PGA synthesis in Bacillus species facilitates its recovery and purification. The molecular weight and yield of γ-PGA are key factors, which influence its purification and recovery cost (Markus et al., 1985; Wu et al., 2010). γ-PGA recovery goals mainly include: (i) concentrating the fermentation broth/extract to obtain a stable solid form of microbial product, which is easy to store, handle, transport, and re-dissolve/dilute for specific applications; (ii) purification to mitigate non-polymer solids (salts or cells) and improve its functional performance, taste, color, and odor; (iii) deactivation of contaminating enzymes; and (iv) alteration of chemical properties to amend functional performance, handling properties of solid dried product, dispersion, and solubility rate (Smith and Pace, 1982). PGA is recovered by centrifuging (20,000×g for 15 min) the fermentation broth, which removes cells and simplifies the purification process. The dilution of highly viscose γ-PGA and pH adjustment to 3 helps in reducing viscosity and removing producing strains (Do et al., 2001).

γ-PGA molecular characterization is necessary to understand its properties and potential applicability (Sung et al., 2005b). Gel permeation chromatography (GPC) is performed to measure the polydispersity and molecular weight of PGA. Polydispersity and average molecular weight alter with the PGA-producing strain and culturing conditions. GPC employs various mobile phases for calibration in reference to different molecular mass standards (Birrer et al., 1994). PGA molecular weight depends on bacterial strains (Shih and Van, 2001), medium components (Birrer et al., 1994; Kunioka and Goto, 1994; Wu et al., 2008), and culturing conditions (Cromwick et al., 1996). The molecular complexity and instability of bacterial cultures complicate the synthesis of highly homogenous PGA. However, Bacillus species produce γ-PGA within a molecular mass range of 10⁵ to 10⁶ Da, and it can differ with the application mode (Shih and Van, 2001). The reduction of molecular mass is a key step in γ-PGA synthesis for drug delivery. Alkaline hydrolysis, ultrasonic degradation of medium composition, and enzymatic or microbial degradation techniques are adopted for PGA molecular mass reduction (Shih and Van, 2001). Ultrasonic degradation is the most effective option to reduce the dispersity and molecular mass of biosynthesized PGA without affecting its chemical composition (Graciela et al., 2000). Richard and Margaritis (2006) used the B. subtilis IFO 3335 strain for the in situ γ-PGA depolymerization in cell-free fermentation broth. GPC and intrinsic viscosity correlations were employed, which revealed a molecular mass reduction from ~46,106 to 5.56104 Da in 144 h. Yao et al. (2009) revealed reduced γ-PGA dispersity as a function of hydrolysis time. Enzymatic degradation is a better technique for achieving the required γ-PGA molecular mass in a controlled way.

Amino acids containing only glutamic acid represent γ-PGA purity (Shih and Van, 2001). Thin-layer chromatography (TLC) or amino acid analyzers are used for amino acid analysis. Amino acid analysis is carried out by hydrolyzing purified γ-PGA with HCl (6 M) at 100°C for several hours in an airtight tube followed by the evaporation of HCl. Then, water is used to hydrolyze the final product, and amino acid content is analyzed by TLC (Yokoi et al., 1995). TLC is performed on a cellulose plate with different solvent systems (butanol/acetic acid/water (3:1:1, w/w/w) and 96% ethanol/water (63:37, w/w)), and ninhydrin (0.2% in acetone) is sprayed for amino acid identification (Yokoi et al., 1995; Shih et al., 2001). Purified γ-PGA is analyzed by HPLC (high-performance liquid chromatography) according to the methodology of Aboulmagd et al. (2000). Briefly, γ-PGA is hydrolyzed in HCl (6 N, 1 mg/100 mL) in a closed and evacuated tube at 95°C for 12 h, lyophilized, dissolved in distilled water (equal volume), and subjected to HPLC. Pure amino acids are used to chromatographically calibrate γ-PGA (Goto and Kunioka, 1992; Aboulmagd et al., 2000; Shih et al., 2001).

γ-PGA has a wide range of applications in pharmaceutical manufacturing including delivery systems, tissue engineering, gene carriers, and therapeutic and immunological effects. It can significantly reduce drug toxicity and improve drug efficiency in combination with other matters (Luo et al., 2016; Zhan et al., 2018; Cai et al., 2023). However, molecular weight is a decisive factor for drug-delivery properties such as the controlled and delayed release of drugs (Luo et al., 2016). Γ-PGA has also been applied as a novel biological adhesive that was formed by its chemical cross-linking with other compounds (Lee et al., 2020). Moreover, γ-PGA serves as a good adjuvant by inducing a higher vaccine antigens’ immunogenicity (Nguyen et al., 2019). Γ-PGA is known to protect probiotics and Caco-2 cells against oxidative damage as well (Csikós et al., 2018).

Proper moisturization and nutrition are vital to the health and beauty of human skin and hair. Over-dryness caused by low humidity is often detrimental to the skin and hair conditions. In winter, low temperature and dry air especially cause the dryness of the skin and hair, deteriorating the skin health conditions and even hardening or damaging the epidermis and electrifying the hair. To prevent the dryness of the skin, hair, and nail, cosmetic products such as skin essence, hand and body lotions, bath soaps, skin and body creams, hair gels, hair shampoos and mousse, and many other personal care products often contain certain moisturizers to provide the necessary moisturizing conditions to the skin and hair, and also to protect and beautify the skin and hair (Ho et al., 2005). γ-PGA and its hydrogel are considered as super moisturizer(s) in cosmetic products and personal care products showed excellent water absorption capacity and long-lasting good water retention, form soft tender smooth thin film with a matrix structure for controlled release function for water and other nutrients in the water, improve the elasticity of the skin, reduce wrinkles by anti-radical activity in the aging process, beautify, improve the health conditions of skin by increasing the natural moisturing factors in the stratum corneum, and reduce transepidermal water loss from the epidermis (Ho et al., 2005). γ- PGA hydrogel is a natural polymer hydrogel, with good biocompatibility and minimal cytotoxicity, it also includes a large number of free carboxyl groups in γ-PGA, which helps the hydrogel absorb water. However, when the water content of γ-PGA increases, its mechanical capacity decreases, hence various materials are frequently mixed to produce composite hydrogels to compensate for its inadequacies (Cai et al., 2023). PGA as moisturizer can enhance the effectiveness of personal care products for better exfoliation, skincare, hair care, and wrinkle-removing properties. PGA chemistry allows its homogenous miscibility and stability in the ingredient matrix of facial creams (Ben-Zur and Goldman, 2007). PGA also forms an elastic, smooth, and self-moisturizing soft film on the skin. It acts as an excellent hydrophilic humectant to enhance the synthesis of natural moisturizing factors including urocanic acid, lactic acid, and pyrrolidone carboxylic acid as compared to soluble collagen and hyaluronic acid. γ-PGA also improves skin elasticity better than hyaluronic and collagen and nourishes the skin for a smooth desirable appearance (Ben-Zur and Goldman, 2007). PGA hydrogel microscopy depicts a multi-baglike structure that can absorb moisture 5,000 times more than its weight. Salt content and pH influence the water-retaining capability of PGA hydrogel, which enhances the moisturizing capability of skin or hair formulations through optimum pH adjustments and electrolyte addition. Moisture release is important in facial masks for skin hydration and wrinkle removal (Ben-Zur and Goldman, 2007). γ-PGA has successfully served as hyaluronidase inhibitor’s active ingredient where it inhibited hyaluronidase-based hyaluronic acid degradation to maintain skin’s elasticity (Sung et al., 2005a). The composition was tested on fifty female participants (30–50 years) where it successfully maintained skin elasticity through the inhibition of hyaluronidase activity and alleviated allergenic reactions by restricting inflammatory cells’ permeability (Sung et al., 2005a). Moreover, a safe formulation of γ-PGA and natural extract (green tea or aloe vera) has been investigated as well. The result revealed a rise in pyrrolidonecarboxylic acid on the skin epidermis without affecting the constancy and quantitative balance of this humectant. PGA is also known to improve hair strength to support the bleaching process by promoting moisture-retaining capability and the formation of a protective barrier, which dilutes the chemical interactions of colorings applied with hair protein (Ben-Zur and Goldman, 2007).

Hyaluronic acid (HA) is a natural polymer and one of the components of polysaccharide extracellular matrix (ECM). Although pure HA is rigid and degrades quickly, it has been widely utilized in various ocular surgeries, can hasten wound healing, and has vast prospects in the cosmetics industry. Notably, PGA’s biodegradability, high water-retaining capacity, and ability to create amide bonds make it a valuable component for polyamide-PGA, outperforming hyaluronic acid in terms of improved moisturizing properties (Li et al., 2014; Cai et al., 2023). Bian et al. (2016) reported that γ-PGA can improve the shear resistance of HA. HA/γ-PGA hydrogel has a rich porous structure. Therefore, this hydrogel is a good material to provide a potential therapeutic method for future medical applications. Recently, an injectable hydrogel was developed through the photopolymerization of methacrylate-functionalized HA and γ-PGA. This hydrogel exhibited superior load-bearing capacity and toughness compared to conventional hydrogels (Ma et al., 2018).

Beneficial nutration effects of γ-PGA have been previously reported. In mice, high-fat diets containing 3% γ-PGA significantly increased the serum HDL-cholesterol and significantly decreased the serum triglycerides (TG) compared to levels observed in the mice fed a high-fat control diet. However, the inhibition of intestinal high-fat absorption is one of mechanisms that improved the lipid metabolism in the mice fed with high-fat diet (Lee et al., 2013). Park et al. (2011) studied the effect of diets containing high molecular weight γ-PGA on adiposity and lipid metabolism in rats, suggesting that dietary supplementation with high molecular weight γ-PGA may act as a hypocholesterolemic agent. γ-PGA can serve as a non-toxic, edible, antimicrobial, and strong water-holding antioxidant in food industries. Thus, it has the potential to replace existing functional food supplements. γ-PGA role as a stabilizer and texture enhancer could promote the quality of wheat gluten (WG), starch, and their products (Xie et al., 2020). γ-PGA can stabilize fermented foods (yogurts) by maintaining microorganism viability, texture, food flavor, and aroma during production, transportation, and selling (Yu et al., 2018). It has been shown that γ-PGA can act as a bitterness-masking agent to curb the bitter taste of quinine, amino acids, minerals, peptides, and caffeine. It is also used as a thickener in fruit juice beverages (Feng et al., 2015). The anti-freezing feature of γ-PGA helps its utility as a cryoprotectant to sustain probiotics viability during freeze-drying (Siaterlis et al., 2009). Low molecular weight (<20,000 Da) γ-PGA possesses better anti-freezing properties than glucose and thus more efficiently protects Lactobacillus paracasei compared to sorbitol, sucrose, and trehalose (Bhat et al., 2013; Zhan et al., 2018; Jang et al., 2019). Multiple studies have demonstrated that 10% γ-PGA can protect Lactobacillus paracasei more effectively than 10% sucrose and offers better protection of lactobacilli during freeze-drying in comparison to sorbitol and trehalose (Siaterlis et al., 2009; Lim et al., 2012). A study was performed regarding the γ-PGA effect on doughnut moisture loss and oil absorption during deep-fat frying. A γ-PGA concentration of 1 g (100 g dough)⁻¹ was found to effectively reduce (five-fold) doughnuts’ oil uptake to 0.2 g (g dough)⁻¹ compared to an oil uptake of 0.7 g (g dough)⁻¹ in regular doughnuts. Overall, γ-PGA-added doughnuts had better taste and appearance than regular doughnuts. Therefore, γ-PGA can be used as a potent oil-reducing agent in deep-fat fried foods. The antioxidant mechanism of γ-PGA was also found to shield the gastrointestinal tract against redox imbalance (Lee et al., 2020). Furthermore, γ-PGA enhances Ca²⁺ bioavailability via effective intestinal absorption. A single dose of γ-PGA was noted to enhance intestinal absorption in post-menopausal women, and it particularly benefited individuals with lower basal absorptive capacity (Tanimoto et al., 2007). Hu et al. (2008) studied γ-PGA-based possible induction of protein crystallization in xylanase, lysozyme, and glucose isomerase. They investigated high-molecular-mass (Na + salt:16106 Da) and low molecular-mass (Na + salt: 26105–46,105 Da) γ-PGA and noted γ-PGA-induced protein precipitation. In recent studies, Tamura et al. (2020) reported that γ-PGA could reduce postprandial glucose rise and plasma glucose levels. In an in vivo emperiment, mice were fed with different levels of a γ-PGA-containing diet or control diet for 91 days to suppress the initial rise in blood glucose levels. They found that blood and plasma glucose levels at 15 min were significantly decreased in the PGA group compared to the control group, suggesting that γ-PGA may effectively prevent metabolic syndrome by lowering blood glucose levels.

γ-PGA has promising agricultural uses as a new environmentally benign fertilizer synergist to boost nutrient uptake by plants. However, its agricultural application is hampered by the low yield and high cost of the production compared to conventional materials, because the production of γ-PGA by fermentation often requires glutamate and other high-cost components as substrates. However, some low-cost feedstocks are urgently required to overcome the economic and long-term barriers to biotechnologically producing γ-PGA (Feng et al., 2017). γ-PGA is particularly beneficial for soil fertility due to strong water retention, water solubility, high cation exchange capacity, high biodegradability, and metal-chelating and elements preserving capability to avoid their loss by rinsing with drainage water. The role of γ-PGA as an environment-friendly fertilizer synergist has increased its importance in plant growth-related soil dynamics (Chen et al., 2005). γ-PGA can significantly promote potassium (K), nitrogen (N), and phosphorus (P) uptake to increase plant biomass. It strengthens nutrient uptake by promoting root activity and biomass. γ-PGA is known to affect nitrogen (N) and carbon (C) metabolism in plants, which is represented by reduced free amino acids, nitrates, and high soluble sugar content (Zhang et al., 2017). Factors such as CO₂ emission and carbon (C) and soil nitrogen (N) loss by leaching can demonstrate the potential of γ-PGA in agriculture. Zhang et al. (2016) reported a γ-PGA-based reduction in soil N leaching loss at an application rate of ≥0.2 g kg⁻¹ soil. γ-PGA exerts only minor effects on the leaching of dissolved organic carbon (DOC), but it can significantly enhance the loss of soil CO₂ gas. Yin et al. (2018) have revealed a significant reduction in soil organic carbon (SOC ≈ 0.4 g kg⁻¹) and total nitrogen (TN ≈ 0.025 g kg⁻¹) after the addition of γ-PGA. Contrarily, high γ-PGA concentrations (0.4 and 0.8 g kg⁻¹ soil) increased the soil TN without affecting SOC content (Zhang et al., 2016). Furthermore, γ-PGA could enhance root activity and biomass by improving water-soluble nutrients’ uptake, and exogenous γ-PGA application can protect seedlings against adverse conditions (Wang et al., 2008; Yin et al., 2018). Moreover, γ-PGA has also been applied under biotic or abiotic stress conditions as an antimicrobial and biocontrol agent (Lee et al., 2018). γ-PGA application as a biological chelating agent could effectively alleviate toxic heavy metals accumulation in farmlands and crops (Pang et al., 2018). Currently, agricultural applications of γ-PGA are limited, but its broad range of applications can be predicted in the future for reducing nutrient loss, improving crop production, promoting fertilizer utilization, and sustaining the ecological function of soil (Zhang et al., 2017).

The biodegradable and non-toxic nature of γ-PGA presents it as an eco-friendly wastewater treatment option. γ-PGA (MW ~ 5.8–6.2 × 10⁶ Da) could perform better than most conventional wastewater flocculants in treatment plants, which operate downstream of food fermentation processing units (Inbaraj et al., 2006). Ben-Zur et al. (2007) revealed that γ-PGA (MW ~ 9.9 × 10⁵ Da) efficiently removed basic dyes (98%) from the aqueous solution at pH 1 and remained reusable. Thus, a γ-PGA-based biodegradable and non-toxic adsorption system offers an eco-friendly solution to the dye industry. Chang et al. (2013) investigated the efficacy of γ-PGA added super-paramagnetic iron oxide NPs (γ-PGA/Fe₃O₄ NPs) for the elimination of heavy metal ions (Ni²⁺, Cr³⁺, Pb²⁺, and Cu²⁺). γ-PGA /Fe₃O₄ NPs demonstrated excellent metal removal efficiency as compared to individual treatments of γ-PGA or Fe₃O₄ NPs indicating its higher potential in wastewater treatment. During a study, B. subtilis 2063-produced γ-PGA (5.86106 Da) exhibited significant flocculating activity and thus can replace conventional synthetic flocculants (high environmental persistence) for wastewater treatment (Bhunia et al., 2012). Bajaj and Singhal (2011a) examined the B. subtilis R 23-derived γ-PGA (6.26106 Da) and revealed its excellent flocculation capability in wastewater treatment plants. Bodnár et al. (2008) investigated a novel complex of biodegradable NPs, B. licheniformis 9945a-derived γ-PGA, and bivalent lead ion. The complex remained stable in aqueous media under neutral, mild, and low-alkaline conditions, and exhibited effective water treatment and heavy metal-binding potential. Poly-γ-glutamic acid flocculant (PGAF)-based flocculation–sedimentation treatment could effectively eliminate SP-stabilized cement particles via gravitational settling of the flocs (Yanagibashi et al., 2019). However, higher ionic strength reduces the removal efficiency of γ-PGA, which might be due to its shrinkage and could be overcome by high intensity and rapid mixing. Several flocculation–sedimentation experiments have established the relationship between ionic strength and PGAF amount. The results suggested a higher efficacy of PGAF for cement suspension purification through flocculation–sedimentation, which could be further enhanced with rapid mixing at lower ionic strength (Yanagibashi et al., 2019). There are several other potential applications of γ-PGA, which are continuously expanding.