A team of researchers recently published a paper in the journal Environmental Research that comprehensively reviewed cellulase enzyme-producing marine actinobacteria in the context of bioenergy generation.
Study: A comprehensive review on strategic study of cellulase producing marine actinobacteria for biofuel applications. Image Credit: Mateusz Kropiwnicki/Shutterstock.com
Background
A substantial amount of cellulose produced by plants every year is available as waste biomass. One of the effective ways to dispose of this biomass is to use it for bioenergy generation to adequately meet growing energy requirements. Microbial cellulase can catalytically convert cellulose biomass into simple sugar units/glucose, the precursor for the production of biofuel.
Marine actinobacteria, one of the common gram-positive bacteria that produces multienzyme cellulase with high pH stability, thermal tolerance, and resistance towards salt concentration and metal ions, can act as a suitable source to obtain highly stable cellulase to metabolize the cellulose.
In this study, researchers comprehensively reviewed different cellulase-producing marine actinobacteria that can facilitate the conversion of lignocellulosic biomass to bioenergy.
Cellulase from Marine Actinobacteria
Actinoalloteichus sp, a strain of marine actinobacteria isolated from marine sediments obtained from Havelock Island, produced cellulase with 873bp molecular size and 14.379 1U/ml enzyme activity. Similarly, Streptomyces albidoflavus (S. albidoflavus) isolated from marine sediments obtained from Nahoon beach produced cellulase with 205 U/g enzyme activity at a pH of 4 and 40 oC optimum temperature.
Streptomycetes sp., Streptomyces sp. NIOTNAVKKMA02, and Nocardiopsis dassonvillei PSY13 isolated from the marine sediments of the south Indian coast, Port Blair bay, and Southern Ocean waters produced cellulase with a high enzyme activity of 8.93 U/ml, 7.75 U/ml, and 6.36 U/mg, respectively.
Cellulase with a molecular size of 110K was produced by Streptomyces actuosus, which was isolated from the gut of Mugil cephalus obtained from the Vellar estuary. Streptomyces variabilis isolated from sediment obtained from the Persian Gulf produced cellulase with a very low enzyme activity of 0.09 1U/ml.
Lignocellulosic Conversion Using Marine Actinobacterial Cellulase
Wheat straw, maize stover, and rice husk are commonly used as cellulose sources for bioethanol production. A recent study on lignocellulosic bioconversion demonstrated the synthesis of 0.78mL g-1 of bioethanol using seagrass cymodocea species as a cellulose biomass source in presence of marine actinobacteria as a cellulase source.
Similarly, the use of pretreated wheat straw and corn cob as a cellulose source resulted in a saccharification yield of 330 mg/g and 320 mg/g, respectively, when magnesium ion was added for the production of cellulase in Streptomyces species-CC48. Additionally, the use of several unexplored lignocellulose biomasses with high cellulose content can provide new sources of cellulose for renewable biofuels.
Strategies to Enhance the Production of Cellulase from Novel Marine Actinobacteria
Genetic Engineering Approach
This approach involves inserting the cellulase gene in a simple model organism to increase cellulase production for a minimal duration. For instance, the endoglucanase 2 overexpression achieved through a genetic engineering approach can considerably enhance the enzyme activity.
Genome Mining Technique
Genome mining is used to screen cellulase from novel actinobacteria and express the mined cellulose in other systems. A genome-based analysis is performed to identify the enzymes and metabolites in the marine-based microorganisms.
Several cellulase genes were identified in the marine Streptomyces xinghaiensis (S. xinghaiensis) using this technique. Moreover, a very high carboxymethyl cellulase (CMCase) activity was observed in S. xinghaiensis compared to the other marine Streptomyces.
Similarly, only seven cellulase coding genes were detected in the model actinobacteria strain of Streptomyces coelicolor, while no such gene was identified in another well-explored strain Streptomyces avermitilis.
Ribosomal and Metabolic Engineering Approaches
Ribosomal engineering tools are typically used to upregulate the cellulase gene by translation to increase the cellulase yield. For instance, a mutation was introduced in the rpsL gene of marine Streptomyces viridochromogenes to produce xylanase that can withstand high temperatures by ribosome engineering.
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Metabolic engineering tools are used to manipulate the metabolic shift and flux to produce the desired quantity of cellulase. For instance, the direct treatment of untreated plant biomass such as switch grass and corn stover by an engineered Thermobifida fusca and bifunctional alcohol dehydrogenase resulted in the highest enzymatic yield due to the metabolic shift of the succinyl-coenzyme A (CoA) pathway to propanoyl-CoA.
Mutagenesis Approach
Mutagenesis is used to achieve overexpression of the cellulase genes through site/chemical directed mutation. For instance, the β-1,4 endoglucanase activities from the Thermotoga maritima TmCel12B were improved using sited directed mutagenesis, with the mutant strains demonstrating an enhanced cellulase activity. Similarly, the directed evolution method was used to increase the exoglycanase and β-glucosidase activities, which resulted in a significant increase in the production of glucose while performing coexpression.
Effect of Solvent, Metal Ions, and Chemical Reagents on the Cellulase
The effect of solvent, chemical reagents, and metal ions directly impacts the agricultural waste to biofuel conversion efficiency. Studies showed an increased cellulase activity when metal ions, such as calcium chloride, mercuric chloride, and cupric chloride, were added to the enzyme, while a moderate activity was observed in the presence of sodium dodecyl sulfate surfactant. Stable cellulase activity was observed in the presence of chemical reagents, such as laundry detergents.
Different Cellulase Screening Methodologies
Qualitative and quantitative analysis of cellulase plays a crucial role in the waste-to-energy conversion process as efficient conversion depends on the quantity and quality of the cellulase. Screening methods such as ammonium bromide flooding, Congo red assay, and filter assay can be used to perform an accurate qualitative analysis of the cellulase.
However, these methods cannot be used for quantitative analysis. Although the DNS assay can be utilized to perform highly accurate qualitative and quantitative analysis of the cellulase, the technique is heat sensitive, which is a major drawback. The Miller method is often preferred as it can perform both qualitative and quantitative analyses and requires a smaller incubation time.
Immobilization Strategy for the Multiple Usage of Enzyme
The extremely high energy demand has necessitated the identification of a simple and economically feasible methodology to convert the lignocellulosic waste to biofuel. Immobilization of cellulase can be considered the most advanced and cost-effective strategy as it relies on trapped actinobacterial cellulase for the large-scale conversion of agricultural biomass into bioethanol. Thus, the immobilization technique promotes the reuse of enzyme and reduces the cost of waste-to-bioethanol conversion.
Crosslinking of enzymes using glutaraldehyde is a commonly used method for the immobilization of cellulase. A 133% increase in the hydrolysis activity was observed when the cellulase was immobilized on the sodium alginate- polyethylene glycol (PEG) blend through crosslinking.
Similarly, a 90% increase in the hydrolysis activity was observed when the cellulase was immobilized on polyaniline/cationic hydrogel. Moreover, 73% hydrolysis activity was retained by the enzyme even after the 9th hydrolysis cycle. Both methods can be used for enzyme hydrolysis.
Metal-organic frameworks (MOFs), such as chitosan@zeolitic imidazolate framework-8 (ZIF-8), which remain extremely stable during different chemical and physical changes, can be potentially used as substrates for cellulase immobilization.
Enzyme Hydrolysis of Cellulose
Cellulose can be utilized as an inexpensive substrate for hydrolysis, while the glucose obtained after the enzyme hydrolysis can be used as a crucial substrate for the production of biofuel. The use of a cheap substrate/cellulose and immobilized enzyme/cellulase can make the hydrolysis process more effective and cost-efficient on an industrial scale.
For instance, 3.48 g/L ethanol was produced when the sugarcane juice substituted medium was hydrolyzed by a cellulase enzyme obtained from the Streptomyces olivaceus. Additionally, cellulase with different specificities can be used in other applications. For instance, cellulase obtained from Streptomycetes sps. displayed metabolic flexibility and was used for biomechanical pumping, while cellulase obtained from S. albidoflavus shows maximum activity during agitation and is used for bioprospecting.
Conclusion and Outlook
To conclude, marine actinobacteria can be used reliably to obtain highly stable cellulase for the production of glucose from cellulose. Immobilization of cellulase through crosslinking with different substrates, such as MOF, can result in the cost-effective production of glucose, which can be used to prepare a high quantity of biofuels inexpensively.
However, the use of marine actinobacteria to produce the enzyme for hydrolysis was not explored extensively, as most of the studies on marine actinobacteria are primarily focused on terrestrial areas. Moreover, the application of actinobacteria to achieve industrial-scale production of enzymes was also not investigated thoroughly. These issues must be addressed to fully realize the potential of cellulase obtained from marine actinobacteria in the biofuel production process.
Source
Selvarajan, E., Govarthan, M., Samuel, M. S. et al. A comprehensive review on strategic study of cellulase producing marine actinobacteria for biofuel applications. Environmental Research 2022. https://doi.org/10.1016/j.envres.2022.114018.
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