Terminalia Chebula (kadukkai), Rosa Sinensis (hibiscus), Palm jaggery (refined sugar), Xanthorrhoeaceae , and Indigofera Tinctoria (neelamari) as per the methods given in the ancient palm leaf of Padmanabhapuram Palace, India. Advanced analytical techniques like Gas chromatography-mass spectroscopy (GC–MS), UV-Spectrophotometer and carbon dioxide quantification were used to study the fermented plant extracts and Fourier transform-infrared spectroscopy (FT-IR), X-ray Diffraction (XRD), Field emission-scanning electron microscopy (FESEM) to study hydrated phases and microstructure of organic lime putty. GC–MS recorded the phytochemical compounds like fatty acids, traces of proteins, polysaccharides and carbohydrates. Fermented kadukkai and neelamari extracts reported as fatty acid, palm jaggery as carbohydrate, hibiscus as polysaccharide and aloevera rich in all the biomolecules. The detection limit of Quantification:0.013 and limit of detection:0.067 for polysaccharides, 0.026 and 0.088 for unsaturated fatty acids was reported through a U.V spectrophotometer for all the herbs. Aloevera and neelamari fermented extracts recorded the CO2 release around 96,000 and 90,000 ppm on 4th day of fermentation, whereas for other herbs it ranged below the recorded readings. Supply of CO2 has initiated the internal carbonation of the lime putty and precipitation of calcite in three different forms aragonite, calcite and vaterite minerals. The addition of organics resulted in high-intensity portlandite peaks and calcium carbonate polymorphs as reported in XRD graphs in agreement with FT-IR analysis. FESEM morphology validated the early formation of carbonate polymorphs, and EDX. has shown that kadukkai lime putty, jaggery lime putty and reference lime putty. mixes have calcium around 35–45%. From the overall results, 3% addition of eco-friendly biopolymers has altered the properties like setting time, water repellency and higher carbonation rate, which is the main reason behind longevity of the structure.The study aims at preparation and characterization of six organic lime putty (hydraulic Lime + fermented plant extract) using regionally available plants namely  Moreover, the ancient materials and the protected archives like literary collections, metal carvings, palm leaf manuscripts, wall paintings and stone inscriptions were the earliest historic references symbolizing the regional styles, agama shastras  and Chitra sutra  before the invention of the paper9. The earliest known palm-leaf manuscript belongs to the 2nd Century A.D of the Gupta regime. Writing on palm leaf continued up to 19th Century A.D, archiving the historical significance of civilization. Heritage is any tangible or intangible values passed on to us from the past. This wisdom hauls various traditional significances like economic, cultural, aesthetic, spiritual and archaeological sources that connect with past civilization and carry their advancements to modern times. Likewise, some ancient sources communicate the knowledge, ranging from the caves to copper plates and from the bark of trees to leaves of various kinds12.The ancient manuscripts are the sources of human history available on various media like stone, clay, tablets, palm leaves, metal leaves, barks, animal skin, cloth, paper etc. Despite all the ancient writing materials, palm leaf was used as the predominant media due to the local availability of palm trees across the Indian subcontinent10. Though many modern innovations have emerged in recent times, not all are holistic. Few recent studies underway understand the ancient structures' holistic construction practices through digital preservation, artificial intelligence, and modern analytical techniques. Starting from the Indus valley providing the evidence on the ancient town planning of Harrapan and Mohenjo-Daro depict the building culture, advanced drainage system and road systems. The construction method has transformed since 300 B.C with the whole new level of Dravidian style of gigantic towers. Long pillared halls, artistic vimanas and common structural features such as domes, slender minarets and arches were influenced from the Islamic and the Persian style of architectures. Every structure has its unique significances and architectural point of view. One such fine example of Brihadeshwara temple, Tanjore, India has the longest vimana of height 216 ft with axial and symmetrical geometry rules with fortified granite structure in the world5. The archaeological archive forum across the countries has documented some of the traditional secrets of old construction practices and preservative methods to understand the designs' scientific methods. It is estimated that there are 300 manuscript libraries all over India and 77 libraries outside India on preserving the archives. Some of the major repositories in India are national archives of India (N.A.I.), Indian national trust for art and cultural heritage (I.N.T.A.C.H.), Indira Gandhi national center for the arts (I.G.N.C.A.), Department of culture and its museum and libraries, state archives and state museums. Mostly the national archives reveal the secrets of regimes, culture, medicinal scripts, trades and civilization13. The ancient construction practices prominently utilized the binders such as clay, mud, limestone, volcanic ash and gypsum in the preparation of mortar since the ancient times, following its setting and hardening properties. Archaeological evidences has shown the usage of Lime started before the prehistoric period in Iran and China14. Romans and Egyptians were known for their use of pozzolanic elements, gypsum and quick Lime in early construction, unfortunately the plasters prolonged to set at conventional period of time. Hence the historic engineers and masons experimented with the locally available organic substances like gum Arabic, animal glue, blood of animals, figs, egg whites and yolks, casein, keratin that served as adhesives substances to enhance the weak mortars15. Francesco milizia16 reported on mixture added with oil to enhance the mortars' impermeability used in aqueducts and cisterns. The carbohydrate-based materials such as sugar, molasses, malt, drum stick gum and arabica gum were incorporated during production to quickly react to form calcium saccharate, which improved the cohesion properties and resistance against environmental forces21. The cactus  extracts were commonly added to mortars in ancient Mexican buildings to resist the water penetration and crack formation on the surfaces by improved mechanical properties due to the cactus' polysaccharide mucilage interaction with portlandite at early stages22. Calcium oxalates  are important biominerals found in nature and are the most abundant organic minerals category identified in sediments and hydrothermal veins. The genesis of calcium oxalates has been identified in many limestone structures as layers, and they act as a barrier to reduce the deterioration of stones due to weathering action. The effective role of jaggery (unrefined sugars), kadukkai , kulamavu (Persea Macrantha) in lime mortars have been analyzed, and it has been found that the addition of fermented organics leads to the formation of weddellite in lime mortar, it has dramatically increased physical and mechanical properties23. Protein-lime was mainly used in ancient masonry construction, clay sculptures, mural paintings, and wall paintings; it's been found that the addition of albumen can bring a series of physicochemical property changes stickiness, water repellency, air-entraining and antimicrobial etc.25.The sixth century C.E. sites of Ellora caves, India, reported on earthen/Lime as a binding material in addition to cannabis Sativa (Hemp). Daulatabad fort, India reported on the addition of local vegetal fibres into mortar providing the information about the hempcrete production technology during Yadava regime26. The use of organic compounds dated back to ancient times and has widely spread across the world to enhance the mortar's strength. The mortar produced with lime putty and cheese adhered extremely well to stone blocks. The most commonly used additives were oxblood, animal fat, dairy products, tree barks, linseed oil, fig juices, lard, eggs, wine, beer, casein and cactus juice etc.27. Based on the regional availability a group of herbs rich in carbohydrate, protein and fat were fermented for few days and used for lime mortars production instead of water. During fermentation, the carbon-di-oxide produced will react with Lime for faster hardening and biomineralize calcium carbonate polymorphs28. Much ancient text talks about the use of plant and animal extracts in construction. One such preserved palm-leaf manuscript from the Padmanabhapuram Palace museum , Neelamari (Indigofera Tinctoria), Aloe vera (Xanthorrhoeaceae) and eggs during construction. Recent studies have used natural organic additives to slow down and accelerate the setting time and increase the mortar's workability. Modern analytical techniques like GC–MS were highly recommended to identify organic substances like polysaccharides and fatty acids. The 4th-century Macedonian tombs (Greece) wall paintings revealed the addition of plant gums and their pigments in mural wall paintings, detected by ion chromatography29. The byzantine tombs pointed out egg white usage in the lime mortar to enhance the mortar's binding and flexural strength, investigated through the HPLC technique30. Despite the above-mentioned recent investigations, to our best knowledge, the determination of organic mineral and its effect on Lime was rarely documented, but this study proposes a multi analytical approach to determine the possible bioactive compounds present in the identified herbs as mentioned in the ancient manuscript and to understand its effective role with lime paste through the sophisticated methods like Gas chromatography and mass spectroscopy (GC–MS), X-Ray diffraction, Fourier transform-infrared spectroscopy, U.V. spectrophotometer, FE-SEM and quantitative methods like protein and fatty acid test methods. The characterized results will help Archaeology department, conservation engineers, architects and N.G.O.s working in the field of conservation to understand the traditional production technology and to produce the simulation mortar to restore the historic structures.The palm leaf manuscripts acknowledged the traditional usages of organics like palm jaggery (Refined sugar), Kadukkai .According to the palm leaf inscriptions and a semi-formal survey among the local people, the locally available healthy plant herbs like Kadukkai, Neelamari, Aloe vera, Hibiscus and Palm jaggery, etc. collected for the experiment, as shown in Fig. 2 release from 0 to 14 days of fermented organics was regularly measured at an interval of 24 h using a wireless CO2 sensor operated by PASCO PS-3208. The sensing element, a pyroelectric device with a quartz reflector at one end of the probe and the other end is the I.R. band filter, absorbs the CO2 Gas in the probe. The concentration of CO2 is measured by non-dispersive infrared technology (N.D.E.R.) with an operating temperature of 0-50º C, the relative humidity of 0–95%, and the detection limit of CO2 concentration ranged between 100 and 1,00,000 ppm32.The amount of COFrom supplementary Fig. 35 . The detection (L.O.D.) and the quantification (L.O.Q.) limits of fatty acids, sugars, lipids, tannins, protein (amino acids) and carboxylic acids were calculated at a significance level of 0.05 and used for the quantitative evaluation of each compounds33.All the 5-ethyl acetate extracted organic samples were injected in an Agilent 7890 A GC system coupled with an MS 5975 selective detector (Agilent Technologies) with a single quadrupole mass spectrometer PTV injector at National Institute of Ocean Technology (N.I.O.T), Pallikaranai, Chennai, Tamil Nadu. The GC–MS operating conditions were set with oven temperature at 50 ºC for 2 min, then 100ºC at 10 ºC/min and finally increased to 200 ºC and been held isothermally for 10 min. The sample injected was 2µL, and the gas carrier was helium with a flow rate of 1 mL/min. All five samples are ionized by electron energy 70 eV. The GC–MS's total running time was 63 min to characterize the organic compounds through the N.I.S.T. 14.L library36.Figure 37  as shown in Supplementary Fig. 2 mg of the fermented solution is taken into a calorimetric tube, and 0.05 ml of 80% phenol is added. Further, 5 ml of concentrated sulfuric acid is added rapidly, the stream of acid being directed against the liquid surface rather than against the test tube's side to obtain good mixing. The tubes are allowed to stand for 10 min. Then they are placed in a water bath for 10 to 20 min at 25-30ºC. The absorbance of the characteristic yellow-orange colour is measured at the scanning range of 540 nm for galactose, hexoses and pentoses using ultra violet-spectrophotometer38.0.25 g of each fermented extract is taken in a 50 ml round-necked volumetric flask. Add 10 ml of 0.2 N sodium methylate to methanol placed on an electric heater for 15 min to reach 100 °C. The solutions should be clear, usually lasting 10 min. The reaction is complete after 15 min, further removing the flask and the condenser from heat until the reflux stops and adding 1% of phenolphthalein solution to the methanol. Adding 1 N sulphuric acid solution to methanol until the solution is colourless, and add 1 ml more. Fit the condenser and boil for another 20 min. Remove the heat source and cool the bottle under running water. Remove the condenser, add 20 ml of saturated sodium chloride aqueous solution, stir, add 5 ml of heptane, plug the flask and shake vigorously for 15 s. Allow it to settle until the two phases have been separated. Repeat the saturated sodium chloride solution until the aqueous layer reaches the lower end of the flask neck, as shown in Supplementary Fig. 39. Fourier transform infrared spectroscopy (FT-IR) was performed using the Bruker Tensor 27IR with KBr pellets to measure the samples' energy absorption. 5 mg of finely ground < 75 µm specimen powder was homogenously mixed with 250 mg of KBr powder until the mixture had the consistency of fine flour and then pressed into a thin 15 mm diameter disc with infrared spectra obtained at 4000–400 cm − 1 range, with a resolution of 4 cm−1 32 scans respectively43. The samples' morphology was observed with a Carl Zeiss Supra 55 FE-SEM. The elemental analyses were performed using an X-ray dispersive spectrometer (E-DAX) resolution of 0.8 nm coupled to F.E.S.E.M. Each sample was coated with gold sputter (90 s) in a vacuum evaporating system. The sample was observed at a magnification range of 3300–10,000 × with low vacuum mode at 20 kV .The mineralogical phases were determined by X-Ray diffraction (XRD) using the Rigaku smart lab II diffractometer according to the diffraction powder method operating at 9 kW and 9 mA with oriented sample holders in polymethyl methacrylate or Si of diameters 25 and 20 mm depending on the amount of material (2–5 mg). The XRD patterns were obtained by scanning at the rate of 1º/minute from 5º to 90º (2 h) and steps of 0.05º (2 h)2 levels. The air bubbling in the airlock container will gradually slow down, becoming more and more infrequent and then stopping up altogether44 (Reddy 2018).Fermentation was performed on the herbs to enhance the chemical reactions induced by microorganisms or the enzymes that split the complex compounds into the simplest substances in the absence of oxygen. This splitting of complex compounds is one reason for the carbonation of Lime. The fermentation periods of the organic herbs were fixed according to greater CO2 ranged between 10,000 and 90,000 ppm during the complete fermentation process shown in Fig. 2 of 96,000 and 90,000 ppm on the 4th day of fermentation. Further, it slowed down its activity as the days progressed. Similarly, the palm jaggery and kadukkai have liberated the considerable amount CO2 of 36,500 and 50,000 ppm on the 4th day on signalling the rise of air bubbles from the bottom, indicates the complete ethanolic transformation activity. But the hibiscus has rapidly liberated the CO2 of 32,000 ppm on the 2nd day and moderately slowing down the emission on the 7th day, indicating the hibiscus has pyruvate into complete alcoholic transformation by splitting the complex compounds into sugars such as glucose, fructose and sucrose products. The fermentation measurements suggested, the liberation of CO2 was found high for neelamari and aloe vera, followed by kadukkai and jaggery; hence all these four organic fermented extracts can be incorporated into the lime mortar on the 4th day to activate the internal carbonation and to strengthen the mortar due to the internal reaction between the lime particles and organically induced CO2 to improve the calcite crystals. But the hibiscus herb showed high liberation on the 2nd day and gradually slowed down the activity, indicating it took 2 days to ferment and extended its transformation till 14 days.The amount of liberation of CO32. Liberation of CO2 is very important for setting and hardening of lime mortars in heritage buildings. In general, lime sets by carbonation by absorption of CO2 from the atmosphere to precipitate the calcium carbonate 2 being about 100 times higher than that of all three CaCO3 polymorphs. Therefore, self-healing will preferentially occur in partially carbonated lime mortar, where the uncarbonated part will provide Ca (OH)2 to fill thin cracks, thereby enhancing durability. Furthermore, peaks of all three different polymorphs of CaCO3, namely vaterite, aragonite and calcite, are found in the XRD pattern. This combination influences the dissolution and the subsequent recrystallization process and thereby contributes to the mortars' self-healing nature, making the mortars more durable50.Since the lime mortars, walls are huge and a few meters thick, the outer portion up to few centimeters gets carbonated because of atmospheric carbon dioxide. However, the internal core mortars remain un-carbonated for decades and prolong the lime mortars' hardening and resulting in slow strength gain. This main drawback of slow setting and strength gain is enhanced by fermented extract into Lime instead of water. The internal supply of COThe 3% organic fermented extracts were screened under the GC–MS Agilent 7890A G.C. system with an MS 5975 selective detector; it detected the various bioactive compounds present in the five fermented extracts unsaturated fatty acids polysaccharides, carbohydrates and proteins substrates. The complete phytochemical compounds detected from the GC–MS analysis is shown in Table T. Chebula) resulted in the form of unsaturated fatty acids compounds like citric acid, hexadecenoic acid, trans-octadecanoic acid, 13-octadecanoic acid and 6-octadecenoic acid with the overall unsaturated fatty acid percentage quantified around 54.1%. Neelamari (Indigofera tinctoria) chromatogram fractioned adipic acid, demecolcine, nalmefene, strychnine, cholestane-6-one, aspidodasycarpine, cyano, rhodopin, lycopene, phosphine with 80.78% in 100 mg/ml concentration34 (Nayar 1999). Palm jaggery (refined sugar) depicted the acetamide, lumicolchicine, pentadecanedioic acid, acetic acid, valine (amino acid), cyclodecasiloxane, pentamide and manopyranose content around 92.85%. The high-level acetic acid observed in palm jaggery reacts with the lime particle forming calcium acetate to promote the hydration properties due to the carboxyl groups to enhance the initial strength and internal voids the mortar54. Sucrose is the most common sugar, made up of α-glucose and β-glucose linked together by a glycosidic bond. The hydroxyl groups make the molecule highly water-soluble. As far as the lime traditions are concerned, the use of sugars has been documented in historic lime mortars in various countries where its interaction with C–S–H has been observed by adsorbing the Ca (OH)2 to increase the binding properties of the mortar. The addition of jaggery to Lime increases the solubility of Lime in water by 84%. Hence, instead of water jaggery extract, water can be added during slaking. It helps in more conversion of calcium oxide (Lime) into calcium hydroxide (portlandite). Jaggery contains 60–85% sucrose, 5–15% glucose and fructose, 0.4% protein and 0.6–1% of minerals9. Hibiscus (Rosa Sinensis) contained hexadecenoic acid, 14-octadecadienoate, tetracontane, fumaric acid (pigment), tritriacontane, 9-octadecene and hentriacontane ranging around 8.37%, resulting in a very low unsaturated fatty acid from the chromatogram. Table Xanthorrhoeaceae) recorded the pentanoic acid, tetradecanoic acid, n-hexadecenoic acid, oleic acid,9-octadecanoic acid and docosanoic acid around 47.72% from the chromatogram53. The reduced unsaturated fatty acid was observed due to the polysaccharide presence  in hibiscus organic fermented extract shown in Table 1231. The higher unsaturated fatty acid was found in palm jaggery, followed by T. chebula and aloe vera fermented extracts, in agreement with CO2 quantified liberation observed. The role of unsaturated fatty acid compounds in the fermented extracts interacts with the lime particles to improve the water-resistance, mechanical strength and enhance the microstructure's formation. Hence the addition of unsaturated fatty acids have at least one double bond between carbon atoms, and they are thus more reactive and produces compact mortar .The ethyl acetate extracted fractions of organic fermented samples revealed the major bioactive compounds, interpreted through the GC–MS NIST 14. L. The representation of bioactive compounds is tabulated with peak concentration (%), retention time (R.T.), molecular formula, molecular weight and weight percentage with 100 mg/ml. Tables 54. Aloe vera phytochemical analysis screened 13.21% of carbohydrate and polysaccharides, which disperses in water, often making a hydro-colloidal system and retain water. Therefore, aloe vera extract's main function is water-retention, exhibiting quick frying and prevents shrinkage crack on the render after hardening. In addition, the viscosity of the extract improves the plasticity of the mortar by applying thin layers of polished finish on the surfaces. Converting into ethanolic fermented extract reduces the specific surface area and pore distribution system on hydrated phases . The sugars peaks were untraceable in the further analysis, which responded with active fatty acid compounds in kadukkai, neelamari and fermented hibiscus extracts. Kadukkai extracts revealed citric acid (6%), hexadecenoic acid (11.21%), octadecenoate (5.37%), 13-octadecenoic acid (10.15%) and 6-octadecenoic acid (16.71%) as shown in Table 56 .The assessment of polysaccharides  through GC–MS analysis of the fermented organic herbs resulted in showing the sugars and fatty acid bioactive compounds from Tables oic acid .15% and oic acid .21%, oct60. The morphology of kadukkai lime paste, aloe vera lime paste, neelamari lime paste mixes has indicated the transformation of calcium hydroxide in forming the carbonate polymorphs at 7 days of testing. The unsaturated fatty acids like citric acid, hexadecenoic acid, trans-octadecanoic acid, 13-octadecanoic acid and 6-octadecenoic acid present in fermented plant extracts have accelerated the formation of microstructure at an early stage and play a role in biological nucleation.The unsaturated fatty acids are the components that have imparted the hydrophobicity to the lime paste because it comprises the highest amount of C–C double bonds. The organics herbs with more amount of monounsaturated fatty acids (oleic acid) are more effective in imparting the hydrophobicity. The recent study on the addition of oleic acid to the lime paste has observed decreased particle size precipitatesMoreover, it regulates the growth of calcium carbonate crystals and leads to smaller particles and a more compact structure. The lime paste subsumed with unsaturated fatty acids has better mechanical properties, water resistivity and weather resistance than common reference lime paste. (Ziyang Liu 2016).14. Yang and Zeng et.al. Revealed that the presence of carbohydrates played a crucial role in the microstructure and consolidation of lime mortars. Carbohydrates act as a template and consolidate the loose structures and result in strength improvement and durability23. Thirumalini et al., in their research work they have analyzed the influence of organically added lime mortar on mechanical and physical properties of lime mortar and revealed that the addition of organics has enhances the compressive strength of lime mortar due to the reduction of a higher amount of larger pores into micropores. The addition of organics in the form of carbohydrates and protein in the lime mortar mix would have also prolonged the reaction and retarded the initial growth to arrest the drying shrinkage cracks on the lime paste.The functions of protein in lime paste include air-entraining, decrease roughness, viscous enhancing performance and environmentally friendly admixtures for modern construction materials. The addition of amino acids  has improved lime paste's performance due to the interfacial activity of the protein-peptide chains and the bio-mineralization effect. Fang et al. discussed that proteinaceous substances' addition improves the consistency, cohesiveness, durability and reduces the risk of cracking of the lime mortar. Simultaneously, it has a negative influence on the compressive strength and surface hardness of the mortar along with much more compact and slows down the carbonation progress of the lime mortar46. From the overall results, the addition of organics has proven that phytochemicals have altered the mineralogical formation and significant advantages such as setting acceleration, water repellency, hydrophobicity, strength enhancement and carbonation rate to produce an eco-friendly bio-organic mortar.The similar presence of calcite and vaterite in the mortar samples with traces of calcium oxalates (weddellite) could contribute to the self-healing of lime mortar in enhancing the crystalline growth high carbonation rate56.This study's phytochemicals investigation has contributed to understanding the interaction mechanism between the lime particles and fermented organics. Although many efforts have been devoted to identifying the original components of the ancient building materials, it is not always possible to find the exact reason for the structures' survivability and longevity. This is especially true for the organic additions, either because they were incorporated in small amounts (below 5%), thus falling short on detecting the possible interaction and type of phytochemicals that have improved the required structural aspects33.The fatty acid methyl esters (FAME) detected the fatty acid esters derived from the transesterification of fats in the presence of methanol and been screened with a U.V. spectrophotometer between 290 and 660 nm. The absorbance produced the concentration of three herbal extracts that was plotted through the linear regression modelling coefficient (> 0.9981) and calculated the limit of quantification (L.O.Q.) and limit of detection (L.O.D.) ranging around 0.026 and 0.088 shown in Supplementary Figs. 6Sa: Standard deviation of the response, b: slope of the calibration curve.2 quantification analysis shown in Table 211. The unsaturated fatty acid like tung oil, olive oil, plant extracted oil, animal glue and casein are the most promising additive for improving the repair mortar durability and hydrophobicity to the molecular structure of the mortar. The addition of fatty acid components improves the binding capacity among the grains of lime particles due to their complex fatty acid chain structure. The past reported literature stated that plants contain fatty acid molecules such as oleic acid and stearic acid. Linoleic acid and minor ester fractions have provided the impervious layer to the surfaces by preventing structure instability. The presence of an appreciable number of di-unsaturated esters of linoleic acid is prone to polymerization reaction when exposed to the outer atmosphere. Drying could be rigid the molecular structure with an increase in mechanical strength60.The fatty acid concentration of kadukkai was 32.91 µg/ml, neelamari and aloe vera around 154.43 and 383.54 µg/ml through the fatty acid methyl esters transesterification method. De architectura by Vitruvius stated that the natural oil and plant extracted fatty acids were among the most common types of water-repellent additives used in ancient mortars for coating in antiquity stucco works. It's highly reactive because it contains a high amount of Oleic acid (26.4 wt%), hexadecenoic acid (22.22 wt%) and octadecenoic, commonly called (linolenic acid) (10.86 wt %) with two and three double bonds, in agreement with the GC–MS chromatograms as shown in Tables 3The Beer lambert law is a linear relationship between the absorbance and the concentration, molar absorption coefficient and optical coefficient of a solution.-1, c: Molar concentration M, l: Optical path length cm.A: Absorbance, ε: Molar absorption coefficient M cm37. Further reagents like neelamari and hibiscus have recorded a moderate sugar concentration level due to unsaturated fatty acid in agreement with the GC–MS analysis performed on the fermented extracts. The GC–MS polysaccharide analysis detected the glycosides, monomers, monosaccharides, mannopyranose, glucopyranose and D-glucose in palm jaggery and aloe vera around 3.13 µg/ml and 11.17 µg/ml as shown in Tables 54.The U.V. spectrophotometric quantification analysis recorded total sugars through the phenol sulphuric reactive solution with the concentration level of 100-500 µg/ml) to evaluate the absorptivity and the wavelength detection limit ranging between (540 nm). Tables 00 µg/ml 1 and portlandite peak at 3641 cm−1 in FT-IR analysis. But peaks in organic incised lime putty samples have revealed the overall formation of high portlandite peaks with low-intensity calcite peaks with traces of vaterite and aragonite peaks are observed. Minor traces of hatrurite peaks formed in insoluble silica present in the organic fermented herbs reacting with lime particle forming calcium hydrate silicates observed in K.L.P., JLP NLP organic lime putty samples validated with silicate spectral vibration observed at 420 and 478 cm−1. The organics incorporated lime putty samples modified their crystalline formation and properties like workability, water repellency, air entrapment, accelerator, and retarding effect with the interaction of biomolecules present in the fermented herbs62. Likewise, the N.L.P., K.L.P., A.L.P. and H.L.P. samples are rich in the presence of unsaturated fatty acid and fraction of sugars compounds, which has produced an internal dissociation between the lime particles by imparting hydrophobicity, making the surface more porous to increase the carbonation resulting in a gradual increase of calcite peaks with traces of fatty acid FT-IR vibration observed at 3392.1 cm−1. The unsaturated fatty acid is mostly prone to hydrophobicity. The formation of calcium oxalates (dehydrated-weddellite) peaks is observed due to oxalic acid (unsaturated acid) in the fermented organic, converting it into calcium oxalates to develop the formation of calcite slowly. The fraction of sucrose, glucose substrates present in the fermented organics tends to form strong bonds between the lime particle to develop crystalline hydrated phases to stabilize the formation of carbonate polymorphs as the days progress. J.L.P., A.L.P. and N.L.P. samples are rich in polysaccharide in agreement with GC–MS analysis. They have improved the early formation of calcite peaks, with possible break down of alcohol and CO2 converting the Ca (OH)2 to calcium alkoxide and calcium carbonate . The presence of alcohol influences the metastable carbonate polymorphs phases by stabilizing them to increase the metastable vaterite and aragonite phases. The high intense portlandite peaks are similarly observed in all the organic samples. Additionally, carbohydrate FT-IR vibration peaks at 1583 cm−1 confirmed the addition of polysaccharides  would act as water reducers, water retention agents and set retarding effects to the organic incorporated samples21. This overall inflated transformation of portlandite peaks to calcite in reaction with internal recarbonation activity due to the organic fermented herbs will modify the amorphous phases to develop the crystallinity in forming the hydrated phases as the day's progress can be achieved with better absorbing of external CO2 through the external environment.After GC–MS analysis, XRD powder diffraction was performed on organic lime putty samples to evaluate their interaction with fermented herbs. Figures −1 and (C–H asymmetric stretching) vibration at 1409 cm−1,874.15 cm−1 (CH2 out of plane bending) show the presence of calcite peaks with traces of silicate vibration depicted at 1004 cm−1 in agreement with mineralogical investigation64. It's been evidenced that all the organically modified lime putty samples have revealed the intense characteristic peak portlandite (O–H stretching) at 3641 cm−1 and initially transformed calcite around 1400–1475 cm−1 assigned to the in-plane bending and symmetric carbonate out of plane bending vibration of calcite and aragonite. Minor peaks of vaterite have also been confirmed at 999 and 998 cm−1 around 900–1080 cm−1. The traces of silicate peaks at 420, and 480 cm−1 indicate the calcium hydrate silicate formation formed hatrurite peaks (C–S–H) in par with XRD analysis. The vaterite formation indicates the eventual transformation of amorphous conditions into calcite crystals under high CO2 pressure and the alkaline conditions of pH (8.5–10.5)67. The recent studies also have reported that vaterite formation is favored in the presence of amino acids and alcohols in addition to fermented organics . The presence of carbohydrates (C–H stretching) at 1583 cm-1 and unsaturated fatty acid peaks at 3394 and 3392 cm−1 being observed in all the modified mixes indicates the role of polysaccharides unsaturated fatty acids chains with the lime particle to transform the amorphous hydrated phases to crystalline calcite crystals. The minor stretching of hydroxyl ions at 3340–3494 cm−1 indicates ethanol, isopropanol, and calcium hydroxide observed in organically modified samples. It is observed that the reference putty (R.M.) sample has revealed well-developed calcite peaks compared to organic incorporated lime putty samples. However, the unsaturated fatty acid has created a porous surface to increase the carbonation and polysaccharides , slowing down the setting time and as waterproofing surfaces to resist the dampness from the surfaces.As a confirmation to the mineralogical investigation, FT-IR analysis was performed on the lime putty sample to investigate the organic, polymers and inorganic materials from the sample wavelength, as shown in Figs. Nevertheless, when the polymer is added as a dry powder, it has to dissolve first before to be able to adsorb on hydrates. Therefore, the hydrates have more time to grow, and the surface area available for adsorption is higher. It is evident that 5% of fermented organics has performed better and can further visualize the high-intensity calcite peaks with higher curing ages.46. The amorphous nature hindered the identification of calcite and vaterite crystals in the organic lime paste. It is observed that the surface morphology of all the organic lime paste has revealed well-developed calcite crystals and reduced pore spaces due to the enhanced carbonation in the presence of organic mineral67. The polysaccharides play a prominent role in the lime paste's microstructure and decarbonization properties. In the presence of organic additives, the initial setting has been prolonged. Hence, the cracks are not observed. It indicates that Ca (OH)2 and organics like protein and carbohydrates will increase the calcite formation and improved mechanical strength as time progresses69.The morphological information of organic lime paste, provided by the field emission microscopy, helped provide valuable information about the reaction compound, allowing us to observe the nature of formation, sizes, texture and distribution system ranging between 3300 and 10,000 X in Figs. 72. Ancient practices have incorporated the extracted oil and fats from plants and animals to waterproof the surface from the external dampness and capillary rise from the structure's ground level. Vitruvius stated that fig juice, rye dough, hogs, lard, curdled milk, blood and egg whites were employed to toughen and regulate the setting qualities. During the prehistoric periods, the moisture and dampness from the rain harm the structural wall and deteriorate the building materials. To counterbalance the problems, the historic structures were finished with permeable lime mortar to expand, contract, and flex with changes in external temperatures and moisture, hardening the structure on absorbing the CO2 from the external environment throughout its service life to increase the serviceability and longevity of the structure.Many ancient structures across the countries have documented various indigenously available organic materials during the production technology. To improve strength and durability, they included starches, flours, gums, oils, tree barks, waxes or plant resins, and animal origins such as animal fats or casein, blood, excrements and urine2. The natural organic additives have activated the internal carbonation and help in carbon sequestration. Researchers reported using organic additives has improved the durability and hardness, reduced the drying shrinkage, resistance to traction, acceleration and deceleration of setting time, adhesiveness, and resistance freeze–thaw cycles, increases the plasticity and workability properties16. Ancient structures exposed to extreme environmental conditions have been subjected to adverse defects and chemical attacks that require repair and replication of similar traditional production technology with indigenous organics to preserve the structure and ancient wisdom.The addition of chemical additives has made the cement mortar more impervious with the nil absorption rate of CO2 emission on anaerobic conditions. The overall release of CO2 confirms that the unsaturated fatty acid compounds have better carbonation with the early transformation of Ca (OH)2 into carbonate polymorphs to form the very compact structure due to hydrophobic and water repellency factors creating a waterproofing surface. Justnes et al., 2004 reported that fatty acid anions present in oils (unsaturated fatty acid) react with calcium hydroxide as follows Eqs. ..2 emissi-) of the unsaturated fatty acids to form hydrophobic salts of Ca (CXXHXXCOO)2; the dissolved CO2 salts carbonates the lime putty to improve the carbonate ions56 stated that fermented plant extracts containing more monosaturated fatty acids like oleic acid, octadec 9-enoic acid and hexadec 9-enoic acid are more effective producing the hydrophobicity to the mortar71 experimented with the effect of oleic acid on the properties of nano-calcium carbonate. They observed a decrease in the precipitates' particle size, which was also confirmed by wang et al. polysaccharides. These were identified in three herbs like kadukkai, palm jaggery and aloe vera in different percentages. The analyzed polysaccharides and proteins like acetamide, lumicolchicine, pentadecane dioic acid, valine (amino acid), cyclodecasiloxane, pentamide and manopyranose are equally classified as water reducers, set retarders and water retention agents that help to retain the moisture content in lime mortar leading to the delay in the setting time68. Polysaccharides shall contain both hydrophobic and hydrophilic segments responsible for the delay in Lime since water molecules could be attached to the parts of hydrogen bonds; being unable to escape this moisturizes the mortar mix. The major drawback of polysaccharide in lime mortar is the induced delay of hydration, and its interlocking mechanism of water molecules inside the mortar imparts slow carbonation (Poinot 2015). The addition of sugars up to 2% has water reduce the effect and reduce risk in cracks induced by dry shrinkage. However, excess addition could affect the mortar's compressive strength and water resistance. However, the usage of organics in mortar has significantly improved the properties like durability, longevity and resistance against external environmental conditions.Calcium ions from calcium hydroxide react with the carboxyl group 72.The ancient sustainable production technology of organic lime mortars and its capacity to sequester carbon dioxide via biomineralization provides valuable inputs to lessen greenhouse gas emissions, causing climate change and variability. Compared to cement production, Lime is substantially adopted in ancient practice because it produced at a lower temperature around 900–1200 °C, resulting in 40% less COThe GC–MS analysis revealed various bioactive compounds such as unsaturated fatty acids, polysaccharides, carbohydrates, and proteins. The limit of quantification (LOQ) and detection limit (LOD) value was found statistically significant for sugar and fatty acid components.Lime putties of Jaggery, aloevera, and kadukkai mixes have significantly improved the formation of calcite with acicular aragonite crystals in addition to polysaccharides materials such as glycosides, monomers, monosaccharides, mannopyranose, glucopyranose valine (amino acids) and D-glucose. In the presence of polysaccharides, the organic lime paste offered the hydrophobic property on offering better resistance against the absorption of the soluble substances to confront the dampness on the surfaces.Hibiscus lime putty rendered the hydrophobic lining in the lime matrix pores in combined action with the effect of fatty acids and sugar monomers, such as hexadecenoic acid n-hexadecenoic acid, 14-octadecadienoate, tetracontane, fumaric acid, 9-octadecene, hentriacontane and rhodopin-glucose.The phytochemicals present in the jaggery lime putty have attributed the combined chemical reaction like water repellency, retarding effect, adhesive properties that show the lime particle interaction to arrest the porosity resisting the shrinkage cracks and developing early mechanical strength with complete carbonation on the internal and external surfaces.2 has initiated the internal carbonation of the lime putty and precipitation of calcite in three different forms aragonite, calcite and vaterite minerals. The XRD depicted the amorphous polymorphs of  peaks, along with traces of CSH products such as hartrurite and larnite at initial stages, indicated the transformation of amorphous calcium silicate hydrate in agreement with FT-IR analysis. FESEM images revealed that the co-existence of amorphous and crystalline calcite enhances the hydration process as more free lime has been available for the reaction. The EDX results depicted the amount of carbon presence up to 24%.Supply of COScientific research decodes the influence of plant extracts in the lime mortars. It provides valuable input to the State Archeology Department, conservation engineers and architects, NGO’s, and to simulate new organic mortars for repointing, rendering, stucco, and waterproof surfaces to restore and safeguard the ancient structures.From the archival information on the ancient palm-leaf manuscript of Padmanabhapuram palace construction, the ancient production methodology has made us capitalize on the importance of the type of building materials and various organic additives like kadukkai, neelamari, hibiscus, aloe vera and palm jaggery and also its role with the binder materials that has resulted in the long-term durability to the structure.Supplementary Information.


Aedes triseriatus, and Aedes japonicus. Larval and water samples were collected from tree holes and used tires at two study sites, and their bacteria characterized through MiSeq sequencing of the 16S rRNA gene. Bacterial richness was highest in Ae. japonicus, intermediate in Ae. triseriatus, and lowest in water samples. Dysgonomonas was the dominant bacterial taxa in Ae. triseriatus larvae; the unclassified Comamonadaceae was dominant in water samples from waste tires, while Mycobacterium and Carnobacterium, dominated Ae. japonicus. The two mosquito species harbored distinct bacterial communities that were different from those of the water samples. The bacterial communities also clustered by habitat type (used tires vs. tree holes) and study site. These findings demonstrate that host species, and the larval sampling environment are important determinants of a significant component of bacterial community composition and diversity in mosquito larvae and that the mosquito body may select for microbes that are generally rare in the larval environment.Mosquito bacterial communities are essential in mosquito biology, and knowing the factors shaping these bacterial communities is critical to their application in mosquito-borne disease control. This study investigated how the larval environment influences the bacterial communities of larval stages of two container-dwelling mosquito species,  In mosquitoes, the bacterial communities hosted by different mosquito species have been extensively described9, and studies on microbial functions has revealed that mosquito-associated bacterial communities, are essential for host nutrition, reproduction, development, defense against parasites and pathogens, and modulation of host immune function15. Mosquito guts host a low diversity of bacteria dominated by a few taxa most of which fall within the four broad phyologeneitic groups, namely Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes19. Most of these are primarily acquired horizontally from the host environment including the larval environment, or in the case of adults, nectar and other dietary sources24. Thus, most of the bacterial communities that colonize mosquitoes are thought to be transient in nature. However, there is also evidence of transtadial transmission from larvae to adult stages among bacterial communities and their co-evolved mosquito host species, as well as vertical transmission from adult females to their offsprings in the case of intracellular bacteria such as Wolbachia26.Metazoan organisms serve as habitats for a variety of microbial taxa including bacteria, fungi, protists, and viruses. These microorganisms may provide critical biological functions in their hosts, and mutualistic interactions between hosts and their microbiota have been described28. However, the contribution of the environment to the acquisition of bacterial communities is not yet fully understood. The interactions between bacterial commensals and their host mosquitoes has implications for mosquito fitness for disease transmission and therefore their application as tools for mosquito-borne disease control29. Thus, it is imperative to develop a better understanding of the factors that influence the succession pattern of bacterial communities in the host mosquitoes. Among the factors that have been shown to influence the composition and structure of mosquito microbiota include host genetics, species, age, sex, diet, stage of development, host sampling location and infection with parasites and pathogens32. The larval environment has also been shown to be an important factor in shaping the mosquito bacterial communities, however, in respect to two medically important mosquito species evaluated in this study, Aedes japonicus and Aedes triseriatus, the patterns of acquisition of bacterial communities from the larval environment has not been fully evaluated34.Bacterial diversity can vary substantially within and between mosquito species, influenced by a host of factors, including the mosquito life stage, dietary sources, feeding behaviour, and the mosquito host environment36. The bacterial composition and abundance in these aquatic larval habitats are driven mainly by the underlying environmental characteristics, precisely the organic detritus composition and their physical and chemical characteristics, which in turn may dictate the bacterial colonization patterns in immature and adult mosquito stages. Many of these habitats are fueled by organic detritus which provides a substrate for bacterial growth that is consumed by mosquito larvae and other macroinvertebrate consumers42. Different detritus types are known to support bacterial communities of varying composition and abundance, and detritus-mediated differences in physical and chemical characteristics of the larval habitats may cause further alterations in bacterial composition and structure in these habitats47. Stemflow and throughfall provide additional sources of inorganic nutrients such as nitrogen, phosphorus, and sulfates, which are key modifiers of the larval water chemistry parameters such as pH, dissolved oxygen, and conductivity, and thus play a key role in stimulating bacterial growth50. Water chemistry parameters vary significantly among larval habitats, seasons, and between locations49. Thus, detritus composition and the water chemistry characteristics of the larval habitats may have significant effects on bacterial communities that are acquired by mosquito larvae. However, we have limited understanding of how characteristics of the larval environment influence the composition and structure of mosquito-associated bacterial communities.Mosquitoes develop in a variety of natural and human-made aquatic habitats of diverse shapes and sizes. These include pools, ponds, stormwater catch basins, river and lake edges, rice fields, tree holes, indoor and outdoor water-holding containers, and discarded tires, among other habitatsAe. triseriatus and Ae. japonicus were used to investigate how the larval environment influences bacterial composition and diversity in mosquito larvae. Aedes japonicus is an invasive species in the United States51. It is not considered a major vector of human pathogens although it has been found infected with several arboviruses including Japanese encephalitis virus (JEV)52, West Nile virus (WNV)53, La Crosse encephalitis virus (LACV)54 and Cache Valley virus (CVV)55. Aedes triseriatus, the primary vector of LACV, is the dominant tree hole mosquito species and co-exists with Ae. japonicus in woodland areas in the eastern and midwestern US57. Larvae of the two mosquito species occur in a variety of natural and man-made container aquatic habitats, but we specifically focused on tree hole and waste tire habitats, both of which are known to serve as ideal larval habitats for diverse mosquito species58. Tree hole and waste tire habitats differ markedly in the quantity, nature, and type of organic detritus they contain as well as in their chemical characteristics and detritus decomposition rates59. These differences are likely to influence the composition of resident bacterial communities and in turn, the pattern of bacterial community colonization in mosquitoes. This study tested the hypotheses that: (1) tree hole and waste tire habitats differ in their environmental characteristics and support different bacterial communities, (2) the bacterial composition of Ae. triseriatus and Ae. japonicus larvae reflect the bacterial communities of their aquatic larval habitats, and (3) water chemistry characteristics of the larval environment are important in shaping the bacterial community composition of the larval environment. This study found that host species, and the larval sampling environment are important determinants of bacterial community structure in mosquito larvae and that the mosquito body likely select for bacterial communities that are generally rare in the larval environment. The larval environment consisting of used tires and tree holes were characterized by differences in water chemistry parameters that also seemed to have a strong effect on the clustering of the dominant bacterial communities by habitat type (used tires or tree holes). Our findings add additional evidence of the importance of larval habitat to the assembly of internal bacterial communities of mosquito species neglected by previous research.In this study, two container-dwelling mosquito species, Aedes triseriatus samples were dominated by Bacteroidetes, while Ae. japonicus samples were dominated by Actinobacteria  yielded 1,250,692 raw sequences (mean ± SE = 4531.49 ± 272.10 per sample). After quality filtering and rarefying the reads to an even depth of 1058 sequences per sample, 192 samples were retained. This data set consisted of 1,188,971 sequences (161 mosquitoes and 31 water) that were clustered into 918 bacterial OTUs. A total of 11 phyla, 25 classes, 91 families, and 123 genera, were detected. Eighteen of the 918 OTUs had an overall abundance of 1% or greater. The highest proportion of the sequences were from phylum Proteobacteria (34.2%). Other phyla included, Actinobacteria (29.1%), Bacteroidetes (23.4%), Firmicutes (9.0%), Chloroflexi (3.3%), with the rest of the phyla having taxonomic abundance of less than 1%. Water samples from most used tires and tree hole habitats were dominated by Betaproteobacteria, Aedes triseriatus samples from used tire and tree hole habitats were dominated by Bacteroidetes. Aedes japonicus from used tire habitats were dominated by Actinobacteria and Alphaproteobacteria . Water samples from used tire habitats were predominated by Proteobacteria. The rest of the taxa were represented in the three sample types at varying proportions but mostly < 10%. In general, only 1–2 major bacterial taxa were dominant in each sample type , an unclassified Actinomycetales (7.5%), Mycobacterium (7.0%), an unclassified Rhizobiales (6.1%) and an unclassified Comamonadaceae (4.4%). Dysgonomonas were dominant in Ae. triseriatus samples from most of the used tire and tree hole habitats. The unclassified Actinomycetales, Mycobacterium and the unclassified Rhizobiales occurred in disproportionally high abundance in Ae. japonicus samples from used tire habitats, both at South Farms and Trelease Woods. The unclassified Comamonadaceae was the dominant bacterial OTU in water samples from most of the used tire habitats at Trelease Woods, while the taxon Limnohabitans was dominant in used tire habitats at South Farms  revealed that up to 75.0% ± 0.03 (mean ± SE) of the expected bacterial OTUs were detected62. Rarefaction analysis showed that Ae. japonicus from used tires from Trelease Woods had the highest OTU richness while water samples from tree holes at South Farms recorded the lowest OTU richness    recovered majority of the bacterial OTUs by the sequencing depth coverage of 1058 sequences Fig. 61. Chao1ess Fig. . Alpha d01) Fig. . At Trel08) Fig. .Figure 3Ae. triseriatus from tree holes (62.5%) compared to the number shared between Ae. triseriatus and water samples (23.4%)  compared to those shared between Ae. triseriatus, Ae. japonicus and water samples (19.1%)  compared to shared OTUs (13.7%)  and samples from Trelease Woods had more OTUs compared to samples from South Farms  OTUs were shared between mosquito larvae and water samples. More OTUs (36.0%) were observed in larval samples compared to water samples (6.4%) Fig. A. At Sou4%) Fig. B. Aedes 1%) Fig. C. At Treype Fig. D. When aype Fig. E. Generatus Fig. C–E. Pool7%) Fig. F. Pooledrms Fig. G. Pooledrms Fig. H.Ae. japonicus, Ae. triseriatus, water samples, at the individual sampling sites at South Farms and Trelease Woods, respectively , and between Ae. japonicus, Ae. triseriatus, water samples, at the individual sampling sites level at South Farms ; but not at Trelease Woods , indicating dispersion effect on the bacterial composition of mosquito and water samples at South Farms, but no dispersion effect on bacterial composition at Trelease Woods. Post-hoc analysis using multiple pairwise comparison of the individual sampling sites revealed significant differences in bacterial community composition between Ae. triseriatus, Ae. japonicus and water sampling from most of the individual sampling sites at South Farms and Trelease Woods, respectively . We suspect that since Dysgonomonas was detected in water samples, albeit at low abundance, Ae. triseriatus and Ae. japonicus larvae potentially acquired this taxon from the larval environment, but the majority are possibly lost during molt from larvae to pupae rendering them undetectable in adult samples. Future studies characterizing the bacterial communities of Ae. triseriatus or Ae. japonicus across life stages could help test this hypothesis. Both facultative and obligate Dysgonomonas adapted to the anoxic mosquito gut environment have been described in both larvae and adults of several mosquito species, including Ae. albopictus, Anopheles gambiae s.l., Culex tarsalis, and An. stephensi77. In termites, Dysgonomonas have been reported to mediate vitamin B12 synthesis78. However, the role of Dysgonomonas in mosquito biology remains to be described and warrants additional attention in future studies.Interestingly, Ae. japonicus and Ae. triseriatus79. Previous research reveals variation among studies in the dominant bacterial taxa of Ae. triseriatus and Ae. japonicus, possibly due to environmental and seasonal differences. A study with larval samples collected from different sites in Champaign, Illinois, revealed the dominance of the bacterium Bacillus cereus in both Ae. triseriatus and Ae. japonicus79. A related study with adult samples reported the dominance of the bacterial genera Gluconobacter and Wolbachia, to a lesser degree in Ae. japonicus, and the bacteria genera, Morganella and Gluconobacter in Ae. triseriatus9. Another study with adults of Ae. japonicus and Ae. triseriatus revealed the dominance of Paenibacillus and Polynucleobacter in newly emerged Ae. japonicus and Ae. triseriatus samples, respectively18. These studies point to the possibility that variations in bacterial community composition among similar species but from different studies may also also be influenced by differences in bacterial colonization pattern in the larvae and adult stages80.Few studies exist that have characterized the bacterial communities of Limnohabitans  was dominant in used tire habitats at South Farms. The family Comamonadaceae is a group of gram-negative aerobic bacteria that metabolize organic acids, including amino acids81. They have previously been isolated from mosquito larvae and their larval environment as well as in the soil and water from natural and industrial environments85. They also have been reported as part of the core bacterial communities of adult mosquito species such as An. gambiae and An. coluzzii, pointing to potential transtadial transmission among conspecifics, and/or horizontal acquisition from the larval environment87. Other studies report the dominance of the members of Comammonadaceae in aquatic habitats without mosquito larvae relative to larval habitats colonized by mosquito larvae, suggesting that members of this family likely serve as a major component of larval food resources83. The genus Limnohabitans is a member of aerobic anoxygenic phototrophic bacterial communities with a cosmopolitan distribution in fresh water—and other aquatic environments89. This genus occurred in our mosquito samples in insignificant fractions, indicating that although it may be acquired horizontally in the larval environment by mosquitoes, it is readily digested and excreted, or it is poorly adapted to colonizing the anoxic mosquito gut environments. Studies characterizing Limnohabitans in other mosquito bacterial studies are also unavailable.An unclassified Comamonadaceae dominated water samples from used tire habitats at Trelease Woods, while the genus Aedes triseriatus larval samples from tree hole and used tire habitats had distinct bacterial communities suggesting a strong influence of larval sampling site on mosquito bacterial communities. Discriminant analysis identified different water chemistry parameters that characterized used tire and tree hole habitats, that also seemed to have a strong effect on the clustering of the dominant bacterial communities by habitat type (used tires or tree holes). The role of the larval environment in exerting an influence on the mosquito bacterial composition has been reported previously90. For example, Kim et al., found strong variations in the bacterial communities within conspecifics (Ae. japonicus and Ae. triseriatus adults) sampled from different sites, but less variation between different genera (Culex and Aedes) sampled from the same site. Discriminant function analysis results revealed that water from tree holes was highly turbid and had higher concentrations of tannins and lignins. Conversely, used tire habitats were characterized by higher concentrations of reactive phosphorus and nitrogen ammonia. The differences in the physical and chemical characteristics of the two habitat types were likely due to differences in detritus types. Previous studies have shown that tree holes and used tire habitats vary significantly in their detritus composition, and these differences may have a dramatic effect on their bacterial composition91. Tree holes at South Farms are naturally more permanent compared to the used tires sampled in this study. Although the detritus composition of the used tire and tree hole habitats were not quantified, some studies suggest that tree holes generally harbor high abundance of both plant and animal detritus accumulated over multiple years compared to used tires, and thus are likely to structure the bacterial community differently to the transient used tire habitats92. The detritus is mostly composed of senescent plant parts, primarily terrestrial leaf detritus, and some input of animal detritus that provide substrate for bacterial growth and hence shape the bacterial community composition of the container habitats94. Additionally, during the sampling period, it rained at least once every week, and this is likely to have increased stemflow into the tree-holes. Stemflow provides soluble nutrient input that stimulates the growth of diverse bacterial communities in the tree-hole habitats95. In contrast, the used tire habitats were more ephemeral having been established and sampled two weeks into the duration of the sampling period, and therefore, were likely to harbor a different mix of detritus leading to different sets of bacterial communities between the used tire and tree hole habitats. We also observed differences in the physical and chemical profile between the used tire and tree hole habitats which may have additionally driven the difference in the bacterial assemblages of the two habitat types.79. These findings suggest that some bacterial communities in mosquito bodies are likely transmitted vertically or horizontally via conspecifics or that some of the microbes occur in very low abundance in the larval environment that could not be detected by our methods. Additionally, bacterial communities in the larval environment occupy different niches including the surface microlayer, the sub-surface, and substrates such as the container walls and detritus surfaces96. It is possible that Ae. triseriatus and Ae. japonicus larvae spent most of their time browsing on either the leaf surfaces or container strips that possibly contained higher bacterial richness compared to the water column. This is supported by previous reports showing that the larvae of container-dwelling mosquitoes, such as Ae. triseriatus, prefer to graze on the leaf substrate and container surfaces over the water column97. We also agitated the larval water prior to sampling disrupting the surface microlayer and the sub-surface, possibly reducing the recovery rates of bacterial communities occupying these niches. There is also a possibility that the 40 mL of water from the larval habitat used in the bacterial analysis was not sufficient to capture a representative sample of the bacterial communities in the larval habitats. However, this volume falls within the standard volumes that have been used in previous studies, but which have shown higher bacterial richness in water samples relative to mosquito samples79.Some bacterial OTUs detected in mosquito larvae were not found in the water samples, and overall, fewer bacterial OTUs were observed in the water samples compared to the mosquito samples. This is contrary to previous studies showing that the larval environment harbors higher bacterial OTU richness compared to the mosquito larvae101. However, it is well established that most mosquito bacterial communities are concentrated in the midgut and are also the most studied group102. Bacterial communities also colonize the external surfaces of mosquito larvae, creating the possibility that some of the bacterial communities that were isolated in our study could be from the external surface. We thoroughly sterilized our whole-body mosquito larval samples according to previously used protocols8, reducing the possibility that the bacterial communities characterized in our whole-body larval samples could have included external body surface bacterial communities. Additionally, the bacterial communities isolated in our larval samples are similar to those previously isolated in other studies with midgut samples103. Additionally, sample coverage was assessed using rarefaction curves at a maximum read depth of 1058 sequences. The rarefaction curves did not plateau fully at this sequencing depth, indicating that higher bacterial richness and diversity could have been recovered with additional sequencing at higher depth. However, rarefying at higher sequence count in this study would have resulted in too many samples being discarded, hence the threshold utilized.Our study is limited by the use of whole-body mosquito larvae as opposed to midgut samples. Therefore, not all the mosquito bacterial communities that were characterized in this study could be assigned to the gut. Apart from the midgut, mosquito bacterial communities have been isolated from other organs of the mosquito body such as the ovaries and salivary glands, among othersUnderstanding the factors that shape the bacterial composition and diversity in mosquitoes is an essential step in their manipulation for bacterial-based mosquito-borne disease control. Our study has shown that while the host environment is an important driver of mosquito bacterial composition, the bacterial composition does not fully reflect the environmental bacterial assemblages. Whereas this study suggests that mosquitoes may select for the suite of bacterial communities that colonize, we are unable to fully infer the source of the higher numbers of bacterial OTUs detected in the mosquito samples compared to the water from the larval habitat. This leaves a gap in our understanding of the source of the bacterial communities that colonize mosquito bodies and additional studies are needed to address this knowledge gap. Future studies may evaluate in controlled experimental conditions the unknown stress factors regulating bacterial diversity and composition in the larval environment, and thus subsequently influencing the bacterial succession pattern of mosquito larvae.Quercus spp.), ash (Fraxinus spp.), hackberry  and maple species (Acer saccharinum Linn.), with a high, closed canopy and moderately dense understory. The site includes two small seasonal ponds which provide suitable habitat for many aquatic and semi-aquatic invertebrates, including mosquitoes. South Farms is a 20.14-acre woodland approximately 2.41 km southeast of UIUC campus. It is composed of low canopy trees consisting mainly of silver maple (Acer saccharinum Linn.), Sycamore , pine (Pinus spp.), oak (Quercus spp.) and patchy grass undergrowth in some sections 104. Approximately 1 year prior to this study, the invasive Amur honeysuckle (Lonicera maackii), that was the dominant shrub in this woodlot was removed.Mosquito sampling was conducted at the Trelease Woods  and South Farms  study sites in Urbana, IL. Trelease Woods is a 71.17-acre deciduous forest situated ~ 9.5 km northeast of the University of Illinois at Urbana-Champaign (UIUC) campus. This is a woodland ecosystem consisting mainly of mature oak  were quantified using Extech pH/Conductivity, and Extech Dissolved Oxygen meters , for dissolved oxygen measurements. Additionally, 50 mL and 40 mL water samples were collected into sterile 50 mL centrifuge tubes and transported to the lab for calorimetric assays, and bacterial community composition analysis, respectively. Water samples were collected from the habitats every mosquito sampling period whenever there was water and mosquitoes in the sampling habitats. Calorimetric assay of turbidity, tannin and lignin, reactive phosphorus and nitrogen ammonia, was conducted in the laboratory using Hach DR/800 Portable Calorimeter  according to the manufacturer’s instructions. Prior to collecting water for bacterial analysis, the contents of the larval habitat were initially agitated using a turkey baster by drawing in and out the water content repeatedly to mix them thoroughly. This was repeated at each sampling site. Following collection of the water samples, approximately 400 mL water samples were drawn from each tire and tree hole habitat using 40 mL-capacity turkey baster and transferred into 500 mL-capacity whirl Pak bags. The turkey basters were cleaned between each sampling interval. In the laboratory, water samples were individually emptied into clean 2 L enamel pans, and fourth instar larvae were sorted and identified to species level using morphological keys106 on a stereomicroscope. Late fourth instar larvae of Ae. triseriatus and Ae. japonicus, the most abundant species in all sample collections, were preserved in − 80 °C freezer until further processing.At South Farms, mosquitoes were sampled from ten used tires and five tree holes. Tree holes that were productive for mosquito larvae were randomly distributed in the study location, typically occurring approximately 10–20 m apart. In tree hole habitats, only 8. Individual whole larvae were suspended in bead solution of PowerSoil DNA Isolation Kit, homogenized using Retsch MM 300 TissueLyser  and genomic DNA extracted using MoBio PowerSoil DNA Isolation Kit  following the manufacturer’s protocol. The final DNA material from each sample was eluted in 100 µL of sterile elution buffer (10 mM Tris–HCl). DNA was quantified using Nanodrop 1000 . Water samples for microbiome analysis were thawed at room temperatures and 30 mL of each sample centrifuged for 20 min at 5000 RPM using Eppendorf Centrifuge 5810R . The supernatant was discarded, and the pellets suspended in bead solution of PowerSoil DNA Isolation Kit and the DNA extracted following the same procedures used in the larval samples. The samples were shipped on Dry Ice to the National Center for Agricultural Utilization Research, Peoria, IL for sequencing. The V3–V4 hypervariable region of bacterial 16S rRNA gene was PCR-amplified using previously published universal primers, 341f, and 806r108. The V3–V4 hypervariable region of the 16S rRNA has been shown to have higher sensitivity in bacterial phylogenetic analysis compared to the rest of the hypervariable regions of the 16S rRNA gene109. The following primer set specific for V3–V4 region of the 16S rRNA gene were used: Forward 5′CCTACGGGNGGCWGCAG; Reverse 5′GACTACHVGGGTATCTAATCC. The primers were incorporated into fusion primers for dual indexing and incorporation of adapters prior to genome sequencing using Illumina MiSeq 110. There were two PCR cycles. The first PCR cycle was used to amplify the sequences using the primers targeting V3–V4 hypervariable region of 16S rRNA gene, using the following PCR conditions: 95 °C for 3 min; 25 cycles of: 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s; 72 °C for 5 min; hold at 4 °C. The second PCR cycle was used to add the indexes using the following PCR conditions: 95 °C for 3 min; 8 cycles of: 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s; 72 °C for 5 min; hold at 4 °C. A negative control sample consisting of DNA extracted from molecular grade water was sequenced with the same protocol to allow detection of any background contamination, but none was detected. PCR amplicons were cleaned and normalized using a SequalPrep normalization plate . The samples were pooled, and the library quantified with a Kapa library quantification kit . The pooled library was mixed with Phix control spike-in of 5% as a sequencing control. The samples were sequenced using an Illumina MiSeq system with a MiSeq V3 2 × 300 bp sequencing kit. The demultiplexed reads were quality-trimmed to Q30 using CLC genomics workbench v8.5 . Read pairing, fixed length trimming and operational taxonomic unit (OTU) clustering was done using CLC Bio Microbial Genomics module  using the reference sequences from the Greengenes ribosomal RNA gene database and OTU assignment at 99% sequence similarity, which is adequate for bacterial identification to the genus level111.Fourth instar larvae were retrieved from − 80 °C freezer, surface-sterilized individually in 70% ethanol for 10 min, five times in sterile water, and once in sterile 1× DPBS solution112 within Rstudio environment version 1.3.959113 statistical package. Bacterial OTUs accounting for < 0.005% of the total number of sequences were removed from the analysis to reduce the problem of artifact OTUs114. From an initial sample size of 276 samples, 84 samples had < 1058 reads and were excluded from further analysis. The 1058 sampling depth was chosen to ensure it is high enough to capture the diversity in samples without eliminating many samples with low sequence counts that represent actual bacterial OTUs. Rarefaction curves also began leveling off at this cut-off point indicating a good compromise 118. Kruskal–Wallis test was used to test for differences in means between treatment combinations of sample type (Ae. japonicus vs. Ae. triseriatus vs. water samples) and sampling habitat, and pairwise Wilcoxon rank sum test with Bonferroni correction used to compute pairwise comparisons of the treatment groups. Beta-diversity measures were conducted using Bray–Curtis dissimilarity index using “phyloseq” package and non-metric multidimensional scaling (NMDS) ordination plots were generated to visualize the results. Permutational ANOVA (PERMANOVA) test was used to test for the main and interactive effect of sample type  and individual habitat type on mosquito bacterial community composition119. PERMANOVA was conducted using the adonis function in the vegan package with 999 permutations120. PERMANOVA is sensitive to within-group variation, therefore, an analysis of multivariate homogeneity (PERMDISP) was conducted using the function betadisper to test for within-group dispersions with 999 permutations119. Post-hoc multiple pairwise comparisons using function “pairwise.adonis” in the vegan package120 was conducted to isolate individual sampling sites that differed significantly in bacterial community composition between Ae. triseriatus, Ae. japonicus and water samples. The analyses were conducted separately for each of the two study locations (South Farms or Trelease Woods). The p-values were Bonferroni-adjusted to reduce type-I error rate. Venn diagrams were created using the R package “limma”121 to visualize OTUs that were shared between the two mosquito species and water samples across habitat types and sampling locations. Canonical discriminant analysis (CDA) was used to classify the water chemistry parameters and the top 1% of the most abundant OTUs by habitat type. CDA separates groups by identifying the linear combination of variables having the highest multiple correlations within the groups63. Standardized canonical coefficients were used to evaluate the relative contribution of each predictor variable to the classification outcome. Prior to running the CDA analysis, multicollinearity diagnosis was done to remove correlated predictor variables and those with variance inflation factor (VIF) > 10 removed122. The analysis was performed in R package “candisc” version 0-8.3123 and the results visualized on CDA plots.All analyses were conducted using R version 4.0.0ise Fig. . SequencSupplementary Information.


Phanerochaete Chrysosporium fungus were evaluated by means of Fourier transformed infrared spectroscopy, X-ray diffraction, thermal analyses and morphology by scanning electron microscopy for different concentration of NaOH, different hydrothermic times at 121 °C/98 kPa and exposition to P. chrysosporium. The most effective treatment was the hydrothermal one at 121 °C and 98.06 kPa for 30 min. Preliminary tensile tests were performed in COPU biocomposites reinforced with 20% CHFWs subjected to the optimized conditions for each distinct type of treatment. The results indicated that the hydrothermal treatment promoted significant enhancement in the fiber/matrix interfacial bond, increasing the tensile strength up to 60% compared to COPU reinforced with in natura CHFWs fibers. It is important to mention that these composites can be applied as plastic wood for household items’ internal parts and in the automobile industry.Natural lignocellulose fibers have been extensively investigated and applied as a reinforcement of polymer composites in industrial applications from food packing to automotive parts. Among the advantages of natural fibers stands their relatively low cost and sustainable characteristics. These are accentuated in the case of residual fibers such as those obtained from coffee husks, an agribusiness waste, usually burnt or disposed into the environment. As composite reinforcement, hydrophilic natural fibers display adhesion problems to the most hydrophobic polymer matrices. This adhesion might be improved with distinct types of fibers surface treatments. In the present work, the effectiveness of three surface treatments applied to coffee husk fiber wastes (CHFW) were investigated, aiming to improve the tensile performance of castor oil-based polyurethane (COPU) biocomposites. The effects of treatments associated with (i) chemical with sodium hydroxide, (ii) physical by temperature and pressure and hydrothermic treatment, and (iii) biological by fermentation with  The environmental awareness movement has motivated a quest for the use of alternative renewable resources. Numerous studies report the use of vegetable fibers, also known as natural lignocellulosic fibers (NLF), as reinforcing fillers in polymeric composites ,2,3,4. NBrazil is the world’s largest exporter of coffee. The National Supply Company—CONAB—forecasts a production of approximately 49 million bags for the 2021 harvest . It is oDifferent combinations between polymeric matrices and coffee husk fibers were recently tested ,11,12. CUsing NLFs has numerous advantages. In addition to the composites’ enhanced mechanical properties, they are biodegradable, noncorrosive, lightweight, inexpensive, abundant, easy to process, have high strength, and good thermal properties ,9. AlthoThe alkaline mercerization technique stands out among the chemical treatments. Indeed, sodium hydroxide (NaOH) solutions trigger the ionization of the hydroxyl groups in the medium, chemically modifying the fiber structure by breaking the hydrogen bonds ,13. ThisPhysical treatments modify the fibers’ structure without changing their chemical composition. Hydrothermal treatments, such as self-hydrolysis, are examples of this method. Autoclave- or reactor-increased temperatures raise steam pressure, which then dissolves part of the material’s hemicellulose and extractives content. Despite NFLs’ wide use and positive results, chemical and physical treatments have the following shortcomings: (i) risky and expensive reagents; (ii) generation of effluents; and (iii) equipment’s high energy consumption. Therefore, biological agent techniques become a promising alternative to other treatments . Under specific medium conditions, fungi and enzymes can be used to degrade lignin and hemicellulose and remove part of the extractives through enzymatic hydrolysis reactions . So, theIn this study, the influence of different chemical, physical, and biological treatments applied on coffee husk fiber wastes (CHFW), as illustrated in The coffee husk fiber waste (CHFW) was supplied, as cost-free residue from the 2019 crop, by the coffee producer Ricardo Potratz located in the city of Alegre, state of Espírito Santo, Brazil, shown in The castor-oil-based polyurethane (COPU) was synthesized from polyol mixed with diphenylmethane diisocyanate, both commercially supplied by Imperveg Polímeros Indústria e Comércio LTDA, São Paulo, Brazil. The synthesis of COPU was performed just before incorporating the CHFW for composite fabrication, following the process further indicated in To evaluate the influence of alkaline concentration on the CHFW surface treatment, three solutions of sodium hydroxide (NaOH) were prepared at concentrations of 5%, 10%, and 20% (% mass/volume) named NaOH 5, NaOH 10, and NaOH 20, respectively. These concentrations were chosen by a previous study that demonstrated that high NaOH concentrations degraded fibers. The proportion of CHFW and alkaline solution used was 1:10 (% mass/volume), the contact time, reaction temperature, and medium agitation remained constant during 2 h, at 70 °C, and 500 rpm, respectively. At the end of this procedure, the fibers were first vacuum filtered and washed with distilled water until the filtrate had a neutral pH. Then they were oven-dried at 50 °C over 24 h to reach a uniform weight.CHFW used in a proportion of 1:20 (% mass/volume) was immersed in distilled water using a 500-mL Erlenmeyer flask. This treatment evaluated the exposure of cellulose molecules with regard to time. The containers were conditioned in a vertical autoclave Phoenix Luferco AV-100  programmed to operate at 121 °C and 98 kPa for the following intervals: 30, 60, and 120 min, named HYD 30, HYD 60, and HYD 120, respectively. After this step, the fibers were vacuum filtered and oven-dried at 50 °C for 24 h to reach a uniform weight.Phanerochaete chrysosporium. The fungus was inoculated in the CHFWs to verify the degree of exposure of the cellulose fibers caused by this microorganism, and 10 g CHFW was added in 8–500 mL distinct Erlenmeyer flasks, previously sterilized in a vertical autoclave. Solid-state fermentation was conducted with the use of The fermentations took place in a laminar flow hood with insertion 0.1 g of inoculum from a solid stock, with a composition of 20:80 (wheat bran: cane bagasse) into each Erlenmeyer flask. To achieve the medium’s ideal moisture content (60%), 15 mL of a saline solution with the composition described by Urbánszki et al. . Every 24 h after the fermentations, two flasks were removed and placed in an autoclave for 30 min to ensure cell death. The material was then vacuum filtered, washed with 250 mL distilled water, and finally oven-dried at 50 °C until the sample showed no mass variation. This treatment was performed at 4 different times, 24, 48, 72, and 96 h, which were identified as BIO 24, BIO 48, BIO 72, and BIO 96, respectively.To determine the content of lignocellulosic materials in fresh CHFW after the treatments, the following analyses were conducted sequentially. Determination of extractive content according to NBR 14853 , using: A is the sample mass; M1 is the pycnometer mass; M2 is the mass of the pycnometer plus the mass of the sample; M3 is the mass of the pycnometer containing sample and acetone; M4 is the mass of the pycnometer filled with only acetone; and V the pycnometer volume.The true density was measured by pycnometry technique using acetone PA  as a fluid. Then, 1.00 g of CHFW was added to 25 mL in the pycnometers. The pycnometers were completely filled, and the mean value of density obtained according to the following equation:−1). The identification of the fibers’ chemical components in fresh and treated CHFW was done using Fourier transform infrared (FTIR) spectroscopy. The spectra were obtained with a Bruker spectrophotometer, model Tensor 27 , using the KBr pellet technique with mid-infrared region scans  was calculated using the Segal method according to Equation (2) [(200) is the maximum intensity of the principal peak with crystallographic plane (2 0 0), which refers to the fraction of crystalline cellulose in the material; IAM is the intensity attributed to 2θ = 18° that represents amorphous cellulose. The CHFW surfaces were observed using scanning electron microscopy (SEM) in secondary electrons in a model JEOL, JSM-IT200 . Samples were previously coated with gold.X-ray diffraction pattern was recorded using Rigaku MiniFlex 600 Diffractometer  with Cu Kα radiation (λ = 1.54 Å). The samples were scanned at range of 2θ from 5° to 70° with a step size and scan rate of 0.05° and 2° mintion (2) :(2)Ic=I2−1 under inert nitrogen atmosphere to evaluate the thermal degradation of CHFWsThermogravimetric analysis (TGA), derivative thermogravimetry (DTG), and differential scanning calorimetry (DSC), were carried out using a LabSys Evo Thermogravimetric Analyzer  from room temperature to 700 °C with a heating rate of 10 °C minCHFW/COPU composites were manufactured with 20 wt.% of reinforcement. First, 46% of prepolymer synthetized from castor-oil-based polyol with diphenylmethane diisocyanate (MDI) were manually mixed with 54% of polyol, also derived from castor oil, for 2 min and placed in a desiccator coupled with a pump vacuum for 1 min. Previously, these components were oven-dried for 1 h at 100 °C to reduce the moisture content of the resins. After mixing, a suitable amount of CHFW was added to obtain 20 wt.% in the composite. This process was made for NaOH 5, BIO 24, HYD 30 and in natura CHFW.The mixture formed was poured into a silicone mold with shapes and dimensions defined by ASTM D3039  and thenPreliminary tensile tests were conducted at room temperature (RT) on five standard  specimenThe chemical and physical characterizations of plant fibers are crucial in understanding these materials’ relationship with the thermal and mechanical properties of polymer composites. The NLFs do not have a fixed composition: their chemical components and corresponding content can vary according to the species, soil conditions, and regional climate . In thisHemicellulose and lignin play an equally important role in vegetable fibers. Lignin molecules provide resistance, impermeability, and rigidity to cells. Hemicelluloses, however, act as an intermediary between cellulose and lignin bonds, being responsible for the growth and development of fibers ,4,5. TheThe beneftis of cellulose’s high degree of polymerization and semicrystalline structure for increasing composites’ stiffness and mechanical strength when added to the polymer matrix are well known ,2,3,4. HDuring mercerization, vegetable fibers are ionized by converting the hydroxyl groups into alkoxides . Among aAmong all hydrothermal treatments, HYD 30 exhibited the highest percentage of cellulose exposure (35.1%). Aguilar et al.  and NitsP. chrysosporium fungus with the CHFW, a maximum exposure of cellulose molecules of 38.8% was observed on the second day (BIO 48). Jayapriya and Vigneswaran [Aspergillus flavus. Increased tensile strength and hardness of high-density polyethylene (HDPE) composites were observed for 20% incorporation of these fibers into the HDPE matrix. On evaluating the contact time of the neswaran  reportedneswaran  showed 7In all treatments performed, the lower extractives and ash content can be explained by their low molecular weight, which eases their elimination . Increas−3 density proved innovative when used in Daimler–Benz automobile polypropylene-based floors. In fact, its high tensile strength, bending and degradation properties make them comparable to glass fibers. Other automotive companies, such as Audi, BMW, Volkswagen, Toyota, and Ford, use various vegetable fibers in their seat backs, interior doors, and in noise-insulating panels, instrument panel support, and even as engine insulation and internal cover [The observed density variations are also associated with the elimination of substances such as ash and extractives. An inherent characteristic of vegetable fibers is their low density, which affords energy savings when used in automotive parts. For example, abaca fiber with an average 1.5 g·cmal cover .−1 indicates hydroxyl groups (OH) associated with the lignocellulosic material, as well as of extractives in the fibers. The second band of absorption, between 2916 and 2358 cm−1, represents the aliphatic and alkyl groups of cellulose and the methyl in hemicellulose and in lignin methoxy. The aromatic compounds in lignin were identified by stretching the C=C double bond by 1608 cm−1. The band detected at 1370 cm−1 is characteristic of the C–OH bond elongation attributed to crystalline cellulose. The absorption bands between 1318 and 1239 cm−1 can be attributed to carboxylic acids, acetyl groups, and esters of the material. The intense absorption band at 1030 cm−1 is caused by CO bonds stretching and by the deformation of alkoxide groups present in cellulose and ester groups present in lignin [A more detailed analysis of the CHFW composition was obtained using FTIR analysis. n lignin ,16,26. −1 and third 2358 cm−1 absorption bands. Mercerization treatments in −1, thereby proving the effectiveness of the method for removing hemicelluloses, as noticed in The analysis of the FTIR spectra generated after all treatments b–d revea−1 are related to an increased lignin content in all materials after the treatments [Changes observed in the fourth absorption band of 1608 cmeatments . In all eatments . It is known that cellulose, unlike hemicellulose and lignin, has a semicrystalline structure due to the interactions of hydrogen bonds and van der Waals forces between its constituent molecules . SpecifiThe in natura CHFW diffractogram  shows twCollazo-Bigliardi at al.  and SungAspergillus flavus fungi. In another study, Ilyas et al. [The removal efficiency of extractive compounds and the greater exposure of cellulose molecules through mercerization treatments were reported by other authors for different types of natural fibers wastes such as banana stalk , garlic s et al.  obtainedThe SEM photomicrographs in An increase in alkaline solution concentration damaged the CHFW surface, as illustrated in Hydrothermal treatment was also able to promote the exposure of cellulose microfibrils, as shown in P. chrysosporium fungi. In general, apart from slight roughness, the analysis of the physical and biological treatments in Small particles distributed on the CHFW surface were observed in the photomicrographs of The thermal stability of NFLs is one of the limiting factors regarding the use of these materials as fillers in polymer composites. Therefore, knowledge of their degradation properties is crucial in evaluating these materials’ ability to withstand industrial processing temperatures . The theThe CHFW shows some decomposition steps of its lignocellulosic because of their cell structure degradation, similar to any typical NLF. Being an amorphous material with a lower degree of polymerization and organization, hemicellulose tends to decompose first between 250 and 350 °C. It is followed by cellulose decomposition at 275–450 °C. The latter, despite its higher degree of polymerization, does not have aromatic ramifications in its structure, thereby adding greater stability to the polymer. This is why the lignin gradually decomposes from 250 to 500 °C ,52. The degradation profiles of the CHFW exhibit mass loss around 5%, These results may be related to the higher Ic’s in For the HYD 30 treatment, the first stage of degradation started at 103 °C, a value higher than that of the in natura CHFW at 94 °C, indicating that those samples may have absorbed more moisture during treatment.2·g−1 [To evaluate the influence of the treatments used on the tensile strength of COPU matrix, biocomposites with 20% CHFW were preliminarily evaluated. Based on the results of the chemical, FTIR, and morphological analyses, one treatment was chosen to represent each of the classes, namely, NaOH 5, HYD 30, and BIO 1, as well as in natura CHFW for the control. These treatments improved the CHFW surface area, as shown in 2·g−1 .The tensile strength results of the biocomposites are shown in SEM images in −1. Coffee husk fiber waste (CHFW) was chosen as a filler of polymeric biocomposites to improve their mechanical properties given its mass production and the considerable amount of this agricultural residue. The characterization of this CHFW confirms this renewable resource’s potential application for biocomposites. The chemical treatment achieved the exposure of cellulose microfibrils through the elimination of amorphous and low molar compounds. As such, it provides exposure of the cellulose molecules and the dissolution of the hemicellulose molecules. This was confirmed by using FTIR analysis, which revealed absorption bands converging to 2358 cmP. chrysosporium fungus promoted the effective exposure of cellulose microfibrils after 24 h (BIO 24), still maintaining relatively smooth CHFW surfaces, and the extractive materials were observed by SEM for exposures after 48, 72, and 96 h. The physical treatments by hydrothermal exposure at 120 °C and pressure of 98 KPa were able to expose the cellulose microfibrils after 30 min (HYD 30). However, hydrothermal times of 60 and 120 min promoted damage to the CHFW particles. The biological treatment with c) for all treatment, indicating that this crystalline fraction can improve the composites’ mechanical and thermal properties. However, the SEM photomicrographs reveal that the ideal chemical treatment is for mercerization with 5% NaOH concentration. Microcracks were observed for 10% and 20% concentrations, indicating that the material has been degraded. Increases in the thermal degradation of the CHFW were detected by TGA. The maximum rate values of the degradation associated with DTG peaks for chemical treatment were as follows: NaOH 5, 337 °C; hydrothermal treatment HYD 30, 347 °C; and biological treatment BIO 24 at 347 °C, which are significantly higher than that of in natura CHFW at 314 °C. The XRD results show an increase of CHFW crystallinity index (IThe preliminary tensile test of castor-oil-based polyurethane matrix biocomposites incorporated with 20 wt.% of in natura and different treated CHFW showed that among the optimized condition, the HYD 30 was the only on that resulted in a significant increase: 60% of the tensile strength in comparison to the others. The CHFW treatments and the preliminary tensile result indicate a potential for a low-cost agricultural residue to be incorporated in castor-oil-based polyurethane as sustainable biocomposites, with potential to be applied as plastic wood for household and automobile parts.