Physicochemical Characterization of Cattle Dung Fibre Under the Hydrothermal Process

Cattle dung bre is a lignocellulosic material abundant and sustainable non-wood source of polymeric components, which can be converted into high added-value products. Hydrothermal treatment of the bre obtained from cow dung was explored using four different temperatures (120, 140, 160 and 180 ◦ C) and incubation times (0 and 120 minutes) at a xed material to water ratio (1:10). The present study resulted in the highest yield of 94% (w/w) that gradually decreased with temperature and incubation time. The physicochemical analysis revealed that hydrothermal treatment resulted in high cellulose, low lignin, and ash content (51.6%, 30.93%, and 6.3%, respectively) at 160 ◦ C for 2 hr incubation time and was appropriate for pulp and paper production. The SEM and X-ray crystallography indicates the treatment resulted in separated brils and a porous structure. Both FTIR studies and chemical characterisation techniques were used to optimize the temperature and duration of hydrothermal treatment. Overall, the study presents the rst report on the extraction of bres from cow dung and their hydrothermal treatment. In perspective, it is possible to achieve the properties required for its industrial-scale conversion to eco-friendly papers by heating the bre under controlled conditions.


Introduction
Paper plays a signi cant role in our daily life. Due to the increasing human population, the demand for paper and its products is increasing daily. The global annual paper and cardboard production was reported to be more than 400 million metric tons in 2020, with China and United States as the primary paper producing countries. However, the said demand for paper is higher than the overall global paper production capacity (Statista report 2020a). Various types of wood (90%) and plant materials (10%) are used to prepare paper, ultimately diminishes forest resources. To reduce the environmental impact, deforestation and maintenance of circular economy, it is imperative to look for alternative materials for the generation of bre and pulp.
Many of the commonly available sources of bre are generally undervalued and ignored. Agricultural waste, and livestock-based waste or bio-residue, are potential sources of bre, especially in any agricultural-driven country.
The global livestock population is around 987.51 million heads, with India's share as 303 million heads, followed by Brazil, the United States, and China in 2020 (Statista report 2020b). India also had the most signi cant number of cows in the world in 2019. The rearing of many cows results in producing a large amount of cow dung. Various bovine-dairy based industries nd it challenging to manage such a large amount of generated animal dung (Kleinman et al., 2019). Researchers have reported that around 2600 million tons of dung is generated and is a source of greenhouse gases like methane and nitrous oxide (Kaur, 2017). This untreated bio-residue, when leached into water bodies and can contaminate the groundwater as well. There is a strong need to develop techniques to valorize this waste material and convert it into value-added products that can help drive the economy of rural and urban areas.
Although manure production from cow dung is already a well-established technique worldwide, it remains unexplored in the domain of bre extraction, which has tremendous potential. Cattles generally chew food through the cuddling process, and undigested food is ejected out through excreta. It has been found that around 70-80% of cellulose becomes a part of dung. Studies have shown that bre with an average length of 0.8-1.3mm and a 900 to 985 (µm) diameter can be extracted from cow dung (Fasake and Dashora, 2021a). The presence of a naturally aligned carbon-carbon bond can endow these naturally occurring bres isolated from bio-residue with high strength and stiffness (Jústiz-smith et al., 2008). The overall qualities of this bre is a vital function of the type of animal species, age, and eating habits (Zhu et al., 2020).
Researchers have initiated studies on the extraction of bres from two different types of Indian-breed cows, i.e. Indigenous cow, a Jersey cow, and a buffalo. The amount of cellulose (29-31.50%) obtained from these bio-residues is comparable to wheat straw (~ 39%), indicates its potential to be used in the production of paper (Fasake and Dashora, 2020b). High cellulose in the biomass furnishes it with suitable properties to make the form strong. In the paper industry, lignin is an undesirable component in the pulp and needs to be removed using a suitable reagent (Kaur et al., 2019). Removal of lignin using chemical and mechanical means can help utilize this pulp to prepare paper and various innovative products like pots, cartons and trays, etc. (M'Hamdi et al., 2017). Hydrolysis, a hot-water treatment (150-250 0 C), is used to solubilise polysaccharides and increase the cellulosic fraction. The generation of acetic acid by hydrolysis of the acetyl group is expected to catalyze the hydrolysis of hemicellulose. Lignin is removed, and the residue is enriched with cellulose (Caparrós et al., 2008). This hydrothermal treatment of pulp does not use expensive chemicals and is thus eco-friendly and cost-effective.
Thus, the present study aims to create cow dung bre and perform its hydrothermal treatment. The chemical characterisation of differently treated samples was performed using glucose, xylose, arabinose, ASL (Acid soluble lignin) and AIL (Acid insoluble lignin), crystallinity and ash content as parameters. Change in surface morphology was investigated using Scanning Electron Microscopy (SEM) analysis. The effect of hydrothermal treatment on the chemical composition of bre was further investigated using Fourier Transform Infrared Spectroscopy (FTIR) analysis. New green treatment has been proposed for the rst time to provide an elaborated outlook for the effective management of cattle dung waste and the successful production of bre for the paper industry.

Sample Collection and preparation
Raw animal dung sample was received from Mandir gaushala, Kishangarh, Maheroli, New Delhi. Under the author's observation, a trained person from gaushala handled animals and the dung collection. The slurry of fresh cow dung was prepared by mixing it with water and subsequently passed through different sieves, as mentioned in (Fasake and Dashora, 2020a). The semi-digested dung bre particles obtained through BSS 20 and BSS 40 sieve were collected and kept at room temperature for one day with overturned occasionally using sterilized wooden sticks. After attaining the safe moisture level, the dung bre material was stored in separate airtight plastic bags under dry conditions for subsequent experiments.

Hydrothermal treatments
Hydrothermal treatment is an environmental-friendly technique for treating cattle dung bre as it does not involve any toxic chemicals. There is no associated equipment corrosion problem with it. Also, the use of water only makes it an economically feasible process. Figure 1 shows the hydrothermal treatment based cellulose-rich bre production scheme most suitable for the paper industry.
The hydrothermal treatment was performed in a laboratory-scale batch reactor procured from Amar equipment, Mumbai, India, with a maximum volume of 2000 mL. The 100 g samples were mixed with 1000 mL of deionized water and heated at designated temperatures (120°C, 140°C, 160°C, and 180°C) with different incubation times (0 min and 120 min) with the heating rate of 4°C/min. As the planned condition was achieved, tap water was immediately opened through the stainless-steel cooling ring inside the reactor to reduce the inside temperature to room temperature. Figure 2 shows the picture of the reactor used for the study. The temperature dropped rapidly at the start of the cooling process, and the cooling time varied depending on the pretreatment temperature. After the reaction, the solid fractions were collected by ltration and thoroughly washed to the neutral pH. The yield percentage was calculated by the initial weight of the sample divided by the nal weight.

Chemical Characterization
The chemical composition of the raw dung bres and hydrothermally treated (HT) samples were determined and compared. The content of sugars in the solid dung bres was measured after substantial acid hydrolysis of the polysaccharides. 300 mg milled samples were treated with 3 mL 72 % (v/v) H 2 SO 4 at 30°C for 1 hour.
The hydrolysate was diluted with 84 ml distilled water and autoclaved at 121°C for 1 hour. The solution was ltered through crucibles. The aliquot will be used to determine acid-soluble lignin (ASL) and carbohydrate.
The Acid insoluble lignin (AIL) was determined based on the lter cake subtracting the ash content after incineration at 575°C for 6 hrs using the National Renewable Energy Laboratory (NREL Protocol, CO, USA, 2012). The content of sugars in the liquid extract was measured after acidic hydrolysis of the polysaccharides. The High-performance liquid chromatography (HPLC) analysis was performed (Agilent 1200 series, Agilent Technologies, Santa Clara, USA) with an Aminex HPX-87P column at 60°C. The 5mM sulfuric acid (5mM) was used eluent at the ow rate of 0.5 ml/min. The components were detected using a RI detector. Ash content was determined by TAPPI -T 211 cm-02 standards method.

Scanning Electron Microscopy (SEM)
The microstructural changes like surface texture and porosity of the sample were characterized by scanning electron microscopy (SEM). The raw and treated material was rst converted into a ne powder and then observed on a model TM-3000 scanning electron microscope (HITACHI, Japan). The bre samples were placed randomly over the silver tape, sprayed and xed pieces on a thin gold coating (Emitech K550X) to minimize its charging because of non-conducting materials. Images were taken at several magni cations ranging from 100× to 6000×.

Fourier Transform Infrared Spectroscopy (FTIR)
Fourier Transform Infrared Spectroscopy studies were performed to understand the change in functional groups in the sample before and after bre hydrolysis. The analysis of the powdered dried sample was performed using Nicolet iS10 FTIR system (Thermo sher Scienti c, Altham, MA, USA). All the spectra were recorded in 400-4000 cm − 1 wave number.

XRD analysis
The crystalline structure of cellulose was determined by X-ray diffraction analysis (Rigaku Ultima IV, Ri, Tokyo, Japan). Both samples were scanned at the diffraction angle ranging from 3 to 60 0 with a scan speed of 2 0 /min. The crystallinity index (CrI) was calculated according to (Segal and Martin, 1959). I 002 and I AM are the intensity of crystalline and amorphous phase localized at 22° and 18° in 2θ.

Statistical Analyses
The experiments were repeated in triplicate, and the results are reported as the mean of replicates with standard deviation (mean ± SD) of the values.

Results And Discussion
Chemical Characterisation Hydrothermal treatment of raw material was performed by placing it in a reactor surrounded by an aqueous medium at an elevated temperature and pressure. According to Liu et al., 2012 the temperature below 100 • C has no hydrolytic effect on the material, and at above 220 • C, cellulose degradation occurs. Thus, the present study was planned in a temperature ranges between 120 to 180 • C for 0 min and 120 min reaction time. After conducting a preliminary optimization experiment, the material to water ratio (1:10) has nalized according to the apparent and bulk density (Fasake and Dashora, 2021c).  Table 1 shows the effect of hydrothermal treatment on the chemical characteristics of raw material. The maximum yield of 93% was obtained at the temperature of 120 0 C with no holding time. It was found to decrease with a further increase in temperature or contact time. The increase in contact time did not help in improving yield. The raw sample exhibited glucose, xylose, arabinose as 37%, 20.1%, and 2.7%, respectively.
No regular pattern on the effect of temperature on xylose and arabinose content was observed.
In contrast, long incubation times noticeably changed the chemical, morphological parameters and yields.
The glucose and xylose contents were constantly varied up to 140°C for 2 hr incubation time and then abruptly changed when temperatures higher than 160°C were used. The increase in temperature and contact time resulted in the lowering of xylose and arabinose content. It might be because of the removal of Page 7/14 extractives and hemicellulose fraction by hydrothermal. The treatment of biomass to a higher temperature (180 0 C) resulted in low yield (33-38%) and glucose content (60%).
Compared to increasing the reaction temperature, increasing incubation time is more e cient in booming cellulose bioconversion. Similar observations were reported by Yu et al. (2010). As shown in Table 1 Raw dung material has a porous structure that includes lumens and pits, as shown in Fig. 3. When dung bres were immersed in the water, initially, the bre surface was socked. Later, due to penetration, bre became wet. The capillaries are helpful to ll the void space and the amorphous regions of the cell wall. The sorption of water into the bre is a complex process. In pulp preparation, water diffuses into the amorphous regions of the cellulose matrix and breaks inter-molecular hydrogen bonds between cellulose surfaces.
Swelling bres increases the volume, while the surface area does not expand (Botkova et al., 2013). This swelling effect increases the inter-molecular distance of the cellulose chains. It also facilitates the diffusion of sugars and oligomers of hemicelluloses to the aqueous medium (Rowell, 2016). The ash content was observed between 6 to 8% in controlled and all hydrothermally treated samples and which is equal and less than the other non-wood materials, such as Bagasse (8.02%), rice straw (20.02%), corn stover (7.82%) etc.
Generally, the lower ash content indicates maximum pulp yield with good quality of the paper. Chemicals play a crucial role during the production process of the paper. The soda and kraft process is the most commonly used method for treating wood and non-wood related materials. Making paper with the help of chemical is not an economically feasible process for cattle dung bre. The lignocellulosic fodder material has processed into cattle's rumen system using bacteria, microbes and get a semi-digested material in the form of fresh dung. One kg of fresh cattle dung gives 10-12% raw bre depending on their diet and other factors reported by Fasake and Dashora 2020.
Yield plays a vital role in any processing industry. The chemical method reported resulted in about 50% yield, which means that about half of the dung bre material was lost during the digestion process at 140°C for 2 hr with only 1% NaOH. In the present studies, the hydrothermal treatment resulted in a yield of 82% at the same temperature and time. Though slightly higher cellulose and lower lignin content were reported in chemically treated samples, composite hydrothermal treated samples are also suitable in the making of paper.
Scanning  Table 1. The unusual behaviour of bres reported for CrI% has observed in FTIR also where peaks crossed each other in the region of 4000-3000 cm -1 .

Conclusion
In conclusion, the analysis of the hydrothermal treatment of bres obtained from cow dung has suggested that lignin removal can be achieved e ciently. No regular pattern of change was observed in the case of xylose and arabinose content with temperature change. The temperature and incubation period increased the retained glucose percentage, which could be due to the simultaneous degradation of the hemicellulose carbohydrates. The decreased amorphous hemicelluloses and lignin contents were also observed. This nding was accompanied by a gradual increase in crystallinity index with temperature. The ash content between 6 to 8% in both controlled and treated samples was less than other non-wood species indicating the maximum pulp yield with excellent paper quality. SEM analysis showed the porous and cracked surfaces of treated samples. Findings of FTIR were similar to the properties observed during chemical characterisation.
Heating for 120°C for 0 hours was regarded as the optimum temperature for the maximum lignin removal and enriching the pulp with cellulosic content. Although chemical treatment may result in higher lignin removal e ciency, economic feasibility and environmental sustainability need special attention in that case.
Hydrothermal treatment of cow dung bre is an e cient process as it serves the purpose of valorising waste dung.

Declarations
Funding: This research did not receive any speci c grant from funding agencies in the public, commercial, or not-for-pro t sectors.
Con icts of interest/Competing interests: The authors declare that they have no con ict of interest.
Availability of data and material: Not applicable  FTIR spectra of dung samples (untreated and hydrothermally treated)