Influence of Inoculums Source and Pretreatment on Biogas Production from Cashew Nut Shells ( Anacardium occidentale )

— Bioenergy recovery from cashew nut shells was investigated throughout using efficient inoculums source and appropriate pretreatment. Physicochemical characteristics of shells and inoculums including pH, volatile fatty acid (VFA, total solid, volatile solid, ash were determined using standard methods. Total anaerobes and methanogenic archaea from inocula were determined by MPP method. Wastewater (WW), sludge from bioreactor (SBR), bovine dung (BD) and mixed inoculums (MIX) were used to evaluate inoculums source in batch system. Biochemical methane potential of pretreated shells was evaluated. Biogas was measurement by liquid displacement. CH 4 and CO 2 were performed by GC. MIX and WW showed high concentration of methanogenic bacteria (2.3 10 2 CFU/mL). Best biomethane levels 70.38% with yields of 55.52 L biogas. (Kg VS) -1 was observed with old shells inoculated by MIX. Low productivity was noted with thermal and biological treatment of old and fresh shells, 11.20 and 0.02 L CH 4 . (Kg VS) -1 , respectively. Inoculums source has significant effect on biogas production. Mixed inoculums exhibited significantly high yields of biomethane. Thermo-biological pretreatment seems to be not appropriate for a better biomethane production. Combination of thermochemical and biological pretreatment could be necessary for best biomethane production yield.


INTRODUCTION
Worsening international food problems have created the need to develop agri-food sector in developing countries. Development of agro-food industries releases an important amount of organic waste or biomass which can be used as a feedstock source for bioenergy and or bioproduction. However, this technological way for biomass valorization remains still underused in largescale condition in these countries. And their outdoor accumulation leads to environment and population health problems as highlighted by several studies (WHO, 2015, Franchitti et al., 2020). Nevertheless, some lab-and pilotscale studies have been carried out for converting into bioenergy, biofuels and bio-based products (Mahdy et al., 2015,Na et al., 2021. Agricultural residues depend on locality. The third largest agricultural product for export, after cotton and sesame, is cashew nut which is still a poorly performing sector marked by low yields, processing and marketing. However, cashew production increased by 13% in 2018 (85,000 tons) and the processing rate from 40% (7,000 tons) (COMMODAFRICA, 2019). In Burkina Faso, cashew nut sector is experiencing increasingly significant development with production estimated at 81,000 tons in 2017 and expected to reach 200,000 tons in 2030 (Somé, 2014). Processing units generate a large amount of waste consisting of approximately 73% hulls and 6% skins (Tagutchou and Naquin, 2012). According to Lacroix (Lacroix, 2003), cashew shell contains a toxic acid, cashew nut shell liquid (CNSL) or balm, which makes production of almonds painful (during shelling). Improper drying procedures can create potentially hazardous situations for environment following release and infiltration of CNSL into soil. This would cause the death of trees in the area and infertility of the soil for several years. Cashew processing units are confronted with recurring energy problems. They use unsustainable energy sources such as wood and butane gas at excessive costs, leading them to resort to cashew shells as fuel in the processing chain, in particular for the embrittlement of nuts, cooking with steaming and drying almonds (Thiombiano et al., 2011). Godjo et al. ( 2015) showed cashew nut shells burning causes significant damage to environment and human health. Cashew nut consists of hard woody shell containing liquid (Cashew Nut Shell Liquid CNSL). CNSL is composed of 70 to 90% anacardic acid, 10 to 18% cardol and about 5% cardanol (Das, Sreelatha, & Ganesh, 2004;Patel, Bandyopadhyay, & Ganesh, 2006). Most cashew nut upgrading work is oriented towards CNSL extraction processes, some only talking about thermochemical treatment, in particular pyrolysis and gasification (Das et al., 2004, Singh et al. 2006, Tsamba et al. 2006). Faced to increasingly growing energy demand in processing units, and environmental problems linked to burning and release of shells into environment, a more ecological recovery of hulls is required. Several authors such as Saka et al. (2009) and Chandel and Singh (2011) showed difficulties during the bioconversion of plant species due to the structure and components of cell walls, which certainly influences digestibility. Indeed, Mahato et al. (2021) was reported possibility of citrus waste biotransformation, bio-waste which is antimicrobial in nature and inhibits fermentation process. The systematic process of bioproducts production from citrus biomass requires pretreatment steps including physical, chemical, physicochemical and biological pretreatment. Leitão et al. (2011) studied anaerobic digestion of cashew bagasse, but found no conclusive results given the complexity of this substrate. Nikiema et al. (2020) indicated the feasibility of biogas production from cashew nut shells and found experimental biochemical methane potential (BMP) was 46.84 CH4 L. (Kg VS) −1 and 1.98 CH4 L. (Kg VS) −1 for old and fresh shells, respectively. However, theoretical values could reach up to 526.206 CH4 L. (Kg VS) −1 and 666.937 CH4 L. (Kg VS) −1 for old and fresh shells, respectively. The presence of certain substances including anacardic acids, cardol and cardanol could explain lower yields observed in experimental study, since these substances constitute a limit to the bioconversion of cashew shells into biogas. Our study aims to contribute to ecological elimination of cashew nut for environment protection and at the same time to agro-resources valorization by bioconversion into bioenergy. Specifically, it aimed at to find out suitable source of inoculum and appropriate way of pretreatment of cashew nut shells for a better biogas production.

Sampling of cashew nut shells and microbial inoculums
The samples required for biogas production have been collected at ANATRANS Company, a cashew scale transformation units, located in Bobo-Dioulasso, Burkina Faso, West Africa. Two types of waste samples were used: eight-year-old shells (OS) and fresh shells (FS) freshly produced. Four (04) types of inoculums were used: wastewater (WW), sludge from bioreactor (SBR), boving dung (BD), mixed (MIX) consisting of three (03) inoculums combination. Slaughterhouse effluents (wastewater and bovine dung) were sampled in anaerobic basin of Ouagadougou refrigerated slaughterhouse (12 ° 24'59 "N; 1 ° 28'29" W). Sludge from biodigester was sampled from biogas production unit of fecal sludge treatment center (CTBV) of Zagtouli in Ouagadougou. Sludges were sampled in 20 L flasks containing nitrogen gas (N2) to maintain anaerobic conditions.

Mechanical pretreatment of cashew nut shells
Old hulls samples were crushed to obtain particles size ≤ 1 mm in diameter while new hulls were grounded to obtain cake ( Figure 1).

Physicochemical characteristics of inoculums
pH was determined a described above. Total solid (TS), ash (As), volatile solid (VS) in sample were determined according to Sakaki (2014) methods. Titration assay method was used to determine volatile fatty acid (VFA) content. A volume of 25 mL of reactor supernatant was collected in a beaker and stirred. Initial pH was read. Using a 1/10 mL burette, a volume of sulfuric acid (0.1 mol. L -1 H2SO4) was poured until pH = 4. The volume poured was noted (V1) then the liquid was boiled 3 min. After cooling, Na2CO3 (0.05 mol. L -1 ) was poured to pH = 7, and volume V2 poured in noted. Tubes showing turbidity are considered positive for total anaerobes.

Thermal and biological pre-treatment of cashew nut shells
Thermal and thermobiological pretreatment was realized using method described by Fadil et al. (2003) and Aissam (2003) for biodegradation of effluents from olive oil production was adapted for the pretreatment of cashew shells. The fungus strains Aspergillus niger isolated from old shells matrices on sabouraud medium with chloramphenicol was used as inoculums for biological pre-treatment. Liquid culture medium (100 mL) with 2% (v/w) of shells placed in 250 mL Erlenmeyer flasks was used for improving aerobic fermentation. Medium was composed of yeast extract (0.1% w/v), (NH4)2SO4 (0.5% w/v), KH2PO4 (0.4% w/v), MgSO4 (0.05% w/v), NaCl (0.05% w/v). 25%, and pH was adjusted to 5. Erlenmeyer flasks were sterilized by autoclaving at 121 °C for 15 minutes. After fungi inoculation, cultures were incubated during 14 days at 30 °C with shaking at 150 rpm. Controls were uninoculated shells. The experiments were carried out in triplicate.

Assessment of biogas and gaseous metabolites production from cashew nut shells anaerobic digestion
Anaerobic digestion was realized on pretreated shells according to batch method described by Angelidaki

Data processing
Statistical analysis of data was realized using XLSAT 7.5.2 software. Analysis of variance (ANOVA) was performed to compare mean values of the different variables using Fisher's tests at probability p = 5%. Principal Component Analysis was carried out for distribution of biogas production variables from new and old hulls, CO2 and CH4 proportions depending on source of inoculums and pretreatment.

Characteristics of feedstock and inoculums
Physicochemical of cashew nut shells feedstock and inoculums characteristics are pictured in Table 1 Figure 2 shows pH variation during the anaerobic digestion process of shells with regard to inoculums source (SBR, WW, BD and MIX). From first days of anaerobic digestion, pH variation trended towards acidity (pH 6.5). Stability was obtained after 10 th day for all inoculums. This stability depends on inoculums source, because with wastewater inoculum cultures continued to acidify after 20 days. This shows a strong activity of acidogenic bacteria of wastewater on old shells.

Biogas production with regard to inoculums source
Biogas and biomethane production ranged according to inoculums source and type of shells as presented in Table  2. A significant difference of biogas and biomethane values was denoted between different inoculums (p < 0.05). Depending on type of shells, mixed inoculums and wastewater have best yields. SBR, BD and MIX inoculums gave best biogas yields for the old shells 64.

Fig. 3-Prediction of the biochemical methane potential of inoculums according to shell types
The typologies of the variables (Biogas, CH4 and CO2) and cultures according to the inoculums and the type of cashew shells on the factorial plans constituted by axes 1 and 2 are presented in Figure 4a and 4b. In this figure, only the variables close to the correlation circle need to be taken into account. In Figure 4a, there are clearly three groups of variables close to the circle (Biogas, CH4 and CO2, so that the projections on the axes F1 and F2 are 93.47%). Indeed, biogas is made up of CH4 and CO2. The representation of cultures as a function of inoculum and type of shell on the two factorial planes described by the axes F1xF2 (Figure 10a) allows them to be compared according to the production of biogas, CH4 and CO2. BDVC crops have a significant production of biogas. MIXOS and WWOS cultures have high yields of CH4. SBROS crops produce a high CO2 production. batch formed by the cultures from the new hulls shows low production of biogas.

Influence of pretreatment on anaerobic digestion of cashew nut shells
The results of pretreatment influence on shells anaerobic digestion are presented in Table 3. Thermo-biological treatment of cultures of old shells shows a significantly high production (p < 0. Berberis amurensis Rupr juice, a plant whose total content of phenolic compounds is much higher high ranging from 261 to 1074 mg / 100 g (Hassanpour and Alizadeh, 2016). Thermal treatment weakens cell structuring and facilitates enzymatic activity through biological treatment. The differences in biogas production with untreated shells could be explained by the fact that treatment forms improve productivity of CO2 contained in biogas. pretreatment which would contribute to production substances toxic to anaerobic bacteria. These pretreatment conditions cause an increase in the content of phenolic compounds and probable release of hydroxyfulfural and fulfural compounds. Saenab et al. (2017) worked on effects of anacardic acid isolated from cashew nut shells on production of methane and other products in rumen fermentation, found that the inhibition of methane production by l Anacardic acid was lower than that of biogas (crude extract of shells). However, as part of the bioactive compounds present inside the biogas, the contribution of anacardic acid to the total reduction of methane amounted to 77.36% while the contribution of other compounds amounted to 22, 64%. The other compounds besides anacardic acid are cardol, cardanol, dimethyl cardol according to Gandhi et al. (2013) and Njuku et al. (2014). Other forms of pretreatment could reduce the levels of these toxic compounds and thus promote the production of biomethane. Whether old or fresh shells, pretreatment is an essential step in increasing accessibility and biodegradation of macromolecules by anaerobic microorganisms. Indeed, Ghaderi-Ghahfarrokhi et al. (2017) investigated the effects of different treatment processes like boiling, autoclaving, roasting and soaking in solutions (water, acetic acid, NaOH and NaCl) on the removal of polyphenolic compounds from varieties of acorns, namely Quercus brantii var. persica and Quercus castaneifolia var. castaneifolia. According to these authors all the processes applied, with the exception of roasting, resulted in a significant decrease (p <0.05) in the polyphenol content. Boiling reduced the polyphenol concentrations by approximately 52%. A considerable reduction in anti-microbial substances could lead to a significant improvement in biomethane production. In a column, the values which have a letter in common do not present a significant difference according to the Fischer test (LSD) at the 5% threshold, TTBOS: Thermal and biological treatment of old shells, TBOS: Biological treatment of old hulls, T-TTBOS: Control-TTBOS, T-TTBFS: Control-TTBFS.

IV. CONCLUSION
The source of inoculums has a significant effect on biogas production from cashew shells. Mixed inoculums exhibited significantly high biomethane productions with old and new cashew shells. Thermobiological pretreatment of old and new shells performed was not effective in biogas production. It would be advisable either to experiment other forms of pretreatment and to find optimal time of biological pretreatment in order to increase yields or to examine agronomic possibility.