Biocatalysis and Agricultural Biotechnology 45 (2022) 102512 Available online 17 October 2022 1878-8181/© 2022 Elsevier Ltd. All rights reserved. Contents lists available at ScienceDirect Biocatalysis and Agricultural Biotechnology journal homepage: www.elsevier.com/locate/bab Optimization of Bacillus subtilis-based fermentation of anaerobic digestate and biohazard-free application in endophyte-assisted hardening of micropropagated plantlets for increasing survivability Md. Abuhena a, b, 1, **** , Jubair Al-Rashid a, c, d, 1 , Md. Faisal Azim a, 1 , Nirmal Chandra Barman b , Md. Niuz Morshed Khan b , Md. Golam Kabir a, b , Noorain Munim Rasul a, b , Zhiyong Huang d, ** , Shahina Akter e, *** , Md. Amdadul Huq f, * a Department of Research & Development, Apex Biofertilizers & Biopesticides Limited, Gobindaganj, 5740, Gaibandha, Bangladesh b Apex Biotechnology Laboratory, Apex Holdings Ltd., East Chandra, Shafipur, Kaliakoir, Gazipur, 1751, Bangladesh c International College, University of Chinese Academy of Sciences. No.80, Zhongguancun East St., Haidian Dist., Beijing, China d Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China e Department of Food Science and Biotechnology, College of BioNano Technology, Gachon University, Seongnam, 461-701, Republic of Korea f Department of Food and Nutrition, College of Biotechnology and Natural Resource, Chung-Ang University, Anseong, Gyeonggi-do, 17546, Republic of Korea ARTICLE INFO Keywords: Anaerobic digestion Digestate Agricultural waste Hardening Bacillus subtilis Serendipita indica Survivability ABSTRACT Anaerobic digestion of agricultural waste helps to decarbonize the energy sector while also in- creasing the use of stabilized residues in agriculture. Although digestate (Dg) contains amino acids, vitamins, humic acids, and micronutrients, using it directly poses an unknown risk to crops due to the presence of pathogens, heavy metals, and excessive ammonia, necessitating processing for agricultural application as fertilizer. The study aimed to minimize the undesired effects of Dg by optimizing fermentation with Bacillus subtilis CW-S and using it as a potential inoculant for en- dophytic-assisted hardening of micropropagated potato plantlets. The single factor test results re- vealed that Dg concentrations of 70%, 80%, and 90% had a greater positive impact on cell con- centration than concentrations of 50%, 60%, and 100%. According to the Box-Behnken design, diluted (80%) Dg produced a higher cell concentration (4.36 × 10 8 CFU/mL) than undiluted (100%) (1.18 × 10 8 CFU/mL). In hardening experiments, Bacillus subtilis CW-S fermented diges- tate (FDg) had a significantly higher plantlet survival rate of 90.25% and a root length of 5.22 (cm) with greater Serendipita indica infection, whereas Dg (unprocessed) had a lower survival rate of 67.88% and a root length of 3.13 (cm) with less Serendipita indica infection. According to the findings, FDg could be employed as a non-threat bioinoculant for widespread agricultural appli- cation. * Corresponding authors.. ** Corresponding author. *** Corresponding authors. **** Corresponding author. Department of Research & Development, Apex Biofertilizers & Biopesticides Limited, Gobindaganj, 5740, Gaibandha, Bangladesh.. E-mail addresses: abbl@apexbiofertilizer.com (Md. Abuhena), jubair@apexbiofertilizer.com, xyjubair@live.com (J. Al-Rashid), mdfaisalazim@gmail.com (Md.F. Azim), nirmal.bge@gmail.com (N. Chandra Barman), niuzkubge12@gmail.com (Md. Niuz Morshed Khan), golam_kabir07@yahoo.com (Md. Golam Kabir), noorain@apexbiofertilizer.com, noorainrasul@yahoo.com (N.M. Rasul), huang_zy@tib.cas.cn (Z. Huang), shahinabristy16@gmail.com (S. Akter), amdadbge100@cau.ac.kr, amdadbge@gmail.com (Md.A. Huq). 1 Equal contributions. https://doi.org/10.1016/j.bcab.2022.102512 Received 2 June 2022; Received in revised form 28 September 2022; Accepted 7 October 2022