Waste Management 159 (2023) 84–92 Contents lists available at ScienceDirect Waste Management journal homepage: www.elsevier.com/locate/wasman Using green waste as substrate to produce biostimulant and biopesticide products through solid-state fermentation Golafarin Ghoreishi, Raquel Barrena *, Xavier Font GICOM research group, Department of Chemical, Biological and Environmental Engineering, Universitat Autònoma de Barcelona, Edifici Q, Carrer de les Sitges, 08193 Bellaterra (Cerdanyola del Vallès), Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: Although the use of green waste as a substrate in different types of microbial bioprocessing has a major impact on Green waste improving green waste valorization, very little information has been provided on this issue. The purpose of this Solid-state fermentation paper is to study the feasibility of using green waste to produce a biostimulant (Indole-3-acetic acid (IAA)) and Trichoderma harzianum biopesticide (conidial spore) through solid-state fermentation. Trichoderma harzianum was selected as the inoc­ Indole-3-acetic acid ulum of the process and the green waste was a mixture of grass clippings and pruning waste. An experiment was designed to study the effect of tryptophan concentration, proportion of grass and pruning waste, and substrate moisture on IAA and spore production. The results show that washing and using phosphate buffer has a beneficial effect on green waste quality in terms of bioproduction. The maximum IAA and spore productions reported in the current study were 101.46 µg g− 1 dry matter and 3.03 × 109 spore g− 1 dry matter, respectively. According to the results, IAA production increases with a higher amount of tryptophan and grass. However, the number of spores increased with lower amounts of tryptophan and grass. The model suggested the following optimized parameters for the production of spores and IAA: tryptophan 0.45 %, grass 61 %, and moisture 74 %. The effect of fermentation time was also studied, and the results show that the maximum IAA and spore production was obtained on days 3 and 7, respectively. 1. Introduction GW has been also reported as a potential source of energy for microbial processes, which has led to the production of marketable products such Sustainable agriculture needs innovative strategies to increase pro­ as enzymes, polyaromatic hydrocarbons (PHAs), and lipids (Cerrone ductivity within the framework of the circular economy. To this end, the et al., 2015; Langsdorf et al., 2021). Recently, GW has even been used as use of plant wastes as the substrate to produce valuable agricultural a substrate to produce bio-hydrogen (Yue et al., 2021). bioproducts is a considerable step towards improving sustainability. Considering the solid structure of GW, solid-state fermentation (SSF) Green waste (GW) is plant-based, and has become one of the most can potentially be one of the primary options for bioproduction. SSF is a abundantly available forms of organic waste (Wang et al., 2021). It is process that occurs in the absence or near absence of free water. To date, categorized among municipal wastes and is generally considered to refer SSF has been utilized to generate marketable bioproducts such as en­ to the biodegradable waste collected from public parks and private zymes, biosurfactants, biopesticides, biostimulants, and many other gardens. It consists of wood, pruned parts of trees and shrubs, grass products that are of use to the industrial, agricultural, and pharmaceu­ clippings, and leaves that are mixed with soil. As urban areas expand, so tical sectors (Ballardo et al., 2017; Jiménez-Peñalver et al., 2019; Mejias too does the number of municipal green spaces. This leads to a larger et al., 2018; Sala et al., 2021). amount of GW that needs to be properly managed (Langsdorf et al., One of the major challenges for using GW as a substrate in SSF for 2021; Reyes-torres et al., 2018). microbial bioproduction is the heterogenicity of its composition. The For many years, GW has mainly been used for composting (Inghels type of plant source, season, and collection and preservation methods et al., 2016; Liguori et al., 2013). More recently, it has also been used as can change the composition of GW and consequently its potential for a substrate for the production of biochar, biogas, and bioethanol (Jesus bioproduction. However, conditioning procedures can minimize the et al., 2017; Karami et al., 2011; Świechowski et al., 2019). However, effects of GW heterogenicity and make the process easier to control * Corresponding author. E-mail address: raquel.barrena@uab.cat (R. Barrena). https://doi.org/10.1016/j.wasman.2023.01.026 Received 16 November 2022; Received in revised form 10 January 2023; Accepted 24 January 2023 Available online 3 February 2023 0956-053X/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). G. Ghoreishi et al. Waste Management 159 (2023) 84–92 (Inghels et al., 2016; Langsdorf et al., 2021). approximately 1 cm length washed with tap water. It was then dried in a Biostimulants have a promising role to play in making agriculture 50 l reactor that was aerated by 5 l min− 1 constant air at 22 ◦ C for 9 days. more sustainable. They stimulate plant growth by improving charac­ The substrate was agitated manually once a day. After drying, all the teristics such as the efficiency of nutrient use, tolerance of (a)biotic substrates were preserved at − 20 ◦ C. The pruning waste was already stress, and availability of confined nutrients in the soil or rhizosphere crushed at the time of collection, and was directly transferred for storage (Baltazar et al., 2021; Carletti et al., 2021). A wide range of substances at − 20 ◦ C. and also microorganisms can be utilized as biostimulants. Examples of Before fermentation, the substrates were sterilized twice at 121 ◦ C non-microbial biostimulants include humic substances, protein hydro­ for 30 min. The moisture was then adjusted using sterilized potassium lyses, and seaweed extract. The most used microbial biostimulants are phosphate buffer (pH = 6.8) to prevent high pH alteration. Pseudomonas spp., Enterobacter spp., Bacillus spp., Rhizobium spp., Streptomyces spp., Trichoderma spp. Aspergillus spp., and arbuscular 2.2. Inoculum mycorrhizal fungi (AMF) (Drobek et al., 2019; Visconti et al., 2020). The European Biostimulant Industry Council (EBIC) has estimated the Trichoderma harzianum (TH) (CECT 2929) provided by the Spanish annual growth of biostimulant products in the market at around 10 to Type Culture Collection (CECT) was the fungus used as inoculum in this 12 % (EBIC, 2020). study. Trichoderma viride (TV) was also used in the time course experi­ Trichoderma strains have predominantly been used as biostimulants ment as inoculum. This strain was provided by the Biological Control due to their capacity to control fungal phytopathogens and improve Subdirectorate (SCB) SENASA Laboratory in Peru. plant tolerance against abiotic stresses. This fungus is a well-known The original strains were kept at − 80 ◦ C in sterile cryovials with 10 biocontrol agent and has been successfully used as a biopesticide, % glycerol. For inoculum preparation, in all tests, TH and TV were especially in the form of conidial spores (Sala et al., 2020; Zhang et al., cultured on malt extract agar and potato extract agar plates, respectively 2022). Moreover, some strains of Trichoderma, including T. harzianum, at room temperature for 6–8 days and diluted to approximately 106 T. virens, and T. atroviride can affect a plant’s root activity, and some of spores g− 1 of dry substrate before inoculating. its metabolic pathways, by releasing metabolites to the rhizosphere. Auxin is a plant hormone that can be produced by plants and microor­ 2.3. Solid-state fermentation ganisms. Specifically, it is one of the metabolites produced by fungi as a signal to interact with them. The plant produces organic compounds that 2.3.1. Experimental setup fungi can use as nutrients to grow and in turn improve the plant’s own The experimental setup included a 0.5-l packed reactor consisting of growth by producing auxin (Keswani et al., 2020; López-Bucio et al., an inlet and outlet to pass the air through the material inside the reactor 2015; Visconti et al., 2020). Indole-3-acetic acid (IAA) is the most with a constant flow rate (20 ml min− 1). The airflow was provided by a common form of auxin. This hormone plays an important role in many mass flowmeter (Mass-Stream D-6311, Bronkhorst, Netherlands). The physiological processes in plants, including apical dominance, tropisms, reactors were kept in a water bath during fermentation to control the shoot elongation, cambial cell division induction, and root initiation temperature. An electrochemical sensor (Alphasense, UK) was used to (Dong et al., 2022; Jahn et al., 2021). Indole-3-acetic acid can be syn­ measure the oxygen content of the output gases. The airflow and oxygen thesized by several pathways in microorganisms. However, its produc­ consumption data were recorded and analyzed by a non-commercial tion generally occurs through tryptophan (Trp)-dependent or non- self-made software based on Arduino®. dependent pathways (Jahn et al., 2021). The consumed oxygen was calculated as a specific oxygen uptake The microbial production of IAA using a standard culture medium as rate (sOUR) to evaluate microbial activity. Cumulative oxygen con­ the substrate and Trp as a precursor, if necessary, has been thoroughly sumption (COC) was also determined by calculating the area below the investigated (Ikram et al., 2018; Numponsak et al., 2018; Shokri and oxygen consumption curve. sOUR was determined using a formula Emtiazi, 2010). However, only a limited number of studies have described in equation (1). addressed organic waste as the main substrate. Bose et al. (2013) used Jatropha seedcake as a substrate and a kind of white rot fungus to P × 32 × 60 × 103 sOUR = F × (0.209 − yO2 ) × (1) produce IAA through submerged fermentation (SmF). Also, in a more R × DW × 103 recent study, auxin was produced by SSF using different agricultural where, sOUR is the specific Oxygen Uptake Rate (g O2 kg− 1 DM h− 1); wastes and microorganisms (Zanoni do Prado et al., 2019). Fungal F is airflow (ml min− 1); yO2 is the oxygen molar fraction in the exhaust biopesticide production through fermentation has also been studied gases (mol O2 mol− 1); P is the constant pressure of the system (101325 recently using Trichoderma spp. and different types of agricultural and Pa); 32 is oxygen molecular weight (g O2 mol− 1 O2); 60 is the conversion biological wastes (de Rezende et al., 2020; Sala et al., 2021). However, factor from minute to hour; 103 is the conversion factor ml to l; R is ideal there is a lack of information on the use of green waste as the substrate of gas constant (8310 Pa l/K mol− 1); DW is the initial dry weight of solids fermentation. in the reactor (g); 103 is the conversion factor g to mg. This study aims to produce indole-3-acetic acid and conidial spores of T. harzianum using GW as substrate through solid-state fermentation. 2.3.2. Experimental design There are two main objectives. The first is to study the potential of green To analyze the effect of different factors on spore and IAA produc­ waste for use as the substrate for bioproduction. And the second is to tion, a Box-Behnken design was conducted with 15 runs. Three numeric analyze the factors involved in IAA and conidia production in order to factors with two responses were selected for the design. The factors had optimize the amount of both IAA and spores in the process. three levels including a maximum, a minimum, and a center point. The selected factors were tryptophan concentration, G to PW ratio, and 2. Materials and methods initial moisture, while the responses were the number of spores and the IAA concentration. 2.1. Substrate To set up the experiment, 15 reactors were loaded with 80 g of wet material. The substrate was conditioned as described above and the The green waste used in this study included grass clippings (G) and fermentation temperature was set at 25 ◦ C following the study by Sala pruning waste (PW) collected from the campus of the Universitat et al. (2020). The reactors were harvested after 7 days and a represen­ Autònoma de Barcelona, Spain. The pruning waste originated from a tative sample was taken for the following analysis: IAA analysis, spore mixture of different trees. count, and respiration indices. Dry matter, pH, and organic matter were The grass used for all tests was first crushed into pieces of also analyzed according to standard procedures (US Composting 85 G. Ghoreishi et al. Waste Management 159 (2023) 84–92 Council, 2002). heterogenicity of the GW and improve the quality of fermentation (Inghels et al., 2016; Langsdorf et al., 2021). In this regard, a large 2.3.3. Time course experiment enough amount of grass and pruning waste was collected for use in all A time course experiment was carried out to investigate the different the experiments in this paper. Also, different conditioning procedures factors during a specific period. To perform a 10-day time course were used for the grass and the way they affect the sporulation of TH was experiment, 10 reactors were filled with 80 g of the wet material that studied. The conditions were as follows: A) Wash the grass and dry it had been conditioned beforehand. All reactors were prepared according with airflow, B) Add phosphate buffer to the grass before starting to the optimized condition estimated in the experimental design. The fermentation, and C) Use conditions A and B together. fermentation temperature was 30 ◦ C. Every day, one reactor was The results of using different conditioning procedures on TH sporu­ sampled and then discarded. The sample was used for spore counts, IAA lation are presented in Fig. 1A. As observed, after washing and drying concentration, pH, dry matter, organic matter, and respiration indices. the grass, no TH growth was detected. The initial and final pH were 6.42 ± 0.02 and 8.23 ± 0.01, respectively. However, after using buffer TH, 2.4. IAA analysis growth reached (3.7 ± 2.4) × 108 spore g− 1 dm and the final pH was 7.57 ± 0.39. The maximum number of conidia was (2.0 ± 1.1) × 109 For IAA extraction, 10 g of the fermented sample was mixed with 50 spore g− 1 dm obtained when the grass was washed, dried, and buffer ml of distillate water in a 500 ml Erlenmeyer and shaken for 20 min at added, and the final pH was 7.46 ± 0.36. The results of the pH mea­ room temperature at 150 rpm. The extract was separated using a fine surement indicate that a pH value higher than 8 can negatively affect the sieve and centrifuged for 15 min at 4 ◦ C and 10000 rpm. The supernatant growth of TH and the buffer could keep the pH in the tolerable range for was filtrated by a 0.22 µm filter and preserved at − 20 ◦ C for analysis. TH. Zhang and Yang (2015) reported an optimum pH range of 6–7 for For the analysis, a Dionex ultimate 3000 HPLC (Dionex, Idstein, TH which is similar to our finding. Germany) was used equipped with an UltiMate 3000 Autosampler It seems that the conditioning also affected the sOUR profiles of the Column Compartment, UltiMate 3000 Photodiode Array Detector, and grass (Fig. 1B). When the grass was only washed, the maximum sOUR Chromeleon software. An LC Kinetex® 5 µm EVO C18 100 Å column was almost 1.5 g O2 kg− 1 dm h− 1 with a lag phase of 18 h. However, in (250 × 4.6 mm) was used. The eluent was isocratic and involved a so­ the two conditions where the buffer was used, the maximum sOUR was lution of 2.5 % acetic acid in ultrapure water, and 80 % acetonitrile in between 2 and 2.5 g O2 kg− 1 dm h− 1 although the lag phases were ultrapure water. The column oven was set at 30 ◦ C and the flow rate was different. When using buffer alone, the lag phase was longer (35 h) while 0.7 ml min− 1. The fluorometric detector was set at 280 nm. The total run when washing and using buffer together the lag phase was 24 h. Here the was 15 min, and the peak was observed at 11 min. To determine the higher sOUR could be related to the higher sporulation of TH. concentration of IAA in the samples, the area of the obtained peak was In summary, these results indicate that washing and drying the grass compared to that of the standard curve. All the IAA analysis tests were and adding buffer can considerably improve the amount of conidia carried out in triplicate from one extract. production. 2.5. Spore count 3.2. Feasibility of TH growth and sporulation on grass and pruning waste To determine fungal spore production, 10 g of the fermented sub­ In order to study TH growth on G and PW, fermentation was con­ strate was added to 50 ml of Tween 80 0.1 % (v/v), mixed manually for ducted using G, PW, and a mixture of G and PW in a ratio 1:1 (w/w) for 30 s, and diluted according to the procedure reported by Cavalcante 9 days. In view of the occurrence of maximum spore production for TH et al. (2008) with some modifications proposed in Sala et al. (2020). In in 5–6 days in a similar study (Sala et al., 2020), a larger span was all the tests, the spores were counted in triplicate using a Neubauer chosen to be sure that the maximum spore production would be ob­ chamber (Brand™ 717805). The spore concentration per g of dry matter tained during the run time. was calculated by the following formula: Fig. 2A shows the spore production at different percentages of G and PW. On day 5, spore counts were the highest for PW ((9.03 ± 0.01) × N EV SWW SporeConcentration = × × (2) 108. However, after 7 days of fermentation the number of spores DF × CV SWW SDM increased on G and the mixture. On day 9, the spore counts reached the where spore concentration is the number of spores per g dry sub­ maximum on G and the mixture, namely (1.16 ± 0.16) × 109 and (1.22 strate (spore g− 1 dm); N is the number of spores counted in the Neubauer ± 0.13) × 109 spore g− 1 dm, respectively. chamber; DF is the dilution factor; CV is the volume of sample in the The sOUR profile of the SSF process of G, PW, and the mixture is Neubauer chamber (ml); EV is the extraction volume (ml); SWW is presented in Fig. 2B. The maximum sOUR was higher for PW than for G sample wet weight (g ww); and SDM is sample dry matter (g dm). and the mixture. However, COC for the mixture was 110.54 g O2 kg− 1 dm h− 1, which is higher than the COC of G and PW (80.98 and 93.09 g 2.6. Statistical analysis O2 kg− 1 dm h− 1, respectively). The time to reach the maximum respi­ ration was between 24 and 30 h for all the substrates. There was no Design Expert 12 (Stat-Ease, Inc, USA) was used to analyze the re­ strong correlation between sporulation and microbial activity (r = sults of the experimental design including ANOVA and to produce an -0.53). In a similar study, Sala et al. (2021) reported sporulation of TH optimization model. All the graphs were plotted using SigmaPlot 12.5 with different agro-industrial substrates, and they did not find a corre­ (Systat Inc. USA). Paired t-test (p < 0.05) was performed using Sigma­ lation between spore production and the biodegradability of the sub­ Plot 12.5 software to compare the means between treatments when strate either. necessary. Taken together, these results suggest that G and PW seem to be suitable substrates for spore production. As it was mentioned before, no 3. Results and discussion evidence of using grass and pruning waste through solid-state fermen­ tation was found. However, other lignocellulosic wastes especially 3.1. The effect of GW conditioning procedures on SSF agricultural wastes have been successfully used as substrate in solid- state fermentation (Marín et al., 2019; Tanruean et al., 2021; Wang As mentioned earlier, the heterogenicity of the GW could be a et al., 2019). Moreover, regarding the low biological activity of the grass problem for its valorization, especially when it is supposed to be used for and the future scale-up of the process, the mixture probably provides bioproduction. A conditioning process can help to reduce the more advantages in this case. Besides, PW can be used as a bulking agent 86 G. Ghoreishi et al. Waste Management 159 (2023) 84–92 Fig. 1. Different conditioning procedures for the grass. A) The effect of conditioning on spore production and pH. B) Respiration profiles for the condition­ ing procedures. Fig. 2. A) Spore counts in different percentages of grass and pruning waste. B) Respiration profile of different percentages of grass and pruning waste. Fig. 3. A) The effect of Trp (0.5 %) on IAA concentration and spores. B) The effect of temperature on spores and IAA production. 87 G. Ghoreishi et al. Waste Management 159 (2023) 84–92 that will make the mixture more porous (Sala et al., 2021; Ballardo et al., Table 1 2017; Mejias et al., 2018). So, in all the following experiments in this The treatments and results of the Box-Behnken experimental design. paper, the mixture of grass and pruning waste was used as the substrate. Tryptophan Grass Moisture Spore IAA To the best of our knowledge, this is the first time that the use of grass or (%) (%) (%) g¡1 dm (µg g-1dm) pruning waste to produce conidia by TH has been reported. 0.25 20 60 (6.14 ± 0.76) × 108 16.40 ± 5.20 0 50 60 (4.51 ± 2.03) × 108 16.32 ± 5.65 3.3. Effect of tryptophan on spore and IAA production 0.25 80 60 (4.10 ± 1.37) × 108 27.61 ± 2.41 0.5 50 60 (6.90 ± 0.22) × 109 13.36 ± 5.37 0.25 50 70 (1.44 ± 0.19) × 109 16.08 ± 6.85 A preliminary experiment was carried out to test the feasibility of 0.25 50 70 (1.81 ± 0.87) × 109 23.14 ± 7.67 IAA production using GW and the effect of Trp concentration on spore 0.25 50 70 (1.64 ± 0.20) × 109 18.30 ± 7.61 and IAA production. Tryptophan in three different concentrations of 0.5, 0 20 70 (1.61 ± 0.45) × 109 11.63 ± 4.22 1, and 1.5 weight % was added as a powder to the substrate (mixture of 0.5 20 70 (1.63 ± 0.45) × 109 13.97 ± 4.24 0 80 70 (9.02 ± 1.62) × 108 17.11 ± 1.60 G and PW 1:1 mixing ratio (w/w)). The number of spores and IAA were 0.5 80 70 (6.50 ± 6.50) × 108 75.78 ± 8.62 checked after 5, 7, and 9 days. As shown in Fig. 3A, TH did not grow with 0.25 80 80 (6.08 ± 3.01) × 108 31.80 ± 8.53 1, and 1.5 weight % of Trp. It appears that IAA was not produced by the 0.25 20 80 (7.94 ± 0.20) × 108 41.07 ± 4.42 fungi either. However, TH growth with 0.5 % of Trp was high. The IAA 0 50 80 (5.85 ± 1.47) × 108 25.93 ± 10.50 produced by TH were 9.2 ± 2.5, 13.5 ± 2.6, and 6.19 ± 1.12 µg g− 1 dry 0.5 50 80 (5.24 ± 1.45) × 108 36.14 ± 11.30 substrate after 5, 7, and 9 days of fermentation, respectively. The numbers of spores were (1.55 × 109 ± 4.45) × 108, (1.83 ± 0.30) × 109, Zanoni do Prado et al. (2019). Conidia production entailed the use of and (1.61 ± 0.43) × 109 spore g− 1 dm on days 5, 7, and 9, respectively. submerged liquid fermentation and produced 1.38 × 108 spore ml− 1 by The results show that the highest concentration of IAA was obtained on Trichoderma spp. In another study, Cavalcante et al. (2008) reported that day 7, while the number of spores peaked on day 9. However, spore T. harzianum produced 2.28 × 109 spore g− 1 dm through SSF. These data counts on day 9 were not significantly different from the spore counts on show that the number of conidia obtained in this experiment is in a day 7 (p-value = 0.52). Hence, these findings suggest that 7 days may be similar range to the other studies. However, there are reports of higher a desirable period of time for fermentation to produce spores and IAA production of IAA using other microorganisms as inoculum. Luziatelli together. The fermentation time used in the study was 5 days for IAA et al. (2021) set up a fermentation in a 2-l stirred tank fermenter using production. Sala et al. (2020) found that the maximum number of Enterobacter spp. P-36 strain and reported a production of 412.49 mg l− 1 conidial spores produced by TH was obtained in 5–6 days. This incon­ IAA. In another study, a yeast (Rhodosporidium paludigenum) produced sistency in fermentation time could be related to the different substrates 2,743.9 mg l− 1 IAA through a fed batch fermentation (Nutaratat et al., and different lag phases in the fermentation. More information on this 2016). issue will be provided in the following sections. To optimize the production of IAA and spores, a prediction model Based on these results, it is proposed that a mixture of grass and was created using the results of the experimental design, a statistical pruning waste can be a good substrate to produce IAA and conidial analysis of which is shown in Table 2. For spore production, a modified spores by TH. It can also be inferred that a higher value of Trp can quadratic model with basic 10 logarithm data transformation was used negatively affect TH growth and sporulation. It was recently reported and the model was significant. The ANOVA analysis shows that the that in several species of Trichoderma, growth and sporulation decreased percentage of grass is the only significant main parameter for spore in a media containing Trp. On the other hand, the results of previous response. All the terms in the model were significant except for trypto­ studies have proposed that Trp can considerably increase the production phan and moisture. This means that although tryptophan and moisture of IAA in microorganisms that have a Trp-dependent pathway of IAA content are not significant parameters in the model, their interactions biosynthesis (Kumla et al., 2020; Mehmood et al., 2019; Reineke et al., significantly affected the response (spore production). The lack of fit was 2008). Following these studies, it can be assumed that the Trichoderma also not significant, and the model can explain 95 % of the variability of strain used in the current study produces IAA through a Trp-dependent the response (R2). The model produced the following regression equa­ pathway. However, higher amounts of Trp decreased TH growth and tion for the response of spore counts: sporulation. Therefore, an optimized concentration of Trp should be found to produce high amounts of spores and IAA together. log10 (spore) = 9.21 + 0.0313X1 − 0.0862X2 + 0.0346X3− 0.0996 X1 X2 (3) − 0.1268X12 − 0.1005X22 − 0.3394X32 3.4. Optimization of IAA and spore production through SSF (experimental design) where X1, X2, and X3 are tryptophan%, grass%, and moisture%, respectively. The high intercept of the model suggests that the variables As mentioned earlier, the effect of three factors on IAA and spore used in the model are not the most significant for the response (spore production was studied by conducting an experimental design. The production). selected factors and the tested levels were as follows: tryptophan con­ centration (0, 0.25 and 0.5 weight %), grass to pruning waste ratio (1:4, Table 2 1:1 and 4:1 (w/w)), and initial moisture (60, 70 and 80 %). The pa­ The p-value of the model and the parameters for spores and IAA. The model rameters and results of this experimental design are presented in created for spore production is modified quadratic, and for IAA production was Table 1. The maximum spore production was (1.81 ± 0.87) × 109 spore linear (p-value < 0.05 is significant). g− 1 dm when tryptophan, G to PW ratio, and moisture were 0.25 weight p-value %, 1:1 (w/w), and 70 %, respectively. However, the highest IAA pro­ Source Spore IAA duction was the 75.77 ± 8.61 µg g− 1 dm obtained with tryptophan 0.5 Model 0.0005 0.0148 %, G: PW 4:1 (w/w), and moisture 70 %. Mehmood et al. (2019) pro­ A-Tryptophan 0.2280 0.0545 B-Grass 0.0083 0.0339 duced 24.2 µg ml− 1 of IAA in vitro by Aspergillus awamori. In a similar C-moisture 0.1875 0.0320 study, an SSF process set up in 250 ml Erlenmeyer flasks containing 10 g AB 0.0206 of the substrate produced 9.83 and 7.15 µg g− 1 dm of IAA by Bacillus A2 0.0083 subtilis and Trichoderma atroviride, respectively through SSF (Zanoni do B2 0.0235 Prado et al., 2019). Considering the working volume in the current C2 < 0.0001 Lack of Fit 0.3500 0.3418 study, the amount of IAA produced is higher than that produced in 88 G. Ghoreishi et al. Waste Management 159 (2023) 84–92 For analysis of IAA production, the data were best fitted in a linear reactors with the same conditions, which were selected according to the model with the inverse transformation to predict the optimum condi­ optimized parameters predicted by the model. Fig. 5A presents the tion. The regression equation was as follows: profile of spore and IAA production by TH over a 10-day period. Inter­ estingly, the maximum IAA production (101.46 ± 4.11 µg g− 1 dm) was 1 = 0.160878 − 0.041623X1 − 0.000360X2 − 0.001187X3 (4) obtained after 3 days of fermentation whereas the number of spores IAA peaked on day 7 ((3.03 ± 0.86) × 109 spore g− 1 dm). The pH tended to where X1, X2, and X3 are tryptophan%, grass%, and moisture%, increase until day 4–5, going from an initial value of 7.06 ± 0.04 to respectively. reach 8.22 ± 0.03. The pH figures were 8.05 ± 0.07 and 7.84 ± 0.05 The model was significant in terms of p-value with a non-significant respectively when the maximum amounts of IAA and spores were pro­ lack of fit. R2 for this model was 60.03 %. All the parameters in the duced. From then they started to decline and by day 10 had fallen to 7.37 model for IAA were significant. ± 0.06. The respiration profile showed that microbial activity peaked on The optimized condition to produce spores and IAA predicted by the days 1–2, the maximum sOUR value being1.46 g O2 kg− 1 dm h− 1. model were as follows: tryptophan 0.45 weight %, G: PW 1.56:1 (w/w), A second time course experiment was carried out to see if a different and moisture 74 %. Fig. 4 shows that when moisture is 74 % and grass strain of Trichoderma produces IAA and conidia similarly to TH using the content is low, the number of spores increases by Trp in the range of optimized conditions predicted by the model. In this experiment, T.viride 0.2–0.5 %. However, increasing G and Trp improves IAA production was used as inoculum with the same condition of the TH time course (Fig. 4). According to the obtained data, higher grass content (low experiment (Fig. 5B). Here, the maximum IAA was obtained on day 1 lignin) in the substrate can increase IAA production. Zanoni do Prado and the maximum spore production was obtained on day 4, being 18.06 et al. (2019) analyzed IAA production by different microorganisms using ± 5.23 and (4.27 ± 0.43) × 109 respectively. As can be seen from the different types of agricultural wastes and also noted that Trichoderma sp. data, TV produced more spores and a lower amount of IAA than TH. produced more IAA when the substrate has a low lignin content. On the However, note that in both strains, IAA concentration decreased other hand, increasing the concentration of Trp led to an increase in IAA significantly when the number of spores started increasing, even though while the number of spores decreased. Giri and Sharma (2020) opti­ no contamination was detected. The pH increased over time, when the mized IAA production in a submerge fermentation using an experi­ inoculum was TV, starting from 7.05 ± 0.03 on the initial day to 8.15 ± mental design and found that maximum production of IAA obtained 0.04 on day 8. At the time of most spores, the pH was 7.52 ± 0.05 while with higher Trp concentration. Taken together, these results show that when IAA reached its maximum, the pH was 7.20 ± 0.05. The maximum the values of the optimized parameters for spores and IAA are not in the sOUR for TV observed on days 1–2 of fermentation is similar to that of same direction. TH (1.24 g O2 kg− 1 dm h− 1). The optimized time of fermentation to produce IAA was 5 days in the study of Nutaratat et al. (2017). They 3.5. Effect of temperature on spore and IAA production studied the effect of different factors including incubation time on IAA production by a strain of Enterobacter sp. To study the effect of temperature on spore and IAA production, The results of the time course experiments for TH and TV showed three fermentations were set up at different temperatures (25, 27.5, and that there was a similar interaction between IAA concentration and the 30 ◦ C). The optimized condition predicted by the model was used for number of spores produced for both strains. This observation might fermentation. The results indicate that at 30 ◦ C, TH produced more IAA suggest a relationship between IAA and sporulation. The different values (25.5 ± 9.1 µg g− 1 dm) (Fig. 3B). However, the number of spores peaked of the optimized parameters for spores and IAA obtained previously by at 25 ◦ C (1.64 ± 0.72) × 109 spore g− 1 dm) and was (9.43 ± 2.47) × 108 the model also support the hypothesis. It was mentioned earlier that IAA spore g− 1 dm at 30 ◦ C. Napitupulu et al. (2019) reported that the opti­ acts as a signal for the interaction between plant-fungi and fungi-fungi mum temperature range for Trichoderma harzianum growth is between (Tomberlin et al., 2017). Manzo-Valencia et al. (2016) described how 25 and 30 ◦ C. However, for IAA production Zanoni do Prado et al. in some fungal species a low concentration of IAA can stimulate spor­ (2019b) set up a fermentation with Aspergillus flavipe at 30 ◦ C. In another ulation, but high amounts of IAA inhibit it. A similar interaction was study, a temperature of 28 ◦ C was used for Trichoderma spp. to grow and reported to occur between plants and fungi when IAA is the mediator produce phytohormones including IAA (Illescas et al., 2021). (Keswani et al., 2020). It has also been reported that Pseudomonas putida and T.atroviride have the ability to degrade excess IAA to reduce the negative effects on plant growth (Gravel et al., 2007). So, the TH and TV 3.6. Effect of fermentation time on IAA and spore production in the current study might have also degraded excess IAA to increase their sporulation. However, the information and evidence on this issue To obtain more information on the fermentation process, a 10-day are limited and more research is required to elucidate the reasons in time course experiment was performed. This involved setting up 10 Fig. 4. Contour profile of the model’s predicted optimized point. The moisture was set at 74 %. 89 G. Ghoreishi et al. Waste Management 159 (2023) 84–92 Fig. 5. Results of a 10-day time course experiment. A) T. harzianum B) T.viride. more detail. of conidia peaked after 7 days of fermentation on a substrate with lower The pH profile for both strains was not similar, but the pH range was grass and lower Trp. Also, when T. viride was used as inoculum, the the same (approximately 7–8.3) and agrees with that reported in other maximum IAA and spores were obtained at different fermentation times. studies. It was mentioned previously that the optimum pH range for T. Therefore, determination of the best fermentation time for the maximum harzianum to produce spores was reported to be between 6 and 7 (Sala IAA and conidia together seems complicated and needs more research. et al., 2020; Zhang and Yang, 2015). For IAA the optimum pH range is Further studies should be conducted to understand more about the also neutral (5–8) in most microorganisms including T.harzianum relationship between IAA production and sporulation in TH. The (Bharucha et al., 2013; Napitupulu et al., 2019). contribution of more factors like carbon and nitrogen source in the Overall, these results suggest that for both strains, the fermentation production of spore and IAA can also be analyzed, and thus provide the time to obtain the maximum amount of IAA and the highest number of basis to scale up production. spores is different. In the conditions studied, for TH, the maximum IAA was produced at days 2–3 of fermentation while the highest number of Declaration of Competing Interest spores was obtained at days 7–9. However, for TV, IAA production peaked on day 1 whereas the number of spores did so on day 4 of The authors declare that they have no known competing financial fermentation. interests or personal relationships that could have appeared to influence the work reported in this paper. 4. Conclusion Data availability Throughout this article, it is found that despite the limitations due to the heterogenicity of green waste (GW), it can be an advantageous Data will be made available on request. substrate to produce biostimulant (IAA) and biopesticide (conidial spore) through solid state fermentation. Two strains of Trichoderma were Acknowledgements able to grow on GW and produced the marketable products. Optimized conditions for T. harzianum showed that the highest amount of IAA was The authors wish to express their gratitude for the financial support obtained after 2–3 days of fermentation when the grass content of from the Ministerio de Ciencia e Innovación, Spain (Project PID2020- substrate and tryptophan (Trp) concentration were higher. The number 114087RB-I00 and Project FertiLab / PLEC2022-009252). 90 G. Ghoreishi et al. Waste Management 159 (2023) 84–92 References Langsdorf, A., Volkmar, M., Holtmann, D., Ulber, R., 2021. Material utilization of green waste: a review on potential valorization methods. Bioresour. Bioprocess 8, 19. https://doi.org/10.1186/s40643-021-00367-5. Ballardo, C., Barrena, R., Artola, A., Sánchez, A., 2017. A novel strategy for producing Liguori, R., Amore, A., Faraco, V., 2013. Waste valorization by biotechnological compost with enhanced biopesticide properties through solid-state fermentation of conversion into added value products. Appl. Microbiol. 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