# Valorization of Residual Babassu Mesocarp Biomass to Obtain Aroma Compounds by Solid-State Fermentation

Tamires dos Anjos, Robert Wojcieszak, Selma Leite, Ivaldo Itabaiana Jr

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Tamires dos Anjos, Robert Wojcieszak, Selma Leite, Ivaldo Itabaiana Jr. Valorization of Residual Babassu Mesocarp Biomass to Obtain Aroma Compounds by Solid-State Fermentation. Microbiology Research, 2024, 15 (3), pp.1386-1405. 10.3390/microbiolres15030093. hal-04786707

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Article

# Valorization of Residual Babassu Mesocarp Biomass to Obtain Aroma Compounds by Solid-State Fermentation

Tamires N. dos Anjos <fnref n="1" />, Robert Wojcieszak <fnref n="2" />,<fnref n="3" /> ID, Selma G. F. Leite <fnref n="1" /> and Ivaldo Itabaiana Jr <fnref n="1" />,* ID

**Abstract:** In this work, solid-state fermentation (SSF) was applied to babassu mesocarp (BM) for the low-cost bioproduction of natural aroma compounds having *Trichoderma harzianum* (IOC 4042) and *Geotrichum candidum* (CCT 1205) as microbial agents. Fermentation was carried out using in natura babassu mesocarp (IN-BM) and defatted babassu mesocarp through soxhlet extraction (DEF-BM) as support, impregnated with hydration solutions of three and seven salts. The compounds produced were analyzed using solid phase microextraction (SPME) and gas chromatography coupled with a mass spectrometer (GC-MS). Among several aroma compounds detected, 6-pentyl-α-pyrone (6-PP)—GRAS 3696, coconut aroma; 2-phenylethanol (2-PE)—GRAS 2858, rose and honey aroma; and hexanal—GRAS 2557, green apple aroma, were the compounds that were detected with the greatest intensity. The highest concentrations (ppm (w/w)) of 6-PP and 2-PE were obtained in DEF-BM using NS7SG (308.17 ± 3.18 and 414.53 ± 1.96), respectively, while for hexanal, the highest concentration (ppm (w/w)) was obtained in IN-BM using NS7SG (210.83 ± 2.14). The results indicate that producing aroma compounds by *G. candidum* and *T. harzianum* through BM SSF is viable, generating value-added compounds.

**Keywords:** 6-pentyl-α-pyrone (6-PP); 2-phenylethanol (2-PE); hexanal; SSF; agro-industrial waste; solid phase microextraction (SPME); biomass valorization; babassu mesocarp

## 1. Introduction

Given the high growth in the world's population and the real possibility of the finite supply of fossil resources, there is a greater demand for food and energy, which has driven industrialization to obtain a better quality of life. The scarcity of these fossil resources, added to the increase in production and the growing emission of greenhouse gases and other aggravating factors, has led humanity to an increasingly intense search for clean and renewable energy sources, as well as alternatives for proper waste management, by organizations [1-4].

In the current Brazilian economic scenario, agribusiness is prominent and crucial. This sector represented 26.6% of Brazil's Gross Domestic Product (GDP) in 2020, the equivalent of 1.98 trillion reais, and emphasized the fundamental role of agribusiness in Brazilian society. Of this percentage of GDP, 70% comes from the agricultural sector [5,6]. It is also worth noting that agribusiness generates employment and guarantees income for Brazilians, considerably boosting the national economy [6].

However, even in the face of the visible economic advantages that agribusiness brings to the Brazilian economy, many negative aspects can be observed, and the most alarming of these is the environmental impact. The agri-food sector generates large quantities of agri-industrial waste annually, generally disposed of inappropriately in the environment, contributing, for example, to an increase in greenhouse gas (GHG) emissions, pollution

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**Citation:** dos Anjos, T.N.; Wojcieszak, R.; Leite, S.G.F.; Itabaiana Jr, I. Valorization of Residual Babassu Mesocarp Biomass to Obtain Aroma Compounds by Solid-State Fermentation. *Microbiol. Res.* **2024**, *15*, 1386-1405. https://doi.org/10.3390/microbiolres15030093

Academic Editor: Patrizia Romano

Received: 5 July 2024

Revised: 22 July 2024

Accepted: 25 July 2024

Published: 29 July 2024

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**Copyright:** © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

<footnote n="1">¹ Department of Biochemical Engineering, School of Chemistry, Federal University of Rio de Janeiro, Rio de Janeiro 21941-909, Brazil; tamires.anjos@eq.ufrj.br (T.N.d.A.); selma@eq.ufrj.br (S.G.F.L.)</footnote>

<footnote n="2">² Université de Lille, CNRS, Centrale Lille, Université d'Artois, UMR 8181-UCCS-Unité de Catalyse et Chimie du Solide, 59000 Lille, France; robert.wojcieszak@univ-lille.fr</footnote>

<footnote n="3">³ Université de Lorraine and CNRS, L2CM UMR 7053, 54000 Nancy, France</footnote>

<footnote>* Correspondence: ivaldo@eq.ufrj.br</footnote>
of rivers and soils, and the proliferation of disease-transmitting vectors [7]. According to the UNEP (United Nations Environment Programme), in the 2024 food waste index report, approximately 1.05 billion metric tons of food from the total food supply were wasted in 2022 [8].

Agro-industrial waste is the waste generated from the processing carried out by agro-industries and includes husks, seeds, bagasse, straw, stones, and oils, among other materials. This waste comes from processing crops such as sugar cane, rice, babassu, grapes, and barley. Most agro-industrial waste is lignocellulosic, composed of cellulose, hemicellulose, and lignin, and industrially essential polymers [9,10]. In addition, this waste is rich in organic matter, which in turn can serve as a source of proteins, carbohydrates, lipids, essential oils, fibers, enzymes, bioactive compounds, organic acids, and other biomolecules, which can be reused by the pharmaceutical, food, and chemical industries [11–13].

The availability of these nutrients in these raw materials provides a favorable environment for the growth of various microorganisms, including filamentous fungi such as *Trichoderma harzianum* and *Geotrichum candidum*. These microorganisms have a great metabolic capacity to produce aroma compounds, since these substances can result from bioconversion processes or are inherent to the secondary metabolism of these microorganisms. These microorganisms are catalogued as GRAS (Generally Recognized as Safe) and their use in the production of aroma compounds allows these molecules to be used in the cosmetics, pharmaceutical, and food industries, for example, and gives the product a natural connotation. Filamentous fungi such as *T. harzianum* and *G. candidum* can use these raw materials to produce high-value products through submerged fermentation (SF) or solid-state fermentation (SSF). However, better yields have been achieved through SSF [14–17].

SSF is one of the biotechnological alternatives for producing aroma compounds. In this process, the microorganism's growth occurs on solid substrates without free liquid. In this type of process, the amount of free water essential for the development of the microorganism can either be adsorbed onto a solid support or even complexed into a solid matrix for controlled release [18]. Although it is a cost-effective method for producing aroma compounds, SSF still presents some limitations, such as the control of some process variables and mass and heat transfer, for example, which also reinforces the need for studies that seek to overcome these challenges [19]. Filamentous fungi are also somewhat limited, as they grow on the surface and may have difficulty penetrating the gaps in the waste bed [20].

Using agro-industrial waste as a solid support for producing aroma compounds is an advantageous maneuver, as it makes it possible to reduce production costs and obtain a "natural" product through an environmentally friendly process. In addition, using this waste reduces the impact caused by this volume of industrial waste on the environment [17,21].

Babassu mesocarp (BM), which comes from the babassu palm (*Attalea speciosa* Mart. ex Spreng), a plant native to the north, northeast, and central-west regions of Brazil, is part of Brazilian biodiversity and is a nutrient-rich agro-industrial residue with the potential to be exploited in the biotechnological production of flavors. Its composition is basically water, carbohydrates (starch and cellulose), proteins, lipids, and mineral salts. The starchy portion is between 52% and 71% starch [22,23].

Babassu coconut, in which BM is found, is structurally composed of four parts: fibrous epicarp (11–13%), mesocarp (20–23%), woody endocarp (57–63%), and kernels (7–9%) [24–26]. Normally, BM residual is obtained after the extraction of babassu oil generated during the separation of the nut from this fruit [27]. This extractive activity generates a large volume of agro-industrial waste to be discarded, which is readily available for reinsertion into the production chain. In 2022, for example, production of this vegetable reached 30,478 tons [28].

According to a survey carried out by Markets and Markets [29], the global market for flavors and fragrances is looking very promising. Consumers are increasingly concerned
about long-term health, and the effects of using artificial ingredients and additives in
various products have made the population more selective when choosing what to take
home. Due to the growing demand for natural ingredients, the global flavor market was
expected to reach USD 17.1 billion by 2023, with a CAGR of 4.8%. By 2027, the global flavor
market is expected to reach USD 21.3 billion, with a CAGR of 3.5% from 2022 to 2027.

Flavor compounds are chemically complex mixtures that can provide or intensify
strong taste and odor sensations, even at low concentrations. They are mainly used in phar-
maceutical, food, cosmetics, and perfume industries to characterize, improve, standardize,
or reconstruct the aroma/flavor of specific products or mask undesirable aromas/flavors
in the processing of these products. Examples of these products are cakes, sweets, cookies,
yoghurts, oral medication, and soaps [17]. These compounds can be obtained through
microbial biotransformation processes and agro-industrial waste fermentation routes, as
well as enzymatic routes such as esterification catalyzed by lipases, resulting in aroma
compounds considered natural. The advantage of these biotechnological strategies is the
selectivity resulting from the various metabolic steps, where a set of enzymes is able to
generate compounds with enantiomeric purity and high conversions, under mild reaction
conditions, potentially reducing the costs of purifying and/or extracting these compounds
from the matrix. The chemical synthesis of aroma compounds is a process extensively
exploited in the pharmaceutical, chemical, and food industries. However, various chem-
ical catalysts can present low selectivity for the formation of the desired compounds, as
well as requiring extreme reaction conditions, such as temperature and pressure, which
necessitates more exhaustive purification steps [18].

Given aroma compounds' high volatility and hydrophobicity, quantifying the molecules produced during fermentation requires extraction techniques that prevent these compounds from being lost or inactivated. For example, solid phase microextraction (SPME) followed by thermal desorption in gas chromatography (GC) is a combination of simple and effective techniques that makes it possible to both retain volatile compounds and preserve them from degradation until the time of chromatographic analysis, and is widely applied to aroma compounds [30].

Our research group has been working with lignocellulosic biomass to produce en-
zymes [3,4]. Given the nutritional characteristics and availability of BM, and given the
metabolic characteristics of *T. harzianum* and *G. candidum*, we decided to investigate the
potential of this waste as a support, and the potential of microorganisms as producers of
aroma compounds. The bioproduction of flavors using other types of lignocellulosic waste
has already been explored in our previous works, which demonstrated the efficiency of
this waste as biomass, using *T. harzianum* for fermentation [17,31–33].

Given the above, producing natural aroma compounds for the consumer market
through biosynthesis, using a process that is less aggressive for the environment and that
makes it possible to reuse agro-industrial waste to reduce costs and volume, is a factor that
makes this study attractive and essential for the current global production scenario. This
work aims to investigate the production of aroma compounds from the SSF of *G. candidum*
(CCT 1205) and *T. harzianum* (IOC 4042) using residual babassu mesocarp. In order to better
observe the influence of the oily residue on metabolism and obtain the compounds studied,
bioprocesses involving babassu mesocarp in natura (IN-BM) and subjected to the Soxhlet
defatted process (DEF-BM) as a support were compared.

**2. Materials and Methods**

## 2.1. *Support Obtaining*

The raw material for the supports was obtained in the rural area of the municipality of Ariquemes, Rondônia—RO, Brazil, specifically from the babassu palm (Figure 1A), a plant that has several bunches of babassu fruit (Figure 1B). Newly fallen babassu fruits (considered ripe) were collected, washed in running water, and manually fractionated to expose their layers and contents: the almond, endocarp, mesocarp, and epicarp (Figure 1C). The raw mesocarp (Figure 1D) was extracted using a spatula and submitted for drying
in the sun for 5 days. Due to its rough granular appearance, it was ground in a Willey
mill TE-680 (Tecnal, Rio de Janeiro, Brazil) with an output particle size of 10 Tyler mesh
(particles smaller than 2.0 mm). After the milling stage, the flour resulting from the
process was sieved through Taylor Bertel® sieves (Bertel Metalúrgica, São Paulo, Brazil)
to standardize the particle size, and the fractions not retained on the 14-mesh Tyler sieve
were used, i.e., particles with diameters smaller than 1.19 mm (Figure 1E). The particle size
range was selected to achieve bioactive compounds' highest productivity in solid-state
fermentation [32].

<figure><img src="image_5.png" /><figcaption>**Figure 1.** Obtaining babassu mesocarp: (A) babassu palm; (B) babassu coconut bunch; (C) sectioned babassu coconut; (D) mesocarp extracted from several babassu coconuts, showing several lumps; (E) crushed and sieved babassu mesocarp with particles smaller than 1.19 mm in diameter. Adapted from Santos and Muniz, 2017 [22].</figcaption></figure>

2.2. *Pre-Treatment of the Supports*

In order to better study the influence of residual oily components and BM extractives
on fermentation and obtain aroma compounds, part of this biomass was subjected to a lipid
extraction process using Soxhlet equipment (Solab®, São Paulo, Brazil) and 95% n-hexane
as a solvent. In this way, the Babassu mesocarp that remained without extraction was called
in natura babassu mesocarp (IN-BM) (Figure 2A), and the biomass after extraction was
called defatted babassu mesocarp (BM-DEF) (Figure 2B).

<figure><img src="image_6.png" /><figcaption>**Figure 2.** Babassu mesocarp after the pre-treatment process: (A) babassu mesocarp washed with distilled water (IN-BM) and (B) babassu mesocarp treated with 95% hexane (DEF-BM).</figcaption></figure>
## 2.3. Surface Characterization of the in Natura and Defatted Support—Scanning Electron Microscopy (SEM) and Textural Analysis (BET)

Scanning electron microscopy (SEM) analysis was carried out using a scanning electron microscope (Hitachi® TM—3030 Plus, Tokyo, Japan) with coupled EDS (Bruker®, Coventry, UK). Before analysis, the samples were pre-treated by drying and metalizing with gold (99.99%) in an metallizer (Emitech® K550, Beaucouzé, France). These analyses were conducted at the Multiuser Technological Characterization Laboratory (LMCT) of the Mineral Technology Centre (CETEM) at UFRJ. Textural analysis was carried out to determine the specific surface area, volume, and average pore diameter of the raw and treated (degreased) support samples. The analyses were conducted at the Green Technologies Laboratory (GREETEC) of the School of Chemistry at UFRJ, using a Surface Area and Porosimetry Analyzer Tristar 3000 Micromeritic® (Dublin, GA, USA).

## 2.4. Microorganisms, Culture Maintenance, and Propagation

The fungi *T. harzianum* IOC 4042 and the yeast *G. candidum* CCT1205 used in this research were kindly provided by the Mycology Department of the Oswaldo Cruz Institute (FIOCRUZ). The choice of microorganisms was based on their ability to produce multiple bioactive compounds.

The culture was maintained in test tubes containing a PDA (potato dextrose agar) medium made up of agar (2% *m/v*), potato extract (20% *m/v*), glucose (2% *m/v*), $CaCO_3$ (0.02% *m/v*), and $MgSO_4$ (0.02% *m/v*), by Martins (2003) [33]. These tubes were pricked and incubated in an oven (Fisher Scientific®, São Paulo, Brazil) for 7 days at 30 °C and then refrigerated at 4 °C.

The propagation of *T. harzianum* was carried out by repicking the microorganism's spores in Petri dishes containing the following medium composition (g/L): malt extract, 10; glucose, 4.0; agar-agar, 13; yeast extract, 0.75, and peptone, 0.75; the pH of the medium was kept at 5.0 according to Ghose (1958). For *G. candidum*, propagation occurred in Petri dishes containing the following medium composition (g/L): glucose, 5.0; agar-agar, 13; yeast extract, 0.50; and peptone, 0.50; the pH was kept at 5.0. Both propagations were incubated in a Fisher Scientific incubator for 7 days at 30 °C.

## 2.5. Preparing the Inoculum and Conducting the Fermentation

The inoculum was prepared from the propagating Petri dishes, from which a spore suspension was produced, which was obtained by scraping the spores of the fungi grown in Petri dishes and incubating for 7 days in a Fisher Scientific oven at 30 °C. The spores were scraped off using a sterile microbiological loop and suspended in saline solution (0.9%), homogenized in a vortex model 45-2810 (Kasvi Basic®, São Paulo, Brazil), and counted. The spores were quantified using a Primo Star model 4155 optical microscope (Zeiss®, Tokyo, Japan).

The bioproduction of aroma compounds was carried out through the SSF of IN-BM and DEF-BM, which were used as inert organic supports in fermentation. The solid-state fermentation was conducted in 20 mL vials, in which 2.0 g of dry support was impregnated with 2.0 mL of hydration solution + 1.0 mL of spore suspension ($1 \times 10^5$ spores/mL). For both biomass conditions, the hydration solutions used were (a) distilled water (DW), (b) nutrient solution (NS), (c) nutrient solution with glucose (NSG), and (d) nutrient solution with sucrose (NSS). Composition of the nutrient solution (mg/g of support): 7 salts—$(NH_4)_2SO_4$, 0.943; yeast extract 1.0; $MgSO_4 \cdot 7H_2O$, 5; $KH_2PO_4$, 1.0; KCl, 0.5; $CaCl_2 \cdot 2H_2O$, 0.008; $FeSO_4 \cdot 7H_2O$, 0.01; $ZnSO_4 \cdot 7H_2O$, 0.001; 3 salts—$(NH_4)_2SO_4$, 0.943, $KH_2PO_4$, 1.0, $MgSO_4 \cdot 7H_2O$, 5.0. Solutions containing 3 or 7 salts plus (separately) 30 (mg/g of support) glucose or 30 (mg/g of support) sucrose were also used. The nutrient solutions were similar to those described by [30]. Both the support and the nutrient solution were autoclaved separately beforehand. The vials were closed with a cotton cloth and kept in a Visome Plus®—VCC300 (São Paulo, Brazil) climate chamber for 7 days at 30 °C and 90% humidity, without shaking. After 7 days of cultivation, the vials were closed carefully and quickly
with a screw cap with a silicone septum, in order to extract the aroma compounds, which, due to this technical maneuver, were kept trapped inside the vials until extraction. The fermentations were carried out in triplicate with a bed height of 1.5 cm.

## 2.6. *Monitoring of the Fermentations on the Supports*

The fermentations with IN-BM and DEF-BM were investigated by eye for the growth of the microorganisms and sensorially (carefully) for the intensity of the aroma. A scale of 0 (−) to 5 (+++++) was used to rate these two variables (microorganism growth and aroma production). This analysis aimed to identify the microorganisms' adaptation to the support and the preliminary identification of aroma formation.

The aroma compounds from the fermentation were extracted using the solid phase microextraction (SPME) technique, using 100 µm thick PDMS (polydimethylsiloxane)-coated fibers to capture the compounds. The extraction took place via headspace after 7 days of fermentation, and to carry out the procedure, 5.0 mL of saline solution (25% *m/v*) was added to the vials, which were subjected to constant magnetic stirring in a water bath at 79 °C for 30 min.

Gas chromatography (GC) was used to detect the aroma compounds captured by the SPME fiber in a CG-AT2014 (Shimadzu® Sao Paulo, Brazil) device. The parameters used in the equipment were initial column temperature of 35 °C for 2 min, then varying by 50 °C/min up to 200 °C. After reaching this temperature, the column varied by 8 °C/min until it reached 225 °C; the injector and detector were kept at 250 °C. The carrier gas used was helium, and the sample was injected at 1.2 mL/min, totaling 8.43 min run/sample. The investigation was carried out with the main aim of identifying the presence of 6-pentyl-α-pyrone (6-PP), which produces a characteristic coconut aroma; 2-phenyl ethanol (2-PE), which makes a characteristic rose and honey aroma; and hexanal, which creates a distinct green apple aroma.

The aromas in the fermentations were quantified using standard curves made from a stock solution of 2000 ppm of the standard 6-pentyl-α-pyrone (6-PP) (purity < 96% Sigma-Aldrich, St. Louis, MO, USA), which was used to prepare dilutions of 5, 10, 50, 100, 200, 300, and 400 ppm. A volume of 2.0 mL of each diluted solution was added (separately) to 2.0 g of each support (IN-BM and DEF-BM) in a ratio of 1:1. The samples were then subjected to GC analysis and the data obtained plotted on a graph giving rise to the equations y = 507.94x – 1064.5 R² = 0.9901 (IN-BM) and y = 442.23x +1536 R² = 0.9971 (DEF-BM). While the standard curves for the aroma 2-PE and hexanal were prepared from stock solutions of 2000 ppm of the standards 2-phenylethanol (purity ≥ 99%, Sigma-Aldrich) and hexanal (purity ≥ 95%, Sigma-Aldrich) (prepared separately), these were used to prepare the dilutions of 5, 10, 50, 100, 200, 300, and 400 ppm. A volume of 1 mL of the diluted 2-FE solution + 1 mL of the diluted hexanal solution was added concomitantly to the supports, which were quantified with all the dilutions, giving rise to the equations in IN-BM y = 578.65x + 2862.9 R² = 0.9986 (2-FE) and y = 514.97x – 741.86 R² = 0.9989 (hexanal); and in DEF-BM y = 533.85x + 2331.7 R² = 0.9958 (2-FE) and y = 470.75x + 1793.5 R² = 0.9964 (hexanal). The results of the analyses are expressed in ppm (per weight of support).

## 2.7. *Statistical Analysis*

Statistical analysis of the data was carried out using Student's *t*-test, using the analysis of two paired means. The aim of this analysis was to check whether there was a significant difference in the production of aromas with the solutions that performed best.
## 2.8. Surface Analysis of the Fermented Support—Scanning Electron Microscopy (SEM) and Analytical Magnifier

The surface analysis of the fermented supports was used to investigate the adherence of the microorganism to the support, and for this purpose, scanning electron microscopy and an analytical magnifying glass were used. As with the textural characterization, scanning electron microscopy (SEM) was carried out using a Hitachi® (TM—3030 Plus) scanning electron microscope with coupled EDS (Bruker®). Before analysis, the samples were pre-treated by drying and metalizing with gold (99.99%) in a K550 metallizer (Emitech®, Beaucouzé, France). The analytical loupe used was the loupe (Zeiss® SteREO Discovery V8, Japan). Both analyses were conducted at the Multiuser Technological Characterization Laboratory (LMCT) of the Mineral Technology Centre (CETEM) at UFRJ.

# 3. Results and Discussion

## 3.1. Surface Characterization of the Raw and Treated Support—Scanning Electron Microscopy (SEM) and Textural Analysis (BET)

The micrographs (Figure 3) show that the BM is porous and irregular, characteristic of lignocellulosic waste. The surface of the residue is made up of smooth agglomerates of heterogeneous sizes and shapes ranging from spherical to ovoid. The aspects observed on the surface of the residue are mainly associated with the richness of starch in the support, which, although oval in shape, can be spherical, round, polygonal, or irregular [34]. This aspect was also reported by Liu et al. (2018) [35]. They obtained similar images while characterizing babassu mesocarp starch, in which it is also possible to observe that degreasing did not generate morphological changes in the residue compared to the raw residue. However, degreasing gave the residue greater porosity. The analysis also showed the growth of microorganisms in the raw and treated (defatted) waste.

<figure><img src="image_7.png" /><figcaption>**Figure 3.** Scanning electron micrographs of babassu mesocarp: (a) in natura babassu mesocarp (IN-BM), (b) defatted babassu mesocarp (DEF-BM).</figcaption></figure>

The residue was analyzed for its surface topography, and the results were obtained from the nitrogen adsorption and desorption porosimetry analysis (77 °K) using the Brunauer, Emmet, and Teller (BET) method. The surface area, total volume, average pore diameter, and average particle size were obtained by analyzing the data provided by the adsorption isotherm. These analyses are critical, especially for SSF, since, as in other processes, they directly influence the diffusion of microbial cells through the support [36]. According to Table 1, the average particle size of IN-BM (0.48 mm) was slightly more significant when compared to that of DEF-BM (0.45 mm). However, it was possible to observe that, regarding the surface area, volume, and pore diameter of DEF-BM (0.3395, 3.22 × 10⁻⁴, and 38.876 Å, respectively), the defatted residue showed slightly higher values for these analyses when compared to IN-BM (0.3042, 3.19 × 10⁻⁴, and 34.273 Å, respectively). Given
these results, the pre-treatment to degrease the waste may have increased the pore volume. According to Lonappan et al. (2016) [37], the pore diameter of the support can be influenced by pre-treatment, thus increasing the effective surface area by reducing the particle size. In addition, smaller substrate particles provide a greater surface area, facilitating the transfer of oxygen and the action of enzymes by microorganisms [38,39]. Porous materials with a pore diameter between 20 and 500 Å are classified as mesoporous [40], and the waste used in this work is classified in this category.

<table><caption>**Table 1.** Textural characterization (BET) of babassu mesocarp with and without treatment.</caption><thead><tr><th>Waste</th><th>Surface Area (m² g⁻¹)</th><th>Pore Volume (cm³ g⁻¹)</th><th>Pore Diameter (Å)</th><th>Average Particle Size (mm)</th></tr></thead><tbody><tr><td>IN-BM</td><td>0.3042</td><td>3.19 × 10⁻⁴</td><td>34.273</td><td>0.48</td></tr><tr><td>DEF-BM</td><td>0.3395</td><td>3.22 × 10⁻⁴</td><td>38.876</td><td>0.45</td></tr></tbody></table>

## 3.2. *Inspection of the Fermentations on the Supports*

To better understand the results of this analysis, the hydration solutions used in the fermentations are replaced by their corresponding acronyms: distilled water (DW), nutrient solution (NS), nutrient solution with three salts (NS3S), nutrient solution with three salts and glucose (NS3SG), nutrient solution with three salts and sucrose (NS3SS), nutrient solution with seven salts (NS7S); nutrient solution with seven salts and glucose (NS7SG), and nutrient solution with seven salts and sucrose (NS7SS). These hydration solutions were used to provide an environment similar to the fungi's natural habitat, so that they could grow. These solutions contain micronutrients that are fundamental to the metabolism of these microorganisms, mainly because they are required as cofactors in various processes.

After 7 days of fermentation, the microorganisms showed some differences in their development on the supports, depending on the hydration solution used in the fermentation (Figure 4). The results showed that both microorganisms showed slight growth on the supports with the DW, showing that the microorganisms could not assimilate the support as a carbon source without other nutrients in the medium. In the fermentation with *T. harzianum*, for example, it was noted that with IN-BM, in addition to the slight growth, the microorganism showed no signs of sporulation and was white. This differs from what was observed with DEF-BM, although the growth was subtle in this fermentation. In the presence of NS hydration solution, the development of the microorganisms on the supports was observed. However, it was still limited, reinforcing the need for external nutrient sources for the organisms to grow on the supports.

Fermentations with NS3S solution showed more pronounced growth of the microorganisms, suggesting that the external sources of (NH₄)₂SO₄, KH₂PO₄, and MgSO₄·7H₂O salts positively influenced the growth of the microorganisms on the supports. This was also observed in the fermentations using NS3SS and NS3SG solutions (separately), with the growth of the microorganisms being more pronounced in the presence of these solutions but with a slight emphasis on the fermentations with NS3SG solution. The combination of salts and a carbon source in this solution may have been more favorable for the growth of the microorganisms. According to Ramos, 2006 [32] and Martins, 2003 [33], this can favor microbial development. Fermentations with the NS7S solution showed higher microorganism growth than that observed with the NS3S solution, but this growth was slightly lower than that shown with NS3SS and NS3SG solutions. This result indicates the external sources of the salts (NH₄)₂SO₄, MgSO₄·7H₂O, KH₂PO₄, KCl, CaCl₂·2H₂O, FeSO₄·7H₂O, ZnSO₄·7H₂O, and nitrogen from the yeast extract favor the development of the microorganisms on the supports. However, the presence of sucrose and glucose in the NS3SS and NS3SG solutions makes the growth of the microorganisms slightly better than in the presence of the NS7S solution. However, this scenario changed when we analyzed the fermentations of the supports in the presence of NS7SS and NS7SG solutions (separately), in which the microorganisms showed the most pronounced growth in the analyses, and
the support with the NS7SG solution showed the best development of the microorganisms. There was also a slight difference in the development of the microorganisms concerning the supports, with the growth of the microorganisms in DEF-BM being slightly better than that in IN-BM. Nutrient solution compositions similar to this study were observed in previous works [17,30,31].

<table><caption>Fermentation of the supports with Geotrichum candidum</caption><thead><tr><th rowspan="3">TYPE OF WASTE</th><th colspan="3">3 SALTS</th><th rowspan="2">DISTILLED WATER</th><th colspan="3">7 SALTS</th></tr><tr><th>NUTRIENT SOLUTION GLUCOSE</th><th>NUTRIENT SOLUTION SUCROSE</th><th>NUTRIENT SOLUTION SUCTION</th><th>NUTRIENT SOLUTION</th><th>NUTRIENT SOLUTION SUCROSE</th><th>NUTRIENT SOLUTION GLUCOSE</th></tr></thead><tbody><tr><td rowspan="2">IN NATURA BABASSU MESOCARP (IN-BM)</td><td>GROWTH: +++<br>AROMA: ++</td><td>GROWTH: +++<br>AROMA: ++</td><td>GROWTH: ++<br>AROMA: +</td><td>GROWTH: +<br>AROMA: -</td><td>GROWTH: ++<br>AROMA: +</td><td>GROWTH: +++<br>AROMA: ++</td><td>GROWTH: +++<br>AROMA: +++</td></tr><tr><td><img src="image_1.png" alt="Image of GROWTH: +++ with aroma: ++"></td><td><img src="image_2.png" alt="Image of GROWTH: +++ with aroma: ++"></td><td><img src="image_3.png" alt="Image of GROWTH: ++ with aroma: +"></td><td><img src="image_4.png" alt="Image of GROWTH: + with aroma: -"></td><td><img src="image_5.png" alt="Image of GROWTH: +++ with aroma: +"></td><td><img src="image_6.png" alt="Image of GROWTH: +++ with aroma: ++"></td><td><img src="image_7.png" alt="Image of GROWTH: ++++ with aroma: +++"></td></tr><tr><td rowspan="2">DEFATTED BABASSU MESOCARP (DEF-BM)</td><td>GROWTH: ++++<br>AROMA: ++++</td><td>GROWTH: +++<br>AROMA: +++</td><td>GROWTH: +++<br>AROMA: ++</td><td>GROWTH: +<br>AROMA: -</td><td>GROWTH: +++<br>AROMA: ++</td><td>GROWTH: +++<br>AROMA: +++</td><td>GROWTH: ++++<br>AROMA: ++++</td></tr><tr><td><img src="image_1.png" alt="Image of GROWTH: ++++ with aroma: +++"></td><td><img src="image_2.png" alt="Image of GROWTH: ++++ with aroma: +++"></td><td><img src="image_3.png" alt="Image of GROWTH: +++ with aroma: ++"></td><td><img src="image_4.png" alt="Image of GROWTH: + with aroma: -"></td><td><img src="image_5.png" alt="Image of GROWTH: +++ with aroma: ++"></td><td><img src="image_6.png" alt="Image of GROWTH: ++++ with aroma: +++"></td><td><img src="image_7.png" alt="Image of GROWTH: ++++ with aroma: +++"></td></tr></tbody></table>

<table><caption>Fermentation of the supports with Trichoderma harzianum</caption><thead><tr><th rowspan="3">TYPE OF WASTE</th><th colspan="3">3 SALTS</th><th rowspan="2">DISTILLED WATER</th><th colspan="3">7 SALTS</th></tr><tr><th>NUTRIENT SOLUTION GLUCOSE</th><th>NUTRIENT SOLUTION SUCROSE</th><th>NUTRIENT SOLUTION SUCTION</th><th>NUTRIENT SOLUTION</th><th>NUTRIENT SOLUTION SUCROSE</th><th>NUTRIENT SOLUTION GLUCOSE</th></tr></thead><tbody><tr><td rowspan="2">IN NATURA BABASSU MESOCARP (IN-BM)</td><td>GROWTH: +++<br>AROMA: ++</td><td>GROWTH: +++<br>AROMA: ++</td><td>GROWTH: ++<br>AROMA: +</td><td>GROWTH: ++<br>AROMA: -</td><td>GROWTH: +++<br>AROMA: +</td><td>GROWTH: +++<br>AROMA: ++</td><td>GROWTH: ++++<br>AROMA: +++</td></tr><tr><td><img src="image_1.png" alt="Image of GROWTH: +++ with aroma: ++"></td><td><img src="image_2.png" alt="Image of GROWTH: +++ with aroma: ++"></td><td><img src="image_3.png" alt="Image of GROWTH: ++ with aroma: +"></td><td><img src="image_4.png" alt="Image of GROWTH: + with aroma: -"></td><td><img src="image_5.png" alt="Image of GROWTH: +++ with aroma: +"></td><td><img src="image_6.png" alt="Image of GROWTH: ++++ with aroma: +++"></td><td><img src="image_7.png" alt="Image of GROWTH: ++++ with aroma: +++"></td></tr><tr><td rowspan="2">DEFATTED BABASSU MESOCARP (DEF-BM)</td><td>GROWTH: ++++<br>AROMA: +++</td><td>GROWTH: ++++<br>AROMA: +++</td><td>GROWTH: +++<br>AROMA: ++</td><td>GROWTH: +<br>AROMA: -</td><td>GROWTH: +++<br>AROMA: ++</td><td>GROWTH: ++++<br>AROMA: +++</td><td>GROWTH: ++++<br>AROMA: ++++</td></tr><tr><td><img src="image_1.png" alt="Image of GROWTH: ++++ with aroma: +++"></td><td><img src="image_2.png" alt="Image of GROWTH: ++++ with aroma: +++"></td><td><img src="image_3.png" alt="Image of GROWTH: +++ with aroma: ++"></td><td><img src="image_4.png" alt="Image of GROWTH: + with aroma: -"></td><td><img src="image_5.png" alt="Image of GROWTH: +++ with aroma: ++"></td><td><img src="image_6.png" alt="Image of GROWTH: ++++ with aroma: +++"></td><td><img src="image_7.png" alt="Image of GROWTH: ++++ with aroma: +++"></td></tr></tbody></table>

**Figure 4.** Visual and sensory inspection of fermentations with *T. harzianum* and *G. candidum* in babassu mesocarp. Both the growth and production of aroma compounds were monitored qualitatively at this stage, where a scale ranging from 0 (–) to 5 (+++++) denotes intensity.
Regarding the intensity of the aromas produced with the supports, we tried to identify the characteristic aromas of certain compounds in the fermentations, such as coconut, honey, almonds, roses, green apples, and green grapes. This investigation occurred due to the possibility of the microorganisms studied producing certain compounds. In the fermentations with *T. harzianum*, it was possible to identify a slight coconut aroma, with the NS3SS, NS3SG, NS7SS, and NS7SG fermentations standing out. Both had very similar aroma intensities. The aroma that stood out in the fermentations with *G. candidum* was green apple, which was more noticeable in the fermentations with NS3SS, NS3SG, NS7SS, and NS7SG. The aromas of roses, honey, or almonds, which this microorganism can also produce, were not sensorially perceptible. This may have been because it was made at the same time as the green apple aroma, which may have suppressed its volatilization due to its high level of intensity.

### 3.3. *Extraction, Quantification, and Identification of Aroma Compounds*

Several studies have sought strategies to obtain the best conditions for producing aroma compounds, whether by optimizing the nutrient medium used in SSF [31], immobilizing the microorganism in a liquid medium [41], or investigating different strains [42], for example. SPME is an effective technique for extracting aroma compounds from the supports, and the conditions standardized by Ramos (2008) [30] proved efficient for this process. Franco and Janzantti (2004) [43] state that volatile compounds are thermolabile and found in deficient concentrations. Any temperature increase during sample preparation can lead to chemical reactions such as rearrangements, cyclizations, and hydrolysis, among other responses. These modifications are capable of altering the original sample.

The data obtained through chromatographic analysis showed that the aroma compounds in the highest concentration in the organic supports were 6-pentyl-α-pyrone (6-PP) (GRAS 3696), produced by *T. harzianum*, and 2-phenylethanol (2-PE) (GRAS 2858) and hexanal (GRAS 2557), both produced by *G. candidum*. 6-PP is an unsaturated 2-lactone whose characteristic aroma resembles coconut and can be used in food and cosmetics [44]. 2-PE, on the other hand, is an aromatic, colorless, and volatile higher alcohol with a characteristic rose and honey aroma and occurs naturally in jasmines, daffodils, hyacinths, and roses [45,46]. It is mainly used in the cosmetics industry. Finally, hexanal is a colorless, volatile aldehyde with a characteristic green apple aroma in some fruits [47,48]. The production of such compounds using babassu mesocarp as a support has not yet been reported in the literature.

The results obtained in the fermentations with *T. harzianum*, using the different nutrient solutions, can be seen in Figure 5 and show that in IN-BM, it was possible to achieve concentrations (ppm (*w*/*w*)) of 6-PP equivalent to 175.76 ± 1.69 using NS7SG and 150.22 ± 0.97 using NS7SS (Figure 5a). In the fermentation with DEF-BM, the same was observed, since using NS7SG, it was possible to reach 308.17 ± 3.18 and with NS7SS, 294.90 ± 2.38 (Figure 5b). The highest concentration (ppm (*w*/*w*)) of 6-PP using the solutions containing seven salts was obtained in the DEF-BM impregnated with NS7SG (308.17 ± 3.18). However, it was possible that both fermentations using sucrose as the carbon source showed concentrations close to the concentration obtained with glucose as the carbon source.

In fermentations with *T. harzianum*, using nutrient solutions containing three salts, IN-BM achieved a concentration of 6-PP aroma (ppm (*w*/*w*)) equivalent to 154.24 ± 2.64 with NS3SG and 125.18 ± 1.25 with NS3SS (Figure 5c). In fermentations with DEF-BM, the concentration of aroma (ppm (*w*/*w*)) achieved with NS3SG was equivalent to 300.34 ± 3.08, and with NS3SS, it was 286.43 ± 2.75 (Figure 5d). Using the nutrient solution with three salts, the highest concentration of 6-PP was obtained in DEF-BM, impregnated with NS3SG (300.34 ± 3.08). However, as in the fermentations using seven salts, the fermentation with three salts and sucrose showed an aroma concentration close to the concentration obtained with glucose as the carbon source.
In his studies, Ramos (2008) [30], seeking to produce the aroma compound 6-PP, used a nutrient solution identical to the one used in this study. The nutrient solution used by Penha (2015) [17] to produce the aroma compounds 6-PP and γ-decalactone was identical to the one used in this study. Ladeira et al. (2010) [31] used a similar nutrient solution in their experiments to produce 6-PP; unlike in the present study, yeast extract was not part of the solution.

<figure><img src="image_8.png" /><figcaption>**Figure 5.** Quantifying the 6 pentyl-alpha-pyrone (6-PP) aroma in solid-state fermentation (SSF) with *T. harzianum*. (a) SSF with greased babassu mesocarp (IN-BM) using hydration solutions with seven salts. (b) SSF with defatted babassu mesocarp (DEF-BM) using hydration solutions with seven salts. (c) SSF with defatted babassu mesocarp (IN-BM) using hydration solutions with three salts. (d) SSF with defatted babassu mesocarp (DEF-BM) using hydration solutions with three salts.</figcaption></figure>

There are a few studies in the literature in which the production of this compound has been explored using agro-industrial waste. The highest concentration of 6-PP found in this work after 7 days of fermentation (308.17 ppm) was higher than that found by Ladeira et al. (2010) [31] and Penha (2012) [49], who obtained concentrations equivalent to 254 ppm and 93 ppm, respectively. The work by Penha et al. (2015) [17] showed a concentration of aroma closest to that obtained in this work but slightly higher, equivalent to 317 ppm. All the studies cited used sugar cane bagasse as support and solid-state fermentation with the filamentous fungus *T. harzianum* for the same fermentation period (7 days). However, much higher results in the production of 6-PP were observed in the works by Ramos (2006) [32] and Calasans (2012) [42], in which the concentration of this aroma was equivalent to 5.05 mg/g (5050 ppm) and 3.78 mg/g (3780 ppm), respectively, using SSF with the same microorganism as in the previous studies and the same cultivation time. However, the support used in the work by Ramos (2008) [30] was green coconut shell
powder, while in the work by Calasans (2012) [42], the support was sugar cane bagasse. These results and data from the literature show that the optimum production for 6-PP was achieved on the seventh day. However, studies such as that by Sahhty-Bagnon et al. (2000) [50], reporting optimum production of this compound (2.8 mg/g (2800 ppm)) at longer fermentation times (10 days), are also found.

Although the results of this study are quite different in terms of 6-PP concentration, they are similar to those obtained in the study by Ramos et al. (2008) [30], where, in a comparison of the effects of nutrient solutions on fermentation, it was observed that the 6-PP concentrations obtained with NSG and NSS were statistically close. A possible explanation for this is that glucose is a monosaccharide and is readily available, unlike sucrose, a disaccharide composed of glucose and fructose molecules, which requires an initial time for carbohydrate hydrolysis [7]. The fermentation of IN-BM and DEF-BM using NS3S and NS7S had the lowest aroma concentration. However, the fermentation containing NS7S was slightly higher than that containing NS3S. The fermentation using DW had the lowest aroma concentration.

According to [50], producing this aroma above 100 ppm can be fungicidal on the strain itself during fermentation, thus inhibiting the production and causing low yields. However, this inhibitory effect was not observed in this work or in the studies by Ladeira et al. (2010) [31] and Penha (2015) [17], who also obtained concentrations of 6-PP above 100 ppm. To minimize the inhibitory effect of this lactone and increase production yield, the author suggests removing 6-PP during fermentation. This approach is also indicated by Häusler and Münch (1997) [51], who proposed using a pervaporation system with a selective membrane to continuously extract the lactone from the culture medium, avoiding the inhibition phenomenon.

The production of the 2-PE aroma can be intensely influenced by the composition of the culture medium [52] and by the culture conditions and microorganisms used in the fermentation [53,54]. The complexity of the nitrogen source and the nature of the carbon source used in the medium can influence the microorganism's metabolism and, consequently, the quantity of by-products that can be formed [55–57].

Regarding the production of 2-PE, using *G. candidum* and a nutrient solution containing seven salts (Figure 6), the highest concentrations (ppm (*w*/*w*)) were observed in the fermentations with NS7SG and NS7SS. IN-BM fermented with NS7SG reached an aroma concentration of 259.76 ± 2.13, and when fermented with NS7SS, it reached a concentration equivalent to 227.66 ± 2.01 (Figure 6a). With DEF-BM, the aroma concentration (ppm (*w*/*w*)) with NS7SG was comparable to 414.53 ± 1.96, and with NS7SS it was equivalent to 389.49 ± 1.54 (Figure 6b). In the fermentation with IN-BM using NS3SG, the aroma concentration (ppm (*w*/*w*)) obtained was 232.34 ± 1.38, and with NS3SS it was 218.89 ± 2.08 (Figure 6c). In fermentation with DEF-BM, the aroma concentration (ppm (*w*/*w*)) achieved with NS3SG was 404.97 ± 2.47, and with NS3SS it was 372.21 ± 2.19 (Figure 6d). Using NS3S and NS7S, the fermentation results in DEF-BM for both conditions were slightly better than those in IN-BM. The aroma production on the support with DW was the lowest, with no significant difference between IN-BM and DEF-BM. These results were very similar to those obtained by Martínez-Ávila et al. (2021) [18] through biotransformation (25.2 mg/g (25,200 ppm)) and higher than those obtained through de novo synthesis (1.7 mg/g (1700 ppm)). These experiments were conducted during 96 h of fermentation, using 95 ± 1 g of residue. They were also similar to the results of Martínez-Ávila et al. (2018) [58], with production equivalent to 18.4 mg/g (18,400) (dry basis) after 72 h of fermentation, using approximately 96 g of residue.
Given the results obtained in this analysis, it can be seen that defatting the residue was relevant to the production of the aroma since the defatted matrix achieved higher concentrations of 2-PE. According to Chreptowicz and Mierzejewska (2018) [59], the ideal output of 2-PE occurs in the presence of the carbon sources glucose and sucrose. However, there are reports in the literature that concentrations of 2-PE in the medium above 4 g/L can be toxic to some microorganisms and inhibit the production process, resulting in a low final concentration [52–60]. Thus, although the final concentration of 2-PE is lower than the concentration suggested to be toxic to microorganisms, quantifying the aroma every 24 h over the 7 days of fermentation can provide data to clarify whether a possible inhibition of production may have occurred during the fermentation of IN-BM [61,62].

<figure><img src="image_9.png" /><figcaption>**Figure 6.** Quantifying the aroma of 2-phenylethanol (2-PE) and hexanal in SSF with *G. candidum*. (a) SSF with IN-BM using hydration solutions with seven salts. (b) SSF with DEF-BM using hydration solutions with seven salts. (c) SSF with IN-BM using hydration solutions with three salts. (d) SSF with DEF-BM using hydration solutions with three salts.</figcaption></figure>

Regarding the acquisition of the aroma compound hexanal via fermentation with *G. candidum* using nutrient solutions containing seven salts (Figure 6), it was observed that with DEF-BM, the highest concentrations of aroma (ppm (*w*/*w*)) were with NS7SG (196.50 ± 1.15) and with NS7SS (188.19 ± 1.26) (Figure 6a). Using IN-BM, the aroma concentration (ppm (*w*/*w*)) achieved with NS7SG was 210.83 ± 2.14, and with NS7SS it was 198.01 ± 2.07 (Figure 6b). When analyzing the fermentations using the nutrient solution composed of three salts, the highest aroma concentrations (ppm (*w*/*w*)) observed using DEF-BM were with NS3SG (192.93 ± 1.24) and with NS3SS (180.70 ± 2.04) (Figure 6c). In IN-BM, the highest aroma concentrations (ppm (*w*/*w*)) achieved were with NS3SG (211.07 ± 2.11) and with NS3SS (196.74 ± 1.19) (Figure 6d). Rossi et al. (2009) [63] obtained 99.6 µmol/L of volatile compounds using the fungus *Ceratocystis fimbriata*; however, the
concentration in the sample was not analyzed. The results obtained for hexanal in this study were higher than those obtained by Carvalho (2011) [64] for the fruity aroma of ethyl hexanoate, which has a characteristic passionfruit odor (10 mg/L (10 ppm) in malt bagasse and around 5 mg/L (5 ppm) in Manipuri). They were also higher than the results achieved by Araújo (2016) [65] for the aroma compound ethyl acetate after 24 h of fermentation (91.92 µmol/L (8.10 ppm), which has a characteristic odor of pears and apples.

In the fermentations using NS3S and NS7S, unlike in the production of the other compounds, there was a slight difference in the output of hexanal since the concentration of the aroma in IN-BM was slightly higher than that in DEF-BM. In this fermentation, degreasing the residue caused a slight reduction in hexanal production. This is probably because this aroma is a bioproduct of lipid degradation, and removing this fraction may have been detrimental. Production using sucrose as a carbon source was slightly closer to production using glucose, as was observed in the output of 6-PP and 2-PE aromas. In this way, producing hexanal aroma with NSS can further reduce production costs with an organic support. All the aroma compounds identified in this work are recognized by FEMA and GRAS status, which makes them safe to use.

The aromas produced in the fermentations were analyzed by comparing the extraction chromatograms with the chromatograms of the commercial standards and data from the literature [66]. The chromatogram in Figure 7 shows that among the aroma compounds with the highest concentration in the samples, hexanal was the compound with the lowest affinity for the column, with a retention time of 2.992 min, while 2-PE and 6-PP were the compounds with the highest affinity for the column, with retention times of 6.283 and 7.410 min, respectively.

<figure><img src="image_10.png" /><figcaption>**Figure 7.** GC-MS chromatogram of the aroma compounds 6-pentyl-alpha-pyrone (6-PP), 2-phenylethanol (2-PE), and hexanal synthesized from babassu mesocarp by SSF.</figcaption></figure>

### 3.4. Statistical Analysis of Data

Based on the experimental information and the data shown in Figures 5 and 6, statistical analyses were carried out using the paired Student's t-test for means, in order to assess whether the presence of three or seven salts combined with sucrose or glucose in the SN of the fermentations showed significant differences in the production of aroma compounds with IN-BM and DEF-BM (for more details see Table S1 in Supplementary Material). Table 2 shows the statistical analysis carried out for two population samples (IN-BM and DEF-BM), containing two variables, namely the presence of three or seven salts accompanied by sucrose or glucose.
Chromatographic analysis of the concentration of the aroma compounds identified showed that fermentations containing the salts and a sugar as a carbon source had the highest concentrations of the aroma compounds investigated. This study therefore opted to statistically analyze only the results of the fermentations NS3SG, NS3SS, NS7SG, and NS7SS.

The data presented by the paired *t*-test showed similarity with a normal distribution and equivalent variance, since the *p*-values obtained were less than 0.05, except for the comparison between the fermentations with IN-BM, using SN7SG and SN7SS to produce hexanal. Therefore, the results of the means test are statistically significant and the null hypothesis is rejected, except for the hexanal production reported, for which the null hypothesis is accepted, there being no significant difference between the two SN types.

<table><caption>**Table 2.** Statistical analysis of fermentations containing nutrient solution with three or seven salts plus glucose or sucrose.</caption><thead><tr><th rowspan="3">Statistical Variable</th><th colspan="10">6-PP</th></tr><tr><th colspan="4">IN-BM</th><th colspan="4">DEF-BM</th></tr><tr><th>NS3SG</th><th>NS3SS</th><th>NS7SG</th><th>NS7SS</th><th>NS3SG</th><th>NS3SS</th><th>NS7SG</th><th>NS7SS</th></tr></thead><tbody><tr><td>Mean</td><td>154.24</td><td>125.18</td><td>175.76</td><td>150.22</td><td>300.34</td><td>286.43</td><td>308.17</td><td>295.89</td></tr><tr><td>Median</td><td>154.76</td><td>125.23</td><td>175.32</td><td>150.40</td><td>301.58</td><td>287.21</td><td>307.86</td><td>295.05</td></tr><tr><td>Variance</td><td>5.42</td><td>1.21</td><td>2.22</td><td>0.74</td><td>7.40</td><td>5.93</td><td>7.86</td><td>4.41</td></tr><tr><td>Standard deviation</td><td>2.64</td><td>1.25</td><td>1.69</td><td>0.97</td><td>3.08</td><td>2.75</td><td>3.18</td><td>2.38</td></tr></tbody><thead><tr><th rowspan="3"></th><th colspan="10">2-PE</th></tr><tr><th colspan="4">IN-BM</th><th colspan="4">DEF-BM</th></tr><tr><th>NS3SG</th><th>NS3SS</th><th>NS7SG</th><th>NS7SS</th><th>NS3SG</th><th>NS3SS</th><th>NS7SG</th><th>NS7SS</th></tr></thead><tbody><tr><td>Mean</td><td>232.33</td><td>218.89</td><td>259.76</td><td>227.66</td><td>357.70</td><td>329.18</td><td>366.02</td><td>344.23</td></tr><tr><td>Median</td><td>231.08</td><td>219.72</td><td>256.17</td><td>229.72</td><td>356.46</td><td>329.59</td><td>366.56</td><td>344.63</td></tr><tr><td>Variance</td><td>54.91</td><td>16.68</td><td>77.98</td><td>74.34</td><td>4.74</td><td>3.74</td><td>12.29</td><td>5.34</td></tr><tr><td>Standard deviation</td><td>1.38</td><td>2.08</td><td>2.13</td><td>2.01</td><td>2.47</td><td>2.19</td><td>1.96</td><td>1.54</td></tr></tbody><thead><tr><th rowspan="3"></th><th colspan="10">HEXANAL</th></tr><tr><th colspan="4">IN-BM</th><th colspan="4">DEF-BM</th></tr><tr><th>NS3SG</th><th>NS3SS</th><th>NS7SG</th><th>NS7SS</th><th>NS3SG</th><th>NS3SS</th><th>NS7SG</th><th>NS7SS</th></tr></thead><tbody><tr><td>Mean</td><td>192.93</td><td>180.70</td><td>196.50</td><td>188.19</td><td>188.88</td><td>176.41</td><td>210.75</td><td>198.02</td></tr><tr><td>Median</td><td>192.69</td><td>180.00</td><td>196.13</td><td>188.06</td><td>189.07</td><td>176.43</td><td>210.75</td><td>197.04</td></tr><tr><td>Variance</td><td>3.94</td><td>4.67</td><td>3.62</td><td>1.26</td><td>3.49</td><td>1.11</td><td>3.60</td><td>3.32</td></tr><tr><td>Standard deviation</td><td>2.11</td><td>2.04</td><td>1.04</td><td>0.93</td><td>1.24</td><td>2.04</td><td>1.15</td><td>1.26</td></tr></tbody></table>

### 3.5. Surface Analysis of the Fermented Supports—Scanning Electron Microscopy (SEM) and Analytical Magnifier

According to the results observed in the analysis of the surface of the fermented supports (Figures 8 and 9), after 7 days of fermentation, it was found that the microorganisms could grow on the supports, forming vegetative structures and filling their cavities. This shows that BM is satisfactory in supporting SSF. The fermentations impregnated with the NS7SG hydration solution were used in this analysis, as they showed the best development of the microorganisms at the end of the fermentation.
<figure><img src="image_11.png" /><figcaption>**Figure 8.** Scanning electron microscopy of BM supports after 7 days of fermentation with *T. harzianum* and *G. candidum*. (a) IN-BM fermented with *G. candidum*; (b) DEF-BM fermented with *G. candidum*; (c) IN-BM fermented with *T. harzianum*; (d) DEF-BM fermented with *T. harzianum*.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**Figure 9.** Surface analysis of BM supports after 7 days of fermentation with *T. harzianum* and *G. candidum*. (a) IN-BM fermented with *G. candidum*; (b) DEF-BM fermented with *G. candidum*; (c) IN-BM fermented with *T. harzianum*; (d) DEF-BM fermented with *T. harzianum*.</figcaption></figure>

According to Carboué et al. (2018) [14], organic supports can provide the ideal environment for developing some species of microorganisms since they have many nutrients that can be consumed during fermentation. According to Ravindran et al. (2018) [67], the physicochemical nature of most lignocellulosic substrates is naturally suited to the SSF process, which favors using agro-industrial waste as supports/matrices.

It was also observed that the fermentations containing the waste in distilled water showed no growth of the microorganisms. This observation points to the need for nutrients outside the biomass for the microorganisms to start producing aromatic compounds.

**4. Conclusions**

This work preliminarily demonstrated that residual babassu mesocarp, an agro-industrial waste generated in large quantities in northern Brazil, can be a cheap and interesting substrate for obtaining aroma compounds through solid-state fermentation by *G. candidum* and *T. harzianum*. The presence of oily waste influenced the production of 6-PP (6-pentl-α-pyrone) and 2-PE (2-phenylethanol), but was not significant for obtaining hexanal, demonstrating that this waste can be molded for microbial metabolism. Among the various nutrient solutions investigated, the solution containing three salts and sucrose was more efficient as a source of carbon and trace elements than those containing glucose, demonstrating that the process can be more economical. Since SSF is a simple technique,
this bioprocess proved to be a viable alternative for the valorization of BM to obtain
compounds with higher added value, such as aroma compounds. Further studies need
to be carried out to investigate the production of these compounds in the absence of
supplementation, as well as the feasibility of scaling up and economic studies.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microbiolres15030093/s1, Table S1: Statistical analysis.

**Author Contributions:** T.N.d.A.: execution, data curation, writing, editing, analysis; R.W.: conceptualization, writing, editing; S.G.F.L.: funding acquisition, writing, editing, consulting; I.I.J.: funding acquisition, conceptualization, consultancy, writing, editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by Brazilian government through Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Grant Numbers: E-203.267/2017 and E26/201.367/2022; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CAPES BR), Grant Numbers 429974/2018-3, 300873/2018-2, 315160/2021-7 and by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The raw data supporting this article's conclusions will be made available by the authors of this article, and supplementary data can be requested upon request.

**Acknowledgments:** The authors would like to thank the Coordination for the Improvement of Higher Education Personnel (CAPES) for financial support, the Green Technologies Laboratory (GREENTECH) of the UFRJ Chemistry School, and the Multiuser Technological Characterization Laboratory (LMCT) of the Mineral Technology Centre (CETEM) for instrumental support.

**Conflicts of Interest:** The authors declare no conflicts of interest.

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