Trichoderma harzianum Enzyme Production in Stirred Solid-State Bioreactors as a Strategy for Valorizing Water Hyacinth

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

See attached report.

Comments for author File: Comments.pdf

Author Response

Comment (abstract section): Provide detailed information about the controlled variables used in the static bed bioreactors, including their respective operating ranges.

Answer: To provide information on the controlled variables in PBCBs, the following paragraph was added:

“Utilizing laboratory-scale packed-bed column bioreactors (PBCB) with a capacity of 8 g of dry mass (g_dm), the effects of temperature (28-36°C) and initial moisture content (65-80%) on microbial growth and enzyme production were evaluated.”.

 

Comment (abstract section): Indicate the highest activity values obtained for both xylanase and cellulase enzymes.

Answer: Enzyme activity values were indicated according to the reviewer's comment.

 

Comment (abstract section): Additionally, describe the type of stirred bioreactor employed—was it a rotating-wall drum, a scraped-wall drum, or another configuration?

Answer: The following paragraph has been added to specify the type of stirred bioreactor utilized in this study:

“The operational conditions for xylanase production were transferred to 6 L bench-scale cross-flow internal stirred bioreactors, packed to 40% capacity with 450 g_dm, where two stirring regimes were evaluated: intermittent and continuous”.

 

Comment (abstract section): For comparison purposes, include the working volumes of both the static and the stirred bioreactors.

Answer: The following paragraphs were added to the abstract to describe the capabilities of the bioreactors used in this study:

“Utilizing laboratory-scale packed-bed column bioreactors (PBCB) with a capacity of 8 g of dry mass (gdm), the effects of temperature (28-36°C) and initial moisture content (65-80%) on microbial growth and enzyme production were evaluated”.

“The operational conditions for xylanase production were transferred to 6 L bench-scale cross-flow internal stirred bioreactors, packed to 40% capacity with 450 g_dm, where two stirring regimes were evaluated: intermittent and continuous”.

These paragraphs replace the text presented in the initial version:

“Laboratory-scale static bed bioreactors were employed to determine the favorable conditions for microbial growth (estimated by respirometry) and enzyme production”.

“The operational conditions for xylanase production were transferred to bench-scale stirred bioreactors, where two stirring regimes were evaluated: intermittent and continuous”.

 

Comment (abstract section): Include a final remark indicating who will benefit from this work and why it is relevant to them.

Answer: The following paragraph has been added to highlight the importance of this work:

“The approaches developed in this study can aid in designing large-scale bioprocesses for the valorization of water hyacinth”.

Comment (introduction section): The introductory portion of this section regarding water hyacinth is overly long and could be condensed into a single paragraph.

Answer: In response to the reviewer's comment, we have condensed the information on water hyacinth into the following paragraph:

"Water hyacinth (Eichhornia crassipes) is an invasive aquatic weed native to the Amazon basin that has spread globally, causing various environmental, economic, and social issues. This weed forms dense mats on water surfaces, blocking light and displacing wildlife, depleting oxygen levels, and reducing aquatic plant life, which negatively impacts fisheries. These mats also limit habitats for water birds and promote breeding grounds for mosquitoes and other disease vectors. Economically, water hyacinth obstructs transport and fishing, damages infrastructure, raises service costs, and disrupts riparian communities. Once established, it is challenging to eradicate water hyacinth".

 

This paragraph replaces the text presented in the initial version:

“Water hyacinth (Eichhornia crassipes) is an aquatic weed that is native to the Amazon basin. Its spread worldwide has led to various environmental, economic, and social issues [1]. This fast-growing, perennial, free-floating macrophyte was initially valued as an ornamental plant due to its attractive purple flowers, having been distributed by gardeners and horticulturists over a century ago [2].

The plant features a network of fibrous roots and fleshy leaves that are essential for photosynthesis. It has two types of stems: one aerial stem that is semi-succulent and vertical, and another submerged rhizome [3]. Water hyacinth forms dense mats on the water's surface that block light from reaching submerged plants. This competition often displaces wildlife forage and habitats, depletes oxygen levels in aquatic ecosystems, reduces phytoplankton, and alters invertebrate community composition, ultimately impacting fisheries. Additionally, these mats reduce the habitats available for water birds and provide breeding grounds for mosquitoes and other disease vectors [4]. The presence of water hyacinth also has significant economic and social consequences. It obstructs water transport and fishing activities, damages infrastructure, raises service costs, and destabilizes riparian communities. Once established, it is challenging to eradicate [5]”.

Comment (introduction section): There is limited discussion on the successful production of enzymes via solid-state fermentation (SSF): details about the media used, the microbial strains involved, and the specific enzymes produced are lacking. Greater emphasis should be given to the xylanase and cellulase enzymes generated by SSF. Specify which types of cellulases are involved (e.g., endoglucanase, FPase, etc.).

Answer: To improve the information about enzyme complexes and their production via SSC, the following text was added to the introduction section:

“Cellulases are an enzyme complex that hydrolyzes cellulose, a polymer composed of different types of enzymes. Endoglucanases (EG) act randomly on the internal β-1,4-glucosidic bonds of the cellulose chain, producing shorter fragments. Cellobiohydrolases (CBH), also known as exoglucanases, target the non-reducing ends of cellulose and release cellobiose, a disaccharide composed of two glucose molecules. Lastly, β-glucosidases (BG) hydrolyze cellobiose and other oligosaccharides to yield glucose, the final product of cellulose degradation [9].

Similarly, xylanases facilitate the degradation of xylan, the second most abundant polysaccharide found in plant cell walls. This process requires a combination of enzymes, including endo-1,4-β-xylanases, which break down the internal β-1,4-glycosidic bonds of xylan, thereby releasing xylooligosaccharides. β-xylosidases further hydrolyze these xylooligosaccharides into xylose, the fundamental monomer of xylan. Accessory enzymes such as α-arabinofuranosidases, α-glucuronidases, and acetylxylan esterases play a crucial role in removing branches and structural modifications from xylan, thereby facilitating its complete degradation [10].

The cost of the carbon source is a crucial factor influencing the final expenses of the enzymatic production process [11]. However, previous studies have demonstrated that efficient production of cellulases and xylanases can be obtained from lignocellulosic waste (straw, bran, bagasse, shoots and forest waste) by solid-state culture (SSC) using filamentous fungi such as Aspergillus flavus [12], A. fumigatus [13], A. niger [14,15], Myceliophthora thermophila [16], Penicillium echinulatum [17], Trichoderma asperellum [18], T. reesei [19,20], and T. harzianum [21]. Since water hyacinth is abundant and low-cost, it is an excellent candidate for enzyme production, which has led to research focused on producing cellulase and xylanase via SSC [22,23]”.

 

Comment (introduction section): The unit used for enzyme activity is U/g. Please clarify whether this refers to the dry mass of the initial substrate or to another mass basis. Ensure consistency throughout the manuscript—note that in line 69, the term "gds" (grams of dry substrate) is used.

Answer: We agree with the reviewer's comment and have modified the units used for enzyme activity to U per gram of dry mass. We also verified the consistency of these units throughout the manuscript.

 

Comment (introduction section): There is no need to provide the standard deviation for the values presented in The Introduction section.

Answer: We have removed the standard deviation in accordance with the reviewer's comment.

 

Comment (introduction section): More attention should be given to the challenges associated with scaling up SSF bioreactors. What types of moving bed bioreactors are available? Which works have employed stirred bioreactors for enzyme production?

Answer: To provide information about mixed-bed bioreactor types, characteristics, and applications in enzyme production, the following paragraph was added to the introduction section:

“Static-bed SSC bioreactors can experience overheating and poor gas exchange as they scale up [27,28]. A viable alternative is the use of mixed-bed bioreactors, which can include rotatory drum and internal stirrer bioreactors (vertical or horizontal) [29]. Both types have been successfully employed in the production of xylanases and cellulases while using low agitation velocities (<2 rpm) to avoid critical damage to the mycelium [24,30]. The mixed action in these bioreactors prevents the agglomeration and shrinkage of substrates [29]. However, in internal stirred bioreactors (ISB), the movement of the propellers favors mixing within the bed, enhancing heat removal and gas exchange [31,32]. Moreover, the incorporation of forced aeration into the bioreactor further enhances heat and mass transfer [33]. These features make ISBs a suitable option for bioprocess intensification [34,35]. Therefore, in this study, a process to produce T. harzianum enzymes in bench-scale ISBs was established. To achieve this, the incubation temperature and initial moisture content of the bed were determined using laboratory-scale packed bed column bioreactors (PBCBs). Following this, the stirring regime was evaluated in the bench-scale ISBs”.

This paragraph replaces the text presented in the initial version:

“Static-bed SSC bioreactors can experience overheating and poor gas exchange as they scale up [27,28]. A viable alternative is the use of mixed-bed bioreactors, which can include an internal stirrer [29]. The stirring action in these bioreactors prevents the agglomeration and shrinkage of substrates, thereby aiding in heat removal and improving gas exchange [30,31]. Additionally, the incorporation of forced aeration into the bioreactor further enhances heat and mass transfer [32]. These features make internal stirred bioreactors (ISB) a suitable option for bioprocess intensification [33,34]. Therefore, in this study, a process to produce T. harzianum enzymes in bench-scale ISBs was established. To achieve this, the incubation temperature and initial moisture content of the bed were determined using laboratory-scale packed bed column bioreactors (PBCBs). Following this, the stirring regime was evaluated in the bench-scale ISBs”.

 

Comment (introduction section): Finally, the conclusion of this section only mentions the ISB, despite the use of a fixed-bed system.

Answer: To highlight the use of packed bed column bioreactors (PBCBs) in our study, the following text has been added to the introduction section:

“To achieve this, the incubation temperature and initial moisture content of the bed were determined using laboratory-scale packed bed column bioreactors (PBCBs). The operating conditions established in the PBCBs were applied to evaluate the stirring regime (intermittent and continuous) in the bench-scale ISBs”.

This paragraph replaces the text presented in the initial version:

“To achieve this, the incubation temperature and initial moisture content of the bed were determined using laboratory-scale packed bed column bioreactors (PBCBs). Following this, the stirring regime was evaluated in the bench-scale ISBs”.

 

Comment (introduction section): A final remark is needed to highlight the broader significance of this work—emphasizing its relevance for researchers and practitioners involved in enzyme production and bioreactor design for solid-state fermentation processes.

Answer: The following paragraph has been added to highlight the importance of this work:

“The approaches developed in this study may aid in designing large-scale bioprocesses that promote the valorization of water hyacinth or other lignocellulosic waste through the production of hydrolytic enzymes”.

 

Comment (Materials and methods section): Is there any reference for the method presented in section 2.2?

Answer: We included a reference to the inoculum production method in the following paragraph:

“The conidia produced in each flask were suspended with magnetic stirring in 20 mL of sterile 0.05% (v/v) Tween 80 solution (Sigma-Aldrich, St. Louis, MO, USA) and used to produce mycelium in submerged culture (SmC) using the method suggested by López-Ramírez et al. [22]”.

This text replaces the previous version, which read: 

“The conidia produced in each flask were suspended with magnetic stirring in 20 mL of sterile 0.05% (v/v) Tween 80 solution (Sigma-Aldrich, St. Louis, MO, USA) and used to produce mycelium in submerged culture (SmC)”.

 

Comment (Materials and methods section): (L.125) Section 2.4 – The information regarding the packed-bed loading is unclear. Typically, dry mass is used as the reference in the SSF literature. It was stated that the bed was loaded with the same wet mass across different moisture contents, which resulted in varying bed heights. This approach turns difficult the comparison of results across treatments.

Answer: To clarify the information regarding packaging in PBCB, the following paragraph has been added to section 2.4 to specify the packed mass on a dry basis:

"Each PCBB was packed with ~8 g of dry mass (gdm), which accounts for 75% of their total capacity".

This paragraph replaces the previous version, which read: 

"Additionally, the expressions for mass have been revised from a wet basis to a dry basis in sections 2.4 and 2.5".

 

Comment (Materials and methods section): The packed bed was jacketed? If not, how the bed temperature was controlled?

Answer: The incubation temperature in the laboratory-scale PBCBs was regulated by immersing them in water baths, while in the bench-scale stirred bioreactor, it was regulated through a water jacket. These specifications were stated as follows:

Line 131, “The bioreactors were then incubated in water baths maintained at the specified temperatures (±1°C) for ~100 h”.

Line 137, “The bioreactors were incubated in a water bath at 30±1°C (determined in the previous stage) for ~100 h”.

Line 150, “The incubation temperature (30 °C) was regulated through a water jacket”.

 

Comment (Materials and methods section): Change the titles of sections 2,4 and 2.5 to Packed-bed experiments and Stirred-bed experiments, respectively.

Answer: We have modified the titles of both sections in accordance with the reviewer's comments.

 

Comment (Materials and methods section): Provide more details of the SB, such as diameter and length. Inform the geometry of the internal shaft.

Answer: To provide more details about the bioreactors used in this study, we added a new figure (Figure 1):

 

Figure 1. Packed bed column bioreactor (a) and internal stirred bioreactor (b). The numbers symbolize the components of both bioreactors. Air-flow inlet (1), air humidifier (2), culture bed (3), air-flow outlet (4), motor and transmission system (5), water jacked (6), stirring device (7), air-flow inlet (8), water inlet (9), air-flow outlet (10), water outlet (11), filling and sampling door (12), and culture bed (13).

 

Comment (Materials and methods section): How the air was introduced within the bed?

Answer: Both bioreactors are equipped with an air-flow inlet that allows a gas stream to be supplied, which then passes through the culture bed. To illustrate this feature of the system, a new figure has been added (Figure 1).

 

Comment (Materials and methods section): Section 2.6- Provide more information on the gas analyzer (brand and sensor type).

Answer: To provide more details about the gas analyzer, the following text was added to section 2.6:

“Carbon dioxide production and oxygen uptake were used to estimate fungal growth indirectly. Online measurements of O₂ and CO₂ concentrations were taken in the gas exhaust at the output of the bioreactor using a gas analyzer designed by Metropolitan Autonomous University [23]. This gas analyzer system is equipped with 15 solenoid valves, a flow meter (Honeywell, USA), CO₂ and O₂ sensors (United Phosphorus, LTD, USA), and a datalogger M6+ (PROTEC, Mexico)”.

This updated version replaces the previous statement, which read:  

“Online measurements of O₂ and CO₂ concentrations were taken in the gas exhaust at the output of the bioreactor using a gas analyzer [23]”.

 

Comment (Materials and methods section): (L 158) The inconsistent use of gds and gwm without a clearly defined criterion creates confusion. If the initial reference for bed loading was based on gwm, it is unclear why gas concentrations are later expressed in mg/gds. Since the moisture content after cultivation is not reported, the reader cannot reliably convert values to gds, making interpretation and comparison of results difficult.

Answer: To enhance consistency and ensure clarity throughout the manuscript, we have standardized the mass units to a dry basis. Furthermore, we have included moisture content analysis for each experimental stage in both the Materials and Methods and Results sections. The following sentences were added to achieve this clarity:

Line 127, “At the end of the culture, substrate moisture content and the activities of xylanase and cellulase were analyzed. The moisture content of the fermented material was determined using an Ohaus moisture analyzer (Ohaus, Model MB23, USA). Three 1-gram samples were taken from each bioreactor for this analysis”.

Line 135, “At the end of the culture, moisture content and enzyme activity were analyzed”.

Line 148, “At the end of the culture, moisture content and xylanase and cellulase activity were analyzed”.

Line 202, “The moisture content and enzyme activity (xylanase and cellulase) datasets were analyzed using the Shapiro-Wilk goodness of fit test”.

Line 248, “The culture beds incubated at different temperatures achieved a final moisture content ranging from 68.84% to 73.46%. No significant differences were observed in the final moisture content among the treatments analyzed (Tukey, α = 0.95)”.

Line 287, “At the end of culture, treatments with initial moisture contents of 65%, 70%, 75%, and 80% reached final moisture contents of 65.96%, 70.84%, 75.86%, and 78.59%, respectively”.

Line 326, “Water hyacinth beds processed under intermittent and continuous stirring regimes achieved final moisture levels of 63.23% and 64.74%, respectively. No significant differences were observed in the final moisture content between the two stirred regimes (Student's t-test, α = 0.95)”.

Comment (Materials and methods section): There are older and more traditional references than Méndez-Gonzáles [24] to inform about this method.

Answer: Following the reviewer's comment, the reference to Méndez-González et al. (2025) was replaced by Martínez-Ramírez et al. (2021).

 

Comment (Materials and methods section): Section 2.7- Since the final moisture content depends on the initial moisture content and on the water produced along the cultivation, the water used for the extraction is not constant. If the extraction water was referred to the gds, such inconsistency wouldn´t happen. By the end of the enzymatic activity determination, it is said that the results will be presented as U/gds. How the reader would convert all the different mass basis into gds?

Answer: Information on the final moisture content of the water hyacinth beds has been added to the manuscript. This addition allows enzyme activity values to be expressed on a dry weight basis.

 

Comment (Results section): (L. 209) What is the meaning of “…determine the process of producing T. harzianum enzymes”?

Answer: To improve the clarity of the paragraph, the following text was added:

“After establishing these operational conditions, the second section examines the effect of the stirring regime employed in bench-scale ISBs on the growth and enzyme production of T. harzianum”.

This text replaces the previous version, which read: 

“After establishing these operational conditions, the second section examines the stirring regime employed in bench-scale ISBs to determine the process of producing T. harzianum enzymes”.

 

Comment (Results section): Replace the title of section 3.1 to “Packed-bed bioreactor experiments”. Replace the title of 3.2 section to “Stirred-Bed bioreactor experiments”.

Answer: Answer: We have modified the titles of both sections in accordance with the reviewer's comment.

 

Comment (Results section): Why was the kinetics of fungal biomass growth determined if it was not correlated with enzyme synthesis?

Answer: In preliminary studies, we noted that peak xylanase activity coincided with maximum CO2 production (CO2max), while cellulase activity remained relatively constant during the culture. Therefore, in this study, we chose not to consider kinetic measurements of enzyme activity.

 

Comment (Results section): The authors should also present the composition of cellulose, hemicellulose, and lignin in the substrate (even if obtained from the literature), as this information is essential to establish a more robust correlation with xylanase and endoglucanase production.

Answer: According to the reviewer's comment, the following paragraph was added, denoting the concentrations of hemicellulose, cellulose, and lignin:

"However, water hyacinth has a high concentration of hemicellulose, ranging from 24.7% to 49.2% on a dry weight basis, and cellulose, ranging from 15.4% to 18.4%, as well as lignin, ranging from 1.1% to 7.0% [6–9], which allows for the integration of its biomass into enzyme production [10]". 

This paragraph replaces the previous version, which read: 

“However, water hyacinth has a high concentration of hemicellulose, cellulose, and lignin [6], which allows for the integration of its biomass into enzyme production [7]”.

Comment (Discussion section): There is limited comparison with previously published results, particularly regarding enzyme activity levels and bioreactor operation.

Answer: To enhance the comparison of the results obtained with those reported previously, the following paragraph was added to the discussion section:

“The xylanase activity achieved by T. harzianum on water hyacinth in ISBs operated under continuous conditions (77.60 ± 4.49 U/g_dm) is higher than that achieved by co-cultures of A. niger and T. ressei in conical flasks (~57.20 U/g_dm) [25]. The differences in xylanase activity could be attributed to factors such as the specific types of microorganisms used, the culture conditions, and the composition of the water hyacinth [10]. In contrast, the xylanase activity reported by López-Ramírez et al. [24] on pine sawdust beds (109.32 ± 4.91 U/g_dm) is slightly higher than what was achieved in this study. Despite using the same microorganism and similar operating conditions, the higher lignin content in pine sawdust likely plays a significant role in promoting the effective synthesis of xylanases. It is essential to note that the hemicellulose content can induce the synthesis of xylanases, while cellulose and its derivatives, such as cellobiose, act as inducers of cellulase [12,53]. Consequently, the varying composition of water hyacinth should be taken into account when implementing this bioprocess”.

 

Comment (Discussion section): Why would lower moisture content favor the production of one enzyme, while higher moisture promotes the other? Which substrate was used by Kumar et al. in their experiments?

Answer: The effect may be related to changes in mass transfer; however, we do not have enough evidence to confirm this possibility. The following paragraph has been added to clarify the culture conditions used by Kumar:

“Kumar et al. [44] observed that in wheat bran beds, maximum cellulase production occurs at an initial moisture content higher (70%) than that required for maximum xylanase production (65%)”.

This paragraph replaces the previous version, which read: 

“Kumar et al. [44] observed that maximum cellulase production occurs at an initial moisture content higher than that required for maximum xylanase production”.

 

Comment (Discussion section): Based on the literature, was it expected that bed stirring would have no impact on the mycelium of a fungus known for producing a large amount of aerial hyphae?

Answer: The stirring speed of 1 rpm utilized in this study was based on findings by López-Ramírez et al. (2018), which indicated that this speed did not negatively affect the mycelium of T. harzianum when using the same type of bioreactor packed with pine sawdust. To enhance clarity in the Discussion section, the following sentence has been added:

"Previously, López-Ramírez et al. [24] demonstrated that T. harzianum does not present mechanical damage in cross-flow ISBs operating at stirring speeds up to 1 rpm and packed with pine sawdust".

 

Comment (Conclusions): (L. 381) Replace “…static bed bioreactors” by “packed-bed bioreactors”. Add a final remark on the usefulness of the results of this work.

Answer: We have replaced “static bed bioreactor” by “packed-bed bioreactors in accordance with the reviewer's comment.

 

Comment (Conclusions): Add a final remark on the usefulness of the results of this work.

Answer: The following paragraph was added to the conclusion to highlight the usefulness of this study:

“The approaches developed in this study can aid in designing large-scale bioprocesses that promote the valorization of water hyacinth, thereby mitigating its adverse effects on freshwater ecosystems”.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have presented a very valuable research work.  They have shown that "Water hyacinth", a invasive weed, can be used for sustainable production of T. harzianum enzymes. The authors have shown how different conditions affect the enzyme production. Why did the authors pick only this particular fungal strain? is it possible to use this weed for cultivation of other fungi?

I would appreciate if authors can include a photograph of the processing of weed and the laboratory-scale static bed bioreactors. It will be of interest to the readers.

How does the DO level affect fermentation and production of enzymes?

I suggest that the authors can use water hyacinth from more than one source to check if they got consistent results and that the poly phenols  and adsorbed heavy metals etc (especially in polluted waters) didnot impact its use as feedstock.

Author Response

Comment: Why did the authors pick only this particular fungal strain?

Answer: To emphasize the significance of using T. harzianum in this study, the following paragraph has been added to the introduction section:

“Previous studies have shown that fungi from the Trichoderma genus can produce cellulases and xylanases when using water hyacinth as a solid substrate. Deshpande et al. [25] utilized a tray bioreactor with a co-culture of Aspergillus niger and Trichoderma reesei growing on water hyacinth, supplemented with Toyoma Ogow medium, whey, and peptone, to achieve xylanase production of 57.20 U per gram of dry mass (g_dm). In another study, Arana-Cuenca et al. [26] screened the enzymatic potential of 100 fungal strains using water hyacinth as the sole solid support. They showed that the Trichoderma harzianum produced 149.30 U/g_dm of xylanase at 108 h of culture and 16.40 U/g_dm of cellulase at 84 hours”.

This paragraph replaces the previous version, which read: 

“Deshpande et al. [25] utilized a tray bioreactor with a co-culture of Aspergillus niger and Trichoderma reesei growing on water hyacinth, supplemented with Toyoma Ogow medium, whey, and peptone, to achieve xylanase production of 57.20 U per gram of dry mass (g_dm). In another study, Arana-Cuenca et al. [26] screened the enzymatic potential of 100 fungal strains using water hyacinth as the sole solid support. They showed that the Trichoderma harzianum produced 149.30 U/g_dm of xylanase at 108 h of culture and 16.40 U/g_dm of cellulase at 84 hours”.

 

Comment: is it possible to use this weed for cultivation of other fungi?

Answer: The following paragraph has been added to highlight some microorganisms used to produce xylanases and cellulases through solid-state culture.

“The cost of the carbon source is a crucial factor influencing the final expenses of the enzymatic production process [14]. However, previous studies have demonstrated that efficient production of cellulases and xylanases can be obtained from lignocellulosic waste (straw, bran, bagasse, shoots and forestwaste) by solid-state culture (SSC) using filamentous fungi such as Aspergillus flavus [15], A. fumigatus [16], A. niger [17,18], Myceliophthora thermophila [19], Penicillium echinulatum [20], Trichoderma asperellum [21], T. reesei [22,23], and T. harzianum [24]. Since water hyacinth is abundant and low-cost [6], it is an excellent candidate for enzyme production, which has led to research focused on producing cellulase and xylanase via SSC”.

 

Comment: I would appreciate if authors can include a photograph of the processing of weed and the laboratory-scale static bed bioreactors. It will be of interest to the readers.

Answer: We appreciate the reviewer's feedback. Unfortunately, we do not have photographic documentation of the process. However, we have added a new figure (Figure 1) that provides details about the bioreactors used in this study. Additionally, we have included a graphical abstract that outlines the steps of the process.

Figure 1

 

Graphical abstract

 

Comment: How does the DO level affect fermentation and production of enzymes?

Answer: Aerobic conditions promote microbial growth. To highlight the significance of oxygen availability, the following paragraphs have been added to the discussion section:

 

“Thus, experiments in ISBs were conducted under operating conditions that favor the generation of xylanases. Under these conditions, the stirring regime was evaluated, and a  of 1.03 was obtained; this value corresponds to the consumption of substrates with an oxidation grade ≥4 under aerobic conditions [45]. This suggests effective consumption of both i) the glucose added to the culture and ii) the glucose released from cellulose chains. In this type of culture, the energy destined to biomass formation is greater than that required for cell maintenance [45,46]. This fact was verified in terms of mass since the values estimated through mathematical modeling corroborate that  [47]”.

“In filamentous fungal cultures, limited oxygen availability affects microbial growth, resulting in reduced CO₂ production [42,48]. In contrast, bench-scale ISB cultures have achieved up to 60% greater CO₂ production and O₂ uptake (Figure 6) compared to PBCBs (Figure 4). Stirring in SSC bioreactors reduces the agglomeration and contraction of substrate particles, thereby improving oxygen distribution and promoting microbial growth [37,48,49]”.

These paragraphs replace the previous version, which read: 

“Under these conditions, an  of 1.03 was obtained; this value corresponds to the consumption of substrates with an oxidation grade ≥4 under aerobic conditions [31]. This suggests effective consumption of both i) the glucose added to the culture and ii) the glucose released from cellulose chains. In this type of culture, the energy destined to biomass formation is greater than that required for cell maintenance [32,33]. This fact was verified in terms of mass since the values estimated through mathematical modeling corroborate that  [34]”.

“In bench-scale ISBs, up to 60% more CO₂ production and O₂ uptake were achieved (Figure 5) than in PBCBs (Figure 3). Stirring in SSC bioreactors reduces the agglomeration and contraction of substrate particles, which improves oxygen distribution and promotes microbial growth [35–37]”.

 

Comment: I suggest that the authors can use water hyacinth from more than one source to check if they got consistent results and that the poly phenols and adsorbed heavy metals etc (especially in polluted waters) did not impact its use as feedstock.

Answer: We appreciate the reviewer's suggestion. The water hyacinth used in this study is sourced from an area with low levels of contaminants. Investigating the effects of polyphenols, heavy metals, and other pollutants will enhance the applicability of this bioprocess in regions where these substances are present.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The article presents a variant of water hyacinth processing for the production of hydrolytic enzymes (xylonase and cellulase). The study is devoted to determining the optimal incubation temperature and initial moisture content using laboratory stirred tank bioreactors (PBCB). Also, stirring modes in laboratory internal stirred bioreactors are considered to determine the process of obtaining T. harzianum enzymes.

There are a number of questions and comments to the study:

1. All measurements were made without comparison with the "control".
2. Was enzyme production a simultaneous process? Is it possible to provide the study design in the Materials and Methods section?
3. In the study, the optimal incubation temperature and initial moisture content for obtaining xylanase and cellulase were determined step by step. After selecting the optimal temperature, were other combinations of temperature (e.g. 32°C) and substrate moisture content tested?
4. In the conclusions to the study on the effect of stirring regimes, the emphasis was placed on xylanase production. What was the reason for choosing only one enzyme variant?
5. Was the chemical composition of the initial plant material and is there a dependence of the enzyme yield on the composition of the water hyacinth biomass?

Author Response

Comment: All measurements were made without comparison with the "control".

Answer: In the experimental stages, the comparison between treatments included a control treatment. When determining the incubation temperature, the control treatment was the one incubated at 30°C. The initial moisture content control was 70%. Furthermore, the yields achieved in the ISB were compared with those obtained in PBCB, which had a moisture content of 65% and was incubated at 30°C. This comparison is detailed in the following paragraph of the Discussion section:

“In bench-scale ISBs, up to 60% more CO₂ production and O₂ uptake were achieved (Figure 6) than in PBCBs (Figure 4). Stirring in SSC bioreactors reduces the agglomeration and contraction of substrate particles, which improves oxygen distribution and promotes microbial growth [37,49,50]. Previously, López-Ramírez et al. [24] demonstrated that T. harzianum does not present mechanical damage in cross-flow ISBs operating at stirring speeds up to 1 rpm and packed with pine sawdust. Similarly, the results obtained in this study indicate that the mycelium of T. harzianum was not affected in any of the agitation regimes, at a rotation speed of 1 rpm. Even operating the ISB under a continuous regime, the CDPR and OUR values were improved. This improvement may be attributed to the creation of void spaces within the bed, since implementing an intermittent regime, the aerial mycelium can lead to the agglomeration of solid particles, which restricts mass and heat transfer [51]. Moreover, implementing continuous agitation increased the bioreactor's mass capacity 56-fold (from 8 to 450 g_dm) without affecting xylanase production”.

 

Comment: Was enzyme production a simultaneous process? Is it possible to provide the study design in the Materials and Methods section?

Answer: The process conditions, including temperature, initial moisture content, and agitation regime, were analyzed independently using a one-way experimental design. To enhance the clarity of this statement, the following paragraphs were added to the Materials and Methods section:

“In the first stage, the incubation temperature was determined using a one-way experimental design that evaluated five levels: 28°C, 30°C, 32°C, 34°C, and 36°C”.

“Once the incubation temperature was selected, the initial moisture content was determined using a one-way experimental design to examine four different moisture content levels”.

“Bench-scale crossflow internal stirred bioreactors (ISBs) were utilized to assess the effect of stirred regime (intermittent and continuous) on microbial growth and enzyme production through a one-way experimental design”.

 

Comment: In the study, the optimal incubation temperature and initial moisture content for obtaining xylanase and cellulase were determined step by step. After selecting the optimal temperature, were other combinations of temperature (e.g. 32°C) and substrate moisture content tested?

Answer: The conditions for the process, including temperature, initial moisture content, and agitation regime, were analyzed individually using a one-way experimental design. Therefore, interactions between the factors were not examined in this study.

 

Comment: In the conclusions to the study on the effect of stirring regimes, the emphasis was placed on xylanase production. What was the reason for choosing only one enzyme variant?

Answer: The cultures in the internal stirred bioreactors (ISBs) were specifically directed at the production of xylanase, as prior research indicates that the production of activated cellulase is significantly diminished when stirring is applied. To enhance clarity, the following paragraph has been included in the discussion section:

"Therefore, as demonstrated in this study, modifying the initial moisture content can direct the culture towards the production of xylanases or cellulases. However, previous research has shown that cellulase activity is sensitive to stirring, resulting in reductions of up to 75%. Thus, experiments in ISBs were conducted under operating conditions that favor the generation of xylanases".

Comment: Was the chemical composition of the initial plant material and is there a dependence of the enzyme yield on the composition of the water hyacinth biomass?

Answer: The composition of water hyacinth plays a crucial role in enzyme production. The following paragraph has been added to the discussion section to highlight the significance of this composition:

“The xylanase activity achieved by T. harzianum on water hyacinth in ISBs operated under continuous conditions (77.60 ± 4.49 U/g_dm) is higher than that achieved by co-cultures of A. niger and T. ressei in conical flasks (~57.20 U/g_dm) [25]. The differences in xylanase activity could be attributed to factors such as the specific types of microorganisms used, the culture conditions, and the composition of the water hyacinth [10]. In contrast, the xylanase activity reported by López-Ramírez et al. [24] on pine sawdust beds (109.32 ± 4.91 U/g_dm) is slightly higher than what was achieved in this study. Despite using the same microorganism and similar operating conditions, the higher lignin content in pine sawdust likely plays a significant role in promoting the effective synthesis of xylanases. It is essential to note that the hemicellulose content can induce the synthesis of xylanases, while cellulose and its derivatives, such as cellobiose, act as inducers of cellulase [12,53]. Consequently, the varying composition of water hyacinth should be taken into account when implementing this bioprocess”.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have revised the manuscript according to the suggestions. However, the term cellulase is too generic and does not accurately represent the specific enzyme that was measured. Since carboxymethylcellulose was used as the substrate in the cellulase activity assay, the enzyme assessed was endoglucanase. Therefore, the term endoglucanase should be used consistently throughout the manuscript. Aside from this issue, the manuscript is suitable for publication.

Author Response

We appreciate the valuable comments from the reviewer, which have greatly enhanced the quality of the manuscript.

Comment: The term cellulase is too generic and does not accurately represent the specific enzyme that was measured. Since carboxymethylcellulose was used as the substrate in the cellulase activity assay, the enzyme assessed was endoglucanase. Therefore, the term endoglucanase should be used consistently throughout the manuscript.

Answer: We agree with the reviewer's comment and have accordingly replaced the term "Cellulases" with "Endoglucanases" throughout the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

The corrected red part in the introduction is too bookish and should be restricted in length. Rather the choice of bioreactors, analytical methods and other water hyacinth based studies may be explained.

The abstract has very long complicated sentences and may be modified.

The results are well explained. The logistic model may be explained in few sentences.

After these subtle changes the manuscript may be accepted

Author Response

We appreciate the valuable comments from the reviewer, which have greatly enhanced the quality of the manuscript.

Comment: The corrected red part in the introduction is too bookish and should be restricted in length. Rather the choice of bioreactors, analytical methods and other water hyacinth based studies may be explained.

Answer: In response to the reviewer's comment, the introduction was shortened by adding a new paragraph.

“Cellulases are a complex of enzymes that hydrolyze cellulose, which includes different types of enzymes: Endoglucanases, cellobiohydrolases, and β-glucosidases [11]. Similarly, xylanases involve a complex that involves endo-1,4-β-xylanases, β-xylosidases, and accessory enzymes, such as α-arabinofuranosidases and α-glucuronidases, which degrade xylan [12]”.

Instead of:

“Cellulases are an enzyme complex that hydrolyzes cellulose, a polymer composed of different types of enzymes. Endoglucanases (EG) act randomly on the internal β-1,4-glucosidic bonds of the cellulose chain, producing shorter fragments. Cellobiohydrolases (CBH), also known as exoglucanases, target the non-reducing ends of cellulose and release cellobiose, a disaccharide composed of two glucose molecules. Lastly, β-glucosidases (BG) hydrolyze cellobiose and other oligosaccharides to yield glucose, the final product of cellulose degradation [12].

Similarly, xylanases facilitate the degradation of xylan, the second most abundant polysaccharide found in plant cell walls. This process requires a combination of enzymes, including endo-1,4-β-xylanases, which break down the internal β-1,4-glycosidic bonds of xylan, thereby releasing xylooligosaccharides. β-xylosidases further hydrolyze these xylooligosaccharides into xylose, the fundamental monomer of xylan. Accessory enzymes such as α-arabinofuranosidases, α-glucuronidases, and acetylxylan esterases play a crucial role in removing branches and structural modifications from xylan, thereby facilitating its complete degradation [13]”.

Information regarding the production of xylanases and endoglucanases, as well as the selection of bioreactors, is addressed in the following paragraphs included in the Introduction section:

“Previous research indicates that fungi from the Trichoderma genus can produce endoglucanases and xylanases when using water hyacinth as a solid substrate. Deshpande et al. [25] utilized a tray bioreactor with a co-culture of Aspergillus niger and Trichoderma reesei growing on water hyacinth, supplemented with Toyoma Ogow medium, whey, and peptone, to achieve xylanase production of 57.20 U per gram of dry mass (g_dm). In another study, Arana-Cuenca et al. [26] screened the enzymatic potential of 100 fungal strains using water hyacinth as the sole solid support. They showed that the Trichoderma harzianum produced 149.30 U/g_dm of xylanase at 108 h of culture and 16.40 U/g_dm of endoglucanase at 84 hours. Notably, both studies utilized static-bed bioreactors”.

“Static-bed SSC bioreactors can experience overheating and poor gas exchange as they scale up [27,28]. A viable alternative is the use of mixed-bed bioreactors, which can include rotatory drum and internal stirrer bioreactors (vertical or horizontal) [29]. Both types have been successfully employed in the production of xylanases and endoglucanases while using low agitation velocities (<2 rpm) to avoid critical damage to the mycelium [24,30]. The mixed action in these bioreactors prevents the agglomeration and shrinkage of substrates [29]. However, in internal stirred bioreactors (ISB), the movement of the propellers favors mixing within the bed, enhancing heat removal and gas exchange [31,32]. Moreover, the incorporation of forced aeration into the bioreactor further enhances heat and mass transfer [33]. These features make ISBs a suitable option for bioprocess intensification [34,35]”.

Comment: The abstract has very long complicated sentences and may be modified.

Answer: The abstract has been revised for clarity and simplicity in response to the reviewer's comments. The updated section now reads:

“Water hyacinth is an invasive weed that can valorize through the production of hydrolytic enzymes via solid-state culture. This study explores the application of Trichoderma harzianum in producing xylanases and endoglucanases on water hyacinth beds. Laboratory-scale packed-bed column bioreactors (PBCBs) with a capacity of 8 grams of dry mass (g_dm) were used to evaluate the effects of temperature (28-36°C) and initial moisture content (65-80%) on microbial growth and enzyme production. High yields of biomass and enzymes were achieved at 30 °C. Moreover, xylanase activity was enhanced in cultures with a moisture content of 65% (~71.24 U/g_dm), and endoglucanase activity at 75-80% moisture (~20.13 U/g_dm). The operational conditions identified for xylanase production were applied to 6 L bench-scale cross-flow internal stirred bioreactors, packed to 40% capacity with 450 g_dm. Two stirring regimes were tested: intermittent and continuous. The results showed that continuous stirring promotes both microbial growth and xylanase activity. In fact, xylanase activity in continuous stirring conditions was comparable to that achieved in PBCBs. Consequently, continuous stirring enables a 56-fold increase in bioreactor capacity without compromising xylanase production. The approaches developed in this study can support the design of large-scale bioprocesses for the valorization of water hyacinth”.

This text replaces the previous version:

“Water hyacinth is an invasive weed that can valorize through the production of hydrolytic enzymes via solid-state culture. This study explores the application of Trichoderma harzianum in producing xylanases and endoglucanases on water hyacinth beds. Utilizing laboratory-scale packed-bed column bioreactors (PBCB) with a capacity of 8 g of dry mass (g_dm), the effects of temperature (28-36°C) and initial moisture content (65-80%) on microbial growth and enzyme production were evaluated. High yields of biomass and enzymes were produced at 30 °C. Moreover, xylanase activity was enhanced in cultures with a moisture content of 65% (~71.24 U/g_dm), and endoglucanase activity at 75-80% moisture (~20.13 U/g_dm). The operational conditions for xylanase production were transferred to 6 L bench-scale cross-flow internal stirred bioreactors, packed to 40% capacity with 450 g_dm, where two stirring regimes were evaluated: intermittent and continuous. The results from the bench-scale stirred bioreactor demonstrated that continuous stirring is preferable to intermittent stirring, as there was no significant difference in xylanase activity compared to that obtained in PBCBs. As a result, continuous stirring enabled a 56-fold increase in bioreactor capacity without compromising xylanase production. The approaches developed in this study can aid in designing large-scale bioprocesses for the valorization of water hyacinth”.

 

Comment: The results are well explained. The logistic model may be explained in few sentences.

Answer: To provide a clearer explanation of the mathematical models used in this study, the following paragraph has been added to the Discussion section:

“The TCDP curves observed during cultivation displayed a sigmoid profile, which the logistic model can accurately represent [41]. This model effectively simulates the TCDP curves, achieving a goodness of fit higher than 99%. By coupling the logistic equation with the Pirt model, a function was obtained to estimate O₂ uptake associated with microbial growth and cell maintenance, expressed in terms of CO₂ production [37]. This function successfully described the TOU curves, achieving a goodness of fit exceeding 98%. The kinetic parameters included in Equations 2 and 4 provide a deeper understanding of the culture process, which helps determine operating conditions and formulate effective scale-up strategies [42,43]”.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is recommended for publication.

Author Response

We appreciate the valuable comments from the reviewer, which have greatly enhanced the quality of the manuscript.