Optimization of Palm Kernel Cake Bioconversion with P. ostreatus: An Efficient Lignocellulosic Biomass Value-Adding Process for Ruminant Feed

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study optimized the formula for fungi biomass production. The topic is valuable and can be practically applied in biomass utilization. The whole study is well designed with sufficient data and sounding conclusions. The manuscript demands minor revision for publication. 

Comments:

Methods: any public No for P. ostreatus?  
Introduction: focus more on resent studies for formula optimization studies, especially for the effects of N-resources.
Results:  more accurate production of fungal biomass production should be listed, such as 8.0 g/L.  More discussion of the transcriptome results should be added, especially for: (1) any metabolic pathways can be found for tentative N metabolic pathway; (2) any metabolites in final products can be correlated with the transcriptome data? (3) any novel genetic features of P. ostreatus.

Author Response

Article: Optimization of Palm Kernel Cake Bioconversion with P. ostreatus: An Efficient Lignocellulosic Biomass Value-Adding Process for Ruminant Feed

Comments and Suggestions for Authors

This study optimized the formula for fungi biomass production. The topic is valuable and can be practically applied in biomass utilization. The whole study is well designed with sufficient data and sounding conclusions. The manuscript demands minor revision for publication. 

Comments:

Methods: any public No for P. ostreatus?  

Response: see Lines 95-96, where the strain number was included: A commercial strain of P. ostreatus (genetically compatible with P. ostreatus PC15_ PC15)

Introduction: focus more on resent studies for formula optimization studies, especially for the effects of N-resources.

Response: Lines 55-62 included this information related to the nitrogen sources: This highlights the importance of supplementing the medium with nitrogenous nutrients for optimal fungal growth and colonization. Nitrogen metabolite repression (NMR) in filamentous fungi—a regulatory system that controls the expression of enzymes needed to utilize various secondary nitrogen sources—is specifically activated under nitrogen-sufficient conditions, especially when ammonium (NH₄⁺) or L-glutamine is present as the nitrogen source [11],[12]. Supplementation with nitrogen-rich sources has been shown to enhance the production of lignolytic enzymes and fungal biomass in P. ostreatus [13].

Results:  more accurate production of fungal biomass production should be listed, such as 8.0 g/L.  

Response: Lines 250–252 specified the biomass production and enzymatic activity: The data indicate that both fungal biomass (2.4 g/L) and laccase enzyme activity (1200 – 1400 U/L) reached the highest values in the mixtures with the highest proportion of palm kernel cake.

More discussion of the transcriptome results should be added, especially for:

(1) any metabolic pathways can be found for tentative N metabolic pathway

Response (1):  A proposed metabolic pathway for nitrogen assimilation was included in Lines 362–381:

 

Table 6 presents a set of KEGG Orthology (KO) genes identified in this study that are potentially involved in the regulation of nitrogen metabolism, particularly in the utilization of urea as a nitrogen source during the growth of P. ostreatus.

 

Table 6. Proposed metabolic pathway for nitrogen assimilation from urea in Pleurotus ostreatus, including key enzymes and corresponding KEGG orthologs (KOs)

 

Step	Protein name	K number	Principal reaction
1	Urease	K01428	Urea → 2 NH₃ + CO₂
2	Glutamine synthetase	K01915	NH₃ + Glutamate + ATP → Glutamine
3	Glutamate synthase (NADH)	K00264	Glutamine + α-Ketoglutarate + NADH → 2 Glutamate
4	Aminotransferases	K01763, K20247	Glutamate + Keto acid → New amino acid → Biomass synthesis

 

Based on the findings, the proposed pathway for urea assimilation in P. ostreatus involves the use of urea as an accessible nitrogen source, which is initially hydrolyzed into ammonia and carbon dioxide by the enzyme urease. The resulting ammonia is then efficiently incorporated into organic molecules through the glutamine synthetase–glutamate synthase (GS-GOGAT) cycle, a high-affinity system essential under low-ammonia conditions. Subsequently, glutamine produced is converted into glutamate, which acts as a key nitrogen donor in transamination reactions catalyzed by aminotransferases. These reactions enable the incorporation of nitrogen into amino acids and other nitrogen-containing compounds, thereby supporting fungal growth and biomass production. The identification of corresponding KEGG orthologs provides additional evidence supporting the functionality of these enzymatic steps in P. ostreatus.

 

(2) any metabolites in final products can be correlated with the transcriptome data?

Response (2): Lines 408–425 associate the identification of transcripts with biologically active secondary metabolites: In parallel, it has been demonstrated that, beyond improving the nutritional composition of the substrate, the fermentation of lignocellulosic material with P. ostreatus enhances the final product with bioactive secondary metabolites. Among these, lovastatin, an inhibitor of the enzyme (3S)-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which plays a key role in the mevalonate pathway for the synthesis of isoprenoid ether lipid precursors involved in the formation of cell membranes in methanogenic archaea [53]. Lovastatin also interferes with the synthesis of coenzyme F420, which participates in electron transport during methanogenesis, thereby contributing to a reduction in methane production [54]. The presence of this metabolite in palm kernel cake (PKC) may be associated with the identification of KEGG orthologs K05607, K02267, and K00507 in the transcriptomic data from this study, corresponding to the enzymatic activities of enoyl-CoA hydra-tase/isomerase, cytochrome c oxidase, and cytochrome b5-like heme/steroid-binding domain proteins, respectively.

Therefore, the enrichment of PKC with anti-methanogenic metabolites may represent a sustainable approach to mitigating enteric methane emissions—a potent greenhouse gas generated during rumen fermentation—without compromising animal nutrition [55],[56]. These results support the potential of PoFPKC as a functional feed supplement for ruminants.

 (3) any novel genetic features of P. ostreatus.

 Response (3): Lines 317–318 (see also Table 5) describe the findings related to the unique domains identified in the P. ostreatus transcriptome, as compared with the literature: The 36 unique domains in the transcriptome suggest specific adaptations to the experimental conditions with palm kernel cake.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

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Comments for author File: Comments.pdf

Author Response

Article: Optimization of Palm Kernel Cake Bioconversion with P. ostreatus: An Efficient Lignocellulosic Biomass Value-Adding Process for Ruminant Feed

The article “Optimization of Palm Kernel Cake Bioconversion with P. ostreatus: An Efficient Lignocellulosic Biomass Value-Adding Process for Ruminant Feed" explores the sustainable transformation of palm kernel cake (PKC), a by-product of the oil palm industry, into a nutritionally enhanced feed ingredient for ruminants through fungal fermentation. The study focuses on optimizing fermentation conditions using Pleurotus ostreatus and urea as an inorganic nitrogen source, significantly improving fungal biomass production, expression of lignocellulolytic genes, and the overall nutritional value of PKC. Regarding potential improvements, I would like to highlight the following aspects:

Q1 – Clarify Experimental Conditions Summarize the fermentation conditions (e.g., glucose and urea concentrations, temperature, agitation speed, incubation times) in a clear table for quick reference. Justify the selected conditions and discuss how variations might impact fungal growth, enzyme expression, and substrate bioconversion efficiency.

Response Q1: Lines 109–110 describe the fermentation conditions under which submerged fermentation (SmF) took place: Submerged culture fermentations (SmF) of 100 mL of culture medium were carried out in 250 mL flasks at 25°C ± 2°C and 150 rpm for 13 days [19]. Conditions employed were selected based on previously published studies by other authors.

Lines 128-139 describe the fermentation conditions under solid- state fermentation: The solid-state fermentation (SSF) systems design was carried out using an experimental extreme vertex mixing design. The C:N ratio of each formulation was adjusted based on the composition that yielded the highest fungal biomass production under SmF, using urea as the selected inorganic nitrogen source. The mixtures were adjusted to 60% moisture content and an initial pH of 5.5, followed by sterilization at 121 °C and 120 kPa for 15 minutes. After sterilization, the substrate was aseptically inoculated at a rate of 4% (dry matter basis) using 4-mm diameter pellets of Pleurotus ostreatus and incubated at 30 °C for 13 days under dark conditions [21]. To facilitate aerobic metabolism during the incubation, the bags were sealed with a semi-permeable adhesive membrane that allowed gas exchange. Upon completion of the incubation period, the fermented material was dried at 50 °C for 48 hours. The components of the mixtures were palm kernel, fiber and hulls, and urea. The amounts established for the blends are shown in Table 3.

 

The selected fermentation conditions for both SmF and SSF were defined based on a prior literature review and laboratory experimentation

Q2 – Expand Practical Validation Include in vivo validation of the fermented PKC by evaluating its effects on digestibility, weight gain, and health parameters in ruminants. This would strengthen the practical relevance of the findings and support the proposed application in animal nutrition.

Q3 – Economic Feasibility Assessment Perform a preliminary economic analysis to assess the viability of scaling up the fermentation process. Include cost estimates for substrates, fermentation equipment, processing, and potential market value of the enriched feed to enhance the industrial applicability of the research.

Q4 – Stability and Storage Evaluation Investigate the nutritional and microbiological stability of the fermented PKC during storage under different conditions (e.g., ambient vs. refrigerated) over time. Providing data on shelf-life would improve the practical application and commercial potential of the product.

Q5 – Analysis of Secondary Metabolites Assess the profile of secondary metabolites produced during fermentation, determining whether any compounds could pose risks or offer additional benefits for animal health. This would ensure the safety and functional quality of the fermented feed. Addressing these aspects would significantly enhance the scientific rigor, practical relevance, and industrial applicability of the study, providing a stronger foundation for the large-scale implementation of Pleurotus ostreatus-mediated palm kernel cake bioconversion in sustainable animal nutrition strategies.

Response Q2-Q5: The current research focused primarily on optimizing the bioconversion of palm kernel cake (PKC) using Pleurotus ostreatus under controlled fermentation conditions, with the goal of improving its nutritional profile and potential for ruminant feeding. The scope was intentionally limited to the optimization of the solid-state and submerged fermentation processes, as well as the characterization of chemical and enzymatic parameters directly related to fungal treatment efficacy.

Q2–Q5, although highly relevant for future applications, were not included in this phase of the study for the following reasons:

• Q2: In Vivo Validation – In vivo trials are essential for confirming practical outcomes such as digestibility, animal performance, and health, and such experiments require additional ethical approvals, animal facilities, and extended timelines. Such experiments are currently being carried out and will be the basis for the construction of a future manuscript in which we will compare results with those reported in this manuscript.
• Q3: Economic Feasibility Assessment – The current study focused on generating fundamental biological and biochemical data necessary to support later economic modeling. Conducting a full cost-benefit analysis, including scale-up simulations, is planned as part of a follow-up study, once technical parameters have been fully validated.
• Q4: Stability and Storage Evaluation – The primary aim was to establish the feasibility and effectiveness of PKC bioconversion. Storage trials, including microbiological shelf-life and nutrient stability under different conditions, will be considered in subsequent work once optimal fermentation conditions have been standardized.
• Q5: Secondary Metabolite Profiling – Although this aspect is critical for ensuring the safety and added value of the fermented product, the current study prioritized enzymatic activity and macronutrient enrichment. Initially, mycotoxin analysis was considered; however, it was excluded from the final scope since Pleurotus ostreatus is not known to produce mycotoxins, and its safety has been well-documented in the literature. Therefore, resources were redirected toward parameters more directly related to the nutritional enhancement of PKC.

In this context, the current research lays the technical and biological groundwork necessary for future in vivo, economic, and safety assessments. The exclusion of Q2–Q5 is thus a strategic decision to allow focused advancement through phased research, with these important dimensions already identified as priorities for subsequent investigation.

 

Author Response File: Author Response.pdf