Plant Proteins as an Alternative Nitrogen Source for Chiral Purity L-Lactic Acid Fermentation from Lignocellulose Feedstock

Round 1

Reviewer 1 Report

The work by Zhang et al. reports an interesting example of the optimization of L-lactic production process, focusing on the choice of the nitrogen source as a key factor influencing the optical purity of the product.

The results are interesting although the manuscript requires a careful revision of the English language (sentences, typos…).

Furthermore, the following points need to be revised/clarified:

-Line 111-112. Looking at the composition of the pretreated wheat straw, it also contains cellulose and xylan, beside fermentable sugars. Are these polysaccharides further hydrolysed during the fermentation thanks to endogenous hydrolytic bacterial activities?

Line 123 and below: which are the pH conditions chosen for each protease? Could this pH influence further cellulase activity and/or microbial growth? This could be the case, since, differently from the acidic treatment, no neutralization step is reported for the enzymatic process. Can the authors clarify this point?

Line 141-142: could the author better explain the need for glucoamylase addition?

Paragraph 3.1 The authors should better introduce the requirement of a detoxification step also in this section.

Line 179-180: check the English grammar

Table 1: Why the dosages of YE + peptone, reagent and industrial grade are reported with such a big error bar (15 ± 10)?

Line 207: Please specify which lactic acid bacterium are the authors referring at. Furthermore, the necessity to hydrolyse the complex nitrogen sources is not related to the lack of the biosynthetic pathway for the synthesis of the essential aminoacids, but to the absence of microbial protease activity. Is this the case? Please clarify this point.

Line 214: as previously commented, please clarify the protease operative conditions.

Line 229: “Significantly” refers to a statistical analysis? Has it been performed?

Line 230-234: the authors explain the observed differences between the two protein hydrolysates assuming that the enzymatic hydrolysis is not sufficient to provide enough nutrients for the growth. Is it possible to calculate the percentage of hydrolysis with respect to the theoretical maximum? Have the authors tried combinations of different proteases to increase the hydrolysis efficiency? Why is the enzymatic hydrolysate not neutralized as in the case of the acid one? Could the pH of the hydrolysate (that is not specified) affect microbial growth?

Figure 1: why do the authors refer to “cellulosic L-lactic”? Which is the cellulose source in this case?

Lines 252-254 The authors should better explain the rational behind the choice of further investigating the selected conditions (3% alkaline protease, 5% sulfuric acid, 5% hydrochloric acid).

Figure 2: Which is the composition of the medium used in the experiments? Is the wheat straw completely hydrolysed? Or further amount of glucose and xylose are released from cellulose and xylan present in the pretreated wheat straw (according to what reported in lines 111-114)?

Lines 267-275: have the authors checked the biomass growth in the experiments reported in Figure 2? This could help to explain a possible nitrogen limitation that in turn determines the lower glucose consumption in sulfuric acid hydrolysate.

Lines 280-282 Check the English grammar of the sentence.

Line 290-291. Please rewrite the sentence, it is not clear.  

Author Response

Reviewer #1:

Question 1: Line 111-112. Looking at the composition of the pretreated wheat straw, it also contains cellulose and xylan, beside fermentable sugars. Are these polysaccharides further hydrolysed during the fermentation thanks to endogenous hydrolytic bacterial activities?

Answer: The L-lactic acid producing strain Pediococcus acidilactici used in this study had been engineered for L-lactic homo-fermentation. The whole genome sequencing (accession number of CP082111.1 in GenBank) showed that this strain did not contain the genes encoding for polysaccharide (cellulose or xylan) hydrolase. The strategy of simultaneous saccharification and co-fermentation (SSCF) was used in this study due to the continuous hydrolysis by commercial cellulase. The corresponding descriptions had been added in Page 3, Lines 100-102, and Page 8, Lines 288-291 as follows:

“P. acidilactici ZY271 can be used for L-lactic homo-fermentation. The whole genome sequencing of P. acidilactici ZY271 was submitted to GenBank with the accession number of CP082111.1.”

“The pre-hydrolysis (~6 h) can only hydrolyze partial cellulose and xylan in biodetoxified wheat straw, the glucose and xylose can be continuously released from cellulose and xylan due to the continuous hydrolysis by commercial cellulase during the fermentation.”

Question 2: Line 123 and below: which are the pH conditions chosen for each protease? Could this pH influence further cellulase activity and/or microbial growth? This could be the case, since, differently from the acidic treatment, no neutralization step is reported for the enzymatic process. Can the authors clarify this point?

Answer: The corresponding descriptions about enzymatic hydrolysis conditions had been added in Pag 3, Lines 132-138 as follows:

“The pH value of mixture was adjusted to 10.0 by adding 5 mol/L NaOH solution when alkaline proteinase was used as catalyst, while the pH value (~5.0) of mixture didn’t need to be adjusted when other proteinase was used as catalyst. The enzymatic hydrolysis was carried at 50 °C, 150 rpm for 24 h. The hydrolysate was then inactivated at 90 °C for 10 min to eliminate the negative effect of protease on cellulase and the growth of subsequent fermentation microorganism. The pH value of enzymatic hydrolyzed cottonseed meal hydrolysate was adjusted to ~5.0 by adding 5 mol/L NaOH solution or 5 mol/L sulfuric acid.”

Question 3: Line 141-142: could the author better explain the need for glucoamylase addition? Paragraph 3.1 The authors should better introduce the requirement of a detoxification step also in this section.

Answer: Our previous study found that Pediococcus acidilactici cells in medium aggregated into small pieces and attached to flask wall during the seed preparation in flasks, then led to the low cell viabilities and practically poor fermentation performance. The cell bridging by polysaccharide, protein, and lipid is considered as the major reasons of flocculation. Therefore, glucoamylase was supplemented during cell cultivation to break the polysaccharide links among the cell aggregations and the cell aggregation disappeared with the uniform cell distribution after glucoamylase addition [1]. The corresponding explanations had been added in Page 4, Lines 152-154 as follows:

“The glucoamylase was added into MRS medium at 1% (w/w) mass ratio to break the polysaccharide links among the cell aggregations, thus prevents cell flocculation.”

The explanations of biodetoxification had been added in Page 3, Lines 95-97 as follows:

“because the weak organic acids and phenolic aldehyde compounds generated from the harsh pretreatment processing severely inhibit consequent fermenting microbes which need to be removed.”

Reference:

[1]  Liu, G.; Sun, J.; Zhang, J.; Tu, Y.; Bao, J. High titer L-lactic acid production from corn stover with minimum wastewater generation and techno-economic evaluation based on Aspen plus modeling. Bioresour. Technol. 2015, 198, 803–810, http://doi/org/10.1016/j.biortech.2015.09.098.

Question 4: Line 179-180: check the English grammar

Answer: The sentence had been revised in Page 5, Lines 195-197 as follows:

“However, one serious problem of using corn steep liquor was that the optical purity of L-lactic acid product was sharply reduced to only around 95%, which doesn’t meet the requirement of optical purity for PLA production [3].”

Question 5: Table 1: Why the dosages of YE + peptone, reagent and industrial grade are reported with such a big error bar (15 ± 10)?

Answer: In Table 1, 15 represents 15 g/L for YE; 10 represents 10 g/L for peptone in hydrolysate. Table 1 had been revised to avoid ambiguity.

Question 6: Line 207: Please specify which lactic acid bacterium are the authors referring at. Furthermore, the necessity to hydrolyse the complex nitrogen sources is not related to the lack of the biosynthetic pathway for the synthesis of the essential aminoacids, but to the absence of microbial protease activity. Is this the case? Please clarify this point.

Answer: The sentence in Page 6, Lines 228-231 had been revised as follows:

“As lactic acid bacteria such as Lactobacillus casei, Lactobacillus rhamnosus, etc., have a limited capacity to synthesize several amino acids [9,12], complex plant protein should be hydrolyzed into its amino acids form before being utilized.”

In addition, the proteinases currently used, which are capable of hydrolyzing the plant protein, mostly need to be operated under specific conditions (specific temperature, pH, etc.), which significantly differ from the fermentation conditions of lactic acid bacteria. Therefore, it is difficult to hydrolyzed plant protein by secreting proteases from the strains themselves.

Question 7: Line 214: as previously commented, please clarify the protease operative conditions.

Answer: The corresponding descriptions about enzymatic hydrolysis conditions had been added in Page 3, Lines 132-138 as follows:

“The pH value of mixture was adjusted to 10.0 by adding 5 mol/L NaOH solution when alkaline proteinase was used as catalyst, while the pH value (~5.0) of mixture didn’t need to be adjusted when other proteinase was used as catalyst. The enzymatic hydrolysis was carried at 50 °C, 150 rpm for 24 h. The hydrolysate was then inactivated at 90 °C for 10 min to eliminate the negative effect of protease on cellulase and the growth of subsequent fermentation microorganism. The pH value of enzymatic hydrolyzed cottonseed meal hydrolysate was adjusted to ~5.0 by adding 5 mol/L NaOH solution or 5 mol/L sulfuric acid.”

Question 8: Line 229: “Significantly” refers to a statistical analysis? Has it been performed?

Answer: The inaccurate word had been deleted.

Question 9: Line 230-234: the authors explain the observed differences between the two protein hydrolysates assuming that the enzymatic hydrolysis is not sufficient to provide enough nutrients for the growth. Is it possible to calculate the percentage of hydrolysis with respect to the theoretical maximum? Have the authors tried combinations of different proteases to increase the hydrolysis efficiency? Why is the enzymatic hydrolysate not neutralized as in the case of the acid one? Could the pH of the hydrolysate (that is not specified) affect microbial growth?

Answer: According to the content of glutamic acid in cottonseed meal and the concentration of glutamic acid in cottonseed meal hydrolysate, the percentages of hydrolysis from protein to amnio acids were approximately 2.0-7.0% for enzymatic hydrolysis, and 4.0%-45.5% for acid hydrolysis, respectively. However, a proportion of protein was hydrolyzed to the short peptides, this hydrolysis percentage will be further determined by ninhydrin method in subsequent studies. Meanwhile, the detailed descriptions of enzymatic hydrolysis had been added in Page 3, Lines 132-138. The enzymatic hydrolyzed cottonseed meal was also neutralized to pH ~5.0, in order to avoid the effect on the growth of strain.

Question 10: Figure 1: why do the authors refer to “cellulosic L-lactic”? Which is the cellulose source in this case?

Answer: In Figure 1, the lactic acid was also produced from wheat straw hydrolysate. The corresponding descriptions had been added in Page 6, Lines 227-228 and Page 7, Lines 267-268 as follows:

“Cellulosic L-lactic acid fermentation performances using cottonseed meal hydrolysate as complex nutrients source were investigated in flasks (Figure 1).”

“Cellulosic L-lactic acid fermentation performance from wheat straw using cottonseed meal hydrolysate as complex nutrients source in flasks.”

Question 11: Lines 252-254 The authors should better explain the rational behind the choice of further investigating the selected conditions (3% alkaline protease, 5% sulfuric acid, 5% hydrochloric acid).

Answer: In Figure 1a, the cottonseed meal hydrolyzed by 3% (w/w) alkaline proteinase #2 showed the best fermentation performance among the enzymatic hydrolysis methods. Although the fermentation using 7% (w/w) hydrochloric acid hydrolyzed cottonseed meal obtained the highest lactic acid titer, the lactic acid titer using 5% (w/w) hydrochloric acid hydrolyzed cottonseed meal was similar to that. To alleviate the materials costs and reactor corrosion, 5% (w/w) hydrochloric acid was selected as the hydrolysis catalyst for further investigation. Meanwhile, the fermentation using 5% (w/w) sulfuric acid hydrolyzed cottonseed meal was also investigated compared to 5% (w/w) hydrochloric acid hydrolysis. Therefore, the L-lactic acid fermentation performances were evaluated by simultaneous saccharification and co-fermentation (SSCF) of wheat straw using cottonseed meal hydrolysates catalyzed by 3% (w/w) alkaline protease #2, 5% (w/w) sulfuric acid solution and 5% (w/w) hydrochloric acid solution (Figure 2). The corresponding explanations had been added in Pages 7-8, Lines 274-285 as follows:

“As shown in Figure 1a, the cottonseed meal hydrolyzed by 3% (w/w) alkaline proteinase #2 showed the best fermentation performance among the enzymatic hydrolysis methods. Although the fermentation using 7% (w/w) hydrochloric acid hydrolyzed cottonseed meal obtained the highest lactic acid titer, the lactic acid titer using 5% (w/w) hydrochloric acid hydrolyzed cottonseed meal was similar to that. To alleviate the salt inhibition, materials costs and reactor corrosion, 5% (w/w) hydrochloric acid was selected as the hydrolysis catalyst for further investigation. Meanwhile, the fermentation using 5% (w/w) sulfuric acid hydrolyzed cottonseed meal was also investigated compared to 5% (w/w) hydrochloric acid hydrolysis. Therefore, the L-lactic acid fermentation performances were evaluated by simultaneous saccharification and co-fermentation (SSCF) of wheat straw using cottonseed meal hydrolysates catalyzed by 3% (w/w) alkaline protease #2, 5% (w/w) sulfuric acid solution and 5% (w/w) hydrochloric acid solution (Figure 2).”

Question 12: Figure 2: Which is the composition of the medium used in the experiments? Is the wheat straw completely hydrolysed? Or further amount of glucose and xylose are released from cellulose and xylan present in the pretreated wheat straw (according to what reported in lines 111-114)?

Answer: The medium used in Figure 2 contains 25% (w/w) solids loading of biodetoxified wheat straw, 20 g/L cottonseed meal (hydrolyzed), 2 g/L of diammonium hydrogen citrate and 0.25 g/L of manganous sulfate monohydrate. We adopted the simultaneous saccharification and co-fermentation (SSCF) for lactic acid. The pre-hydrolysis (~6 h) can only hydrolyze partial cellulose and xylan in biodetoxified wheat straw, the glucose and xylose can be continuously released from cellulose and xylan due to the continuous hydrolysis by commercial cellulase during the fermentation. The corresponding explanations had been added in Page 7, Lines 271-272, Page 8, Lines 286-288, Page 8, Lines 300-301, and Page 9, Lines 338-340 as follows:

“Other fermentation nutrients included 2 g/L of diammonium hydrogen citrate and 0.25 g/L of manganous sulfate monohydrate.”

“The pre-hydrolysis (~6 h) can only hydrolyze partial cellulose and xylan in biodetoxified wheat straw, the glucose and xylose can be continuously released from cellulose and xylan due to the continuous hydrolysis by commercial cellulase during the fermentation.”

“Other fermentation nutrients included 2 g/L of diammonium hydrogen citrate and 0.25 g/L of manganous sulfate monohydrate.”

“Other fermentation nutrients included 20 g/L of cottonseed meal hydrolyzed by 5% (w/w) sulfuric acid, and 0.25 g/L of manganous sulfate monohydrate.”

Question 13: Lines 267-275: have the authors checked the biomass growth in the experiments reported in Figure 2? This could help to explain a possible nitrogen limitation that in turn determines the lower glucose consumption in sulfuric acid hydrolysate.

Answer: In fact, the biomass in high solids loading wheat straw hydrolysate was difficult to be measured due to the interferences of insoluble solids. It was reported that lactic acid production depends strictly on cell growth, as fermentation is associated with cell growth. The lower cell biomass using sulfuric acid hydrolyzed cottonseed meal leads to lower glucose consumption and lactic acid titer. The corresponding explanations had been added in Page 8, Lines 311-316 as follows:

“Furthermore, the cell biomass in high solids loading wheat straw hydrolysate was difficult to be measured due to the interferences of insoluble solids. It was reported that lactic acid production depends strictly on cell growth [10]. The lower glucose consumption rate and lactic acid titer using sulfuric acid hydrolyzed cottonseed meal as complex nutrients indicated that lower cell biomass was obtained compared to that using hydrochloric acid hydrolyzed cottonseed meal.”

Question 14: Lines 280-282 Check the English grammar of the sentence.

Answer: The sentence had been revised in Page 9, Lines 320-322 as follows:

“The other reason is that the hydrochloric acid is difficult to be removed in the subsequent purification, while sulfuric acid is easily formed the precipitate calcium sulfate after neutralization.”

Question 15: Line 290-291. Please rewrite the sentence, it is not clear.

Answer: The sentence had been revised in Page 9, Lines 330-333 as follows:

“Generally, the complete removal of residual non-glucose sugars from lactic acid broth to polymer grade monomer would cause great difficulties. The low residual sugars level in this study were satisfied to prepare L-lactic acid monomer for PLA polymerization [34].”

In addition, some other minor changes did not list here but highlight in red in the revised manuscript.

Reviewer 2 Report

1)Section 2.1: Was the wheat straw analyzed in this study? They can be different in different varieties and may depend on location, nutrition, weather, etc.

2)L78-81: What was the composition of cottonseed and soybean meal? Not indicated in the text?

3)L94: This is not clear to the audience: ‘…was applied to the biodetoxification of acid pretreated wheat straw [20].               

4)L111: There was no water wash to remove acid from the treated biomass?

5)What method was used for analysis of biomass and treated one? Not explained.

6)L112: Glucose was in the treated biomass in free form? It is part of the structure of cellulose.

7)L125: What was the original activity of protease? Not indicated.

8)L126: The enzymes and optimal conditions should be included in the text.

9)L133: Which acid is this? Not indicated.

10)L145: What brand of bioreactor is this? Manufacturer?

11)L147L What was the cell content/concentration in the seed? Or what was the cell load to the hydrolysate for fermentation process?

12)It is not clear where and when the plan protein was added to the medium?

13)L152: A brief description of HPLC analysis is required.

14)Were the expenses of enzymatic hydrolysis and material cost considered in cost analysis?

15)It is not clear how technoeconomic analysis was conducted?

16)What was the aeration in lactic acid production? Should be indicated under graphs.

17)The results should be compared at the end with some commercial or well known cases.

Author Response

Reviewer #2:

Question 1: Section 2.1: Was the wheat straw analyzed in this study? They can be different in different varieties and may depend on location, nutrition, weather, etc.

Answer: The main compositions of raw wheat straw had been determined by NREL protocol, which include 34.31 ± 0.14% of cellulose, 21.30 ± 1.78% of xylan, 22.12 ± 0.07% of lignin, and 10.63 ± 0.28% of ash on dry base. The corresponding descriptions had been shown in Page 2, Lines 65-68 as follows:

“The raw wheat straw was harvested in Nanyang city, Henan province, China, June, 2022. The main compositions of raw wheat straw include 34.31 ± 0.14% of cellulose, 21.30 ± 1.78% of xylan, 22.12 ± 0.07% of lignin, and 10.63 ± 0.28% of ash on dry base, which were determined by two-step hydrolysis method [18,19].”

Question 2: L78-81: What was the composition of cottonseed and soybean meal? Not indicated in the text?

Answer: The protein content of cottonseed meal and soybean meal were determined by semi-automatic Kjeldahl apparatus, which is 54.5 ± 1.1% and 45.6% ± 1.0% on dry matter, respectively. The cellulose and hemicellulose contents in cottonseed meal and soybean meal were determined by two-step hydrolysis method, which are 5.0 ± 0.3% and 5.9 ± 1.1%, 10.1 ± 0.3% and 10.9 ± 0.6% on dry matter, respectively. The corresponding descriptions had been added in Page 5, Lines 223-225 as follows:

“Note: The cellulose and hemicellulose contents in cottonseed meal and soybean meal were also determined by two-step hydrolysis method, which are 5.0 ± 0.3% and 5.9 ± 1.1% for cottonseed meal, 10.1 ± 0.3% and 10.9 ± 0.6% for soybean meal on dry matter, respectively.”

Question 3: L94: This is not clear to the audience: ‘…was applied to the biodetoxification of acid pretreated wheat straw [20].

Answer: The corresponding descriptions had been added in Page 3, Lines 94-97 as follows:

“The fungus Paecilomyces variotii FN89 (CGMCC 17665) was applied to the biodetoxification of acid pretreated wheat straw [20], because the weak organic acids and phenolic aldehyde compounds generated from the harsh pretreatment processing severely inhibit consequent fermenting microbes which need to be removed.”

Question 4: L111: There was no water wash to remove acid from the treated biomass?

Answer: The washing step would cause the loss of free sugars in pretreated wheat straw, therefore there was no washing step after pretreatment. Sulfuric acid was used as catalyst for pretreatment. The sulfuric acid was neutralized by CaCO₃ powder to form calcium sulfate precipitation. The pretreated wheat straw doesn’t need to be washed. The corresponding descriptions had been added in Page 3, Lines 120-122 as follows:

“The sulfuric acid catalyzed was neutralized to calcium sulfate precipitation. The pretreated wheat straw doesn’t need to be washed. The neutralized pretreated wheat straw was then aerobically biodetoxified by P. variotii FN89.”

Question 5: What method was used for analysis of biomass and treated one? Not explained.

Answer: The main compositions of raw wheat straw and pretreatment wheat straw were determined by two-step hydrolysis method from NREL protocols [2,3]. The corresponding descriptions had been added in Page 2, Lines 67-68 and Page 3, Lines 115-116 as follows:

“which were determined by two-step hydrolysis method from NREL protocols [18,19].”

“were also determined by two-step hydrolysis method.”

References:

[2] Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C. Determination of sugars, byproducts, and degradation products in liquid fraction process samples. National Renewable Energy Laboratory. NREL/TP-510–42623. 2008.

[3] Sluiter, A.; Hames, B.; Scarlata, C.; Sluiter, J.; Templeton, D. Determination of structural carbohydrates and lignin in biomass national renewable. National Renewable Energy Laboratory. NREL/TP-510–42623. 2012.

Question 6: L112: Glucose was in the treated biomass in free form? It is part of the structure of cellulose.

Answer: During the dilute acid pretreatment process, most of xylan and small partial of cellulose was hydrolyzed into corresponding oligosaccharides and monosaccharides. The over-degradation of monosaccharides would result in inhibitors generation (glucose to HMF; xylose to furfural).

Question 7: L125: What was the original activity of protease? Not indicated.

Answer: The original activity of protease had been shown in the section of Materials and methods 2.2. The three kinds of neutral protease were purchased from Novozymes (Beijing, China) (neutral protease #1), SUNSON Industry Group CO., Ltd. (Beijing, China) (neutral protease #2), Vland Biotech INC. (Qingdao, China) (neutral protease #3), respectively, with the enzymatic activity of 0.8 AU/g, 5Í10⁴ U/g and 10Í10⁴ U/g according to the manufacture’s instruction. The two kinds of alkali protease were purchased from SUNSON Industry Group CO., Ltd. (Beijing, China) (alkali protease #1) and Vland Biotech INC. (Qingdao, Shandong, China) (alkali protease #2) with the same enzymatic activity of 20Í10⁴ U/g. The papain and trypsin were purchased from Sunson Industry Co., Beijing, China with the enzymatic activity of 10Í10⁴ U/g and 4Í10⁴ U/g.

Question 8: L126: The enzymes and optimal conditions should be included in the text.

Answer: The corresponding descriptions about enzymatic hydrolysis conditions had been added in Page 3, Lines 132-138 as follows:

“The pH value of mixture was adjusted to 10.0 by adding 5 mol/L NaOH solution when alkaline proteinase was used as catalyst, while the pH value (~5.0) of mixture didn’t need to be adjusted when other proteinase was used as catalyst. The enzymatic hydrolysis was carried at 50 °C, 150 rpm for 24 h. The hydrolysate was then inactivated at 90 °C for 10 min to eliminate the negative effect of protease on cellulase and the growth of subsequent fermentation microorganism. The pH value of enzymatic hydrolyzed cottonseed meal hydrolysate was adjusted to ~5.0 by adding 5 mol/L NaOH solution or 5 mol/L sulfuric acid.”

Question 9: L133: Which acid is this? Not indicated.

Answer: The tested acid included oxalic acid, hydrochloric acid, and sulfuric acid. The corresponding descriptions had been added in Page 3, Lines 143-144 as follows:

“The tested acids included oxalic acid, hydrochloric acid, and sulfuric acid.”

Question 10: L145: What brand of bioreactor is this? Manufacturer?

Answer: The information of bioreactor manufacture had been added in Page 4, Lines 163-165 as follows:

“The fermentation pH in 5-L bioreactor (BaoXing Bioengineering Equipment Co., Ltd, Shanghai, China) was maintained at 5.5 by automatic regulation with 25% (w/w) of Ca(OH)₂ slurry.”

Question 11: L147L What was the cell content/concentration in the seed? Or what was the cell load to the hydrolysate for fermentation process?

Answer: The OD₆₀₀ values of seed culture are between 3.5 and 5.0. The corresponding descriptions had been added in Page 4, Lines 154-155 as follows:

“The OD₆₀₀ values of seed culture are between 3.5-5.0.”

Question 12: It is not clear where and when the plan protein was added to the medium?

Answer: The plant meal hydrolysate was added after the pre-hydrolysis of wheat straw but before the inoculation. The corresponding descriptions had been added in Page 4, Lines 159-161 as follows:

“After the pre-hydrolysis, the neutralized plant meal hydrolysate was added at 10% (w/w) mass ratio, equivalent to 20 g/L of plant meal. Then the seed was inoculated at the ratio of 20% (w/w).”

Question 13: L152: A brief description of HPLC analysis is required.

Answer: The corresponding descriptions about HPLC method had been added in Page 4, Lines 167-170 as follows:

“Glucose, xylose, lactic acid, acetic acid, HMF and furfural was determined by Shimadzu HPLC system equipped with a Bio-rad Aminex HPX-87H column and RID-10A detector. Twenty microliters of the sample were subjected and analyzed at 60 °C using 5 mM H₂SO₄ as eluent with the flow rate of 0.6 mL/min [23].”

Question 14: Were the expenses of enzymatic hydrolysis and material cost considered in cost analysis?

Answer: The cellulase used in this cost analysis was produced onsite according to the report of NREL. The cost of cellulase is $4.34/kg protein. The prices of other materials had been added in Page 10, Lines 359-360 as follows:

“The price of material was updated and shown in Table 5.”

“Table 5. The prices of materials used in techno-economic evaluations.”

Question 15: It is not clear how technoeconomic analysis was conducted?

Answer: The preliminary techno-economic evaluations were based on the operation of biorefinery plant with annual processing capacity of 300,000 metric tons of dry lignocellulose feedstock. The process model, total capital investment, and discounted cash flow rate of return analysis were cited from the previously established lactic acid production processing based on dry biorefinery technology with the cost updates of materials and equipment [1].

References:

[1]  Liu, G.; Sun, J.; Zhang, J.; Tu, Y.; Bao, J. High titer L-lactic acid production from corn stover with minimum wastewater generation and techno-economic evaluation based on Aspen plus modeling. Bioresour. Technol. 2015, 198, 803–810, http://doi/org/10.1016/j.biortech.2015.09.098.

Question 16: What was the aeration in lactic acid production? Should be indicated under graphs.

Answer: The lactic acid fermentation in this study is anaerobic fermentation process, which doesn’t need aeration. The corresponding descriptions had been added in Page 4, Lines 161-162 as follows:

“The anaerobic fermentation was conducted at 42 °C, 150 rpm for 72 h without aeration.”

Question 17: The results should be compared at the end with some commercial or well known cases.

Answer: Due to the significant differences of substrates, biorefining processings, microbes, etc., between different reported cases, it is difficult to make an objective comparison. We firstly compared the MESP of lactic acid in our current studies, suggesting that the replacement of YE and peptone by cottonseed meal hydrolysate can reduce the production costs by 74.9%. And then we compared the MESP of cellulosic lactic acid (using cottonseed meal hydrolysate as complex nutrients) in this study to the cost of starch-based lactic acid production, showing that the cellulosic lactic acid production processing (Case 3, $0.813/kg) was comparable and competitive to that from food crops ($0.833 or $2.50/kg) [4,5]. The corresponding descriptions had been shown in Pages 10, Lines 364-374.

References:

[4]  Åkerberg, C.; Zacchi, G. An economic evaluation of the fermentative production of lactic acid from wheat flour. Bioresour. Technol. 2000, 75, 119–126, http://doi/org/10.1016/S0960–8524(00)00057–2.

[5]  González, M.I.; Álvarez, S.; Riera, F.; Álvarez, R. Economic evaluation of an integrated process for lactic acid production from ultrafiltered whey. J. Food Eng. 2007, 80, 553–561, http://doi/org/10.1016/j.jfoodeng.2006.06.021.

In addition, some other minor changes did not list here but highlight in red in the revised manuscript.

Round 2

Reviewer 1 Report

The authors addressed all the reviewer's comments.

Reviewer 2 Report

The comments are addressed carefully. The manuscript is in good shape for publication.