Acid lactic din xilooligozaharide cu Rhizopus oryzae.pdf

8/9/2019 Acid lactic din xilooligozaharide cu Rhizopus oryzae.pdf

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Biochemical Engineering Journal 94 (2015) 92–99

Contents lists available at ScienceDirect

Biochemical Engineering Journal

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b e j

Simultaneous saccharification and fermentation of xylo-oligosaccharides manufacturing waste residue for l-lactic acidproduction by Rhizopus oryzae

Li Zhang a,b, Xin Li a, Qiang Yong a,∗, Shang-Tian Yang b,∗∗, Jia Ouyang a, Shiyuan Yu a

a College of Chemical Engineering, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, PR Chinab William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 West 19th Avenue, Columbus, OH 43210, USA

a r t i c l e i n f o

Article history:

Received 12 May 2014

Received in revised form

21 November 2014

Accepted 26 November 2014

Available online 27 November 2014

Keywords:

Cellulose

Fermentation

Filamentous fungi

Lactic acid

Rhizopus oryzae

Xylo-oligosaccharides

a b s t r a c t

High substrate cost and low lactic acid yield are the most pressing concerns in fermentative production

of l-lactic acid by Rhizopus oryzae. In this study, waste residue from corncob after xylo-oligosaccharides

(XOS) manufacturing was used as an alternative abundant, renewable, and inexpensive substrate for

l-lactic acid production. After enzymatic hydrolysis, both glucose and xylose in the hydrolysate were

converted to 34.0g L −1 of l -lactic acid, equivalent to a yield of 0.34 g g−1 dry waste residue, by R. oryzae

in separate hydrolysis and fermentation. In contrast, a higher l-lactic acid titer (60.3 g L −1) and yield

(0.60gg−1 dry waste residue) were achieved in simultaneous saccharification and fermentation (SSF)

with 10% (w/v) substrate loading at 40 ◦C, demonstrating, for the first time, the feasibility of l-lactic acid

production from XOS manufacturing waste residues. The SSF process for l-lactic acid production from

XOS wasteresidueswas also demonstratedin a 5-L stirred-tank bioreactor,although further optimization

would be necessary.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Lactic acid is a commonly occurring organic acid that can be

produced biologically from renewable carbohydrates, and is valu-

able due to its wide use in food and related industries [1]. In

addition, a variety of useful chemicals, including plastics, fibers,

solvents, and oxygenated chemicals, can be produced from lactic

acid derived from renewable feedstocks by sustainable biotech-

nological routes [2]. More recently, bio-based l-lactic acid has

attractedincreasingattentionfor itsuse as a startingmaterialin the

synthesis of poly-lactic acid (PLA) polymers, which are biodegrad-

able and biocompatible with wide applications that conventional

petroleum-based plastics such as polyesters are not suitable or

unfavorable due to environmental concerns [3]. Today, lactic acidproduced in fermentation has become one of the most promising

feedstock monomers in the chemical industry.

Abbreviations: R. oryzae,Rhizopusoryzae;XOS, xylo-oligosaccharides;SHF, sep-

arate hydrolysis and fermentation; SSF, simultaneous saccharification

and fermentation.∗ Corresponding author. Tel.:+86 25 85427587; fax: +86 25 85427587.∗∗ Corresponding author. Tel.: +1 614 2926611; fax: +1 614 2923769.

E-mail addresses: [email protected] (Q. Yong), [email protected] (S.-T. Yang).

Current industrial production of lactic acid uses homolactic acid

bacteria, mainly Lactobacillus spp., cultured in enriched (complex)

media with glucose as substrate, which produce l(+)- or d(−)-form

of lactic acid with high product yield and productivity, but usually

sufferfrom highraw material and purification costs [1]. In contrast,

the filamentous fungus Rhizopus oryzae can use relatively inexpen-

sive polysaccharides (e.g., starch) in simple media with minimal

nutrient supplementation and is easy to separate by filtration after

fermentation, and is thus, advantageous for its potential to reduce

lactic acid production cost [4–8]. It produces optically pure l-lactic

acid, which is the desirableform for foodand pharmaceutical appli-

cations, and can also use xylose [9], the main sugar component in

hemicellulose, as substrate. Compared to bacteria, it can grow well

at a wider temperature range (up to 40◦C)andpH range (from 4 to9) [5]. Moreover, chitosan present in the fungal mycelia is a high-

value product, and the fungal biomass and byproducts can be used

in animal feeds to improve quality [4].

The current sugar and starch-based feedstock for lactic acid

fermentation accounts for more than 30–40% of the total produc-

tion cost [4]. Recent research on lactic acid production has thus

focused on abundant, low-cost lignocellulosic feedstocks [4,10,11].

More than 20 million tons of corncobs are available annually

in China [12]. Currently, a large amount of this is used to pro-

duce xylo-oligosaccharides (XOS) [13,14] from the hemicellulosic

http://dx.doi.org/10.1016/j.bej.2014.11.020

1369-703X/© 2014 Elsevier B.V. All rights reserved.


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L. Zhang et al. / Biochemical Engineering Journal 94 (2015) 92–99 93

materials, generating large quantities of waste residues, mainly

cellulosic materials, which would cause major environmental, and

economic problems if not properly treated or utilized. Because of

its low cost, high enzymatic digestibility due to low lignin content,

small particle size, and environmental benefits of the re-utilization

of this industrial waste, XOS manufacturing waste residues can

be considered as an attractive alternative substrate for l-lactic

acid production. Compared to corncobs and other lignocellulosic

biomass, XOS waste residues with most of xylan removed contain

mainlyglucose with a lowcontent ofxylose,and thus, wouldbe bet-

ter for lactic acid fermentation since most microorganisms cannot

use xylose efficiently [4].

The goal of this study was to develop an economical fer-

mentation process for l-lactic acid production from XOS waste

residues by R. oryzae. Unlike the conventional separate hydroly-

sis and fermentation (SHF) process, simultaneous saccharification,

and fermentation (SSF) can synchronize enzymatic hydrolysis and

microbial fermentationin a single step,thus offeringvariousadvan-

tages, including increased productivity and reduced processing

time due to reduced product (glucose) inhibition on cellulose

hydrolysis [11,15,16]. In this work, both SSF and SHF were stud-

ied and compared for lactic acid production from XOS waste

residues. The hydrolysis and fermentation of XOS waste residues

were studied at different solid loadings and temperatures, and theresults are reported in this paper. To our knowledge, this is the

first study demonstrating high-titer, high-yield, and cost-effective

l-lactic acid production from lignocellulosic biomass in an SSF

process.

2. Materials and methods

2.1. XOS waste residue

The XOS waste residue derived from alkali-pretreated corncobs

was kindly provided by Jiangsu Kangwei Biologic Co., Ltd. Milled

corncobs was stewed in 12 m3 alkali extraction tank containing

7% (w/v) sodium hydroxide at 85–90◦C for 1 h. The liquid fraction,

with a high content of hemicellulose was removed by vacuum fil-

tration for further production of XOS; the solid waste residue was

first soaked in water with a solid/liquid ratio of 1:10 (w/v), and

then neutralized with 72% (w/w) sulfuric acid to adjust the pH to

5.0–5.5, followed by removing the water with vacuum filtration to

obtain the solid XOS waste residue. The solid fraction was stored

in plastic bags at 4 ◦C until use. Before treatments, the corncobs

contained (%, dry weight basis) ∼40% cellulose and ∼31% hemicel-

lulose. After treatments, the solid waste residues contained 65.5%

cellulose and 22.2% hemicellulose or 72.8% glucose, 23.3% xylose,

and 1.9% arabinose.

2.2. Microorganism and cultivation

R. oryzae NLX-M-1 was obtained from Institute of Biochemi-

cal Engineering, Nanjing Forestry University, Nanjing, China. The

preculture medium consisted of (g L −1): 50 glucose, 3 (NH4)2SO4,

0.75 MgSO4·7H2O, 0.20 ZnSO4·7H2O, and 0.30 KH2PO4, which was

found to be optimal with glucose as the carbon source. All media

were sterilized by autoclaving at 121 ◦C, 15 psig for 30 min. The

strain was first cultured on potato-dextrose agar slants at 30 ◦C

for 3–5 days to generate spores. The preculture used to seed the

fermentation was prepared in 250-mL Erlenmeyerflasks, each con-

taining 50 mL preculture medium and 10g L −1 CaCO3, inoculated

witha sporesuspension containing 106 spores mL −1, and incubated

at 30 ◦C for 12 h in a rotary shaker agitated at 170rpm.

2.3. SHF for lactic acid production

Cellic® CTec2 (Novozymes), a cellulase complex consisting of

aggressive cellulases, -glucosidases and hemicellulase, was usedfor the degradation of cellulose and hemicellulose to fermentable

sugars. Enzymatic hydrolysis trials were performed with the sub-

strate loadings at 5%, 10%, 15%, and 20% (w/v). Unless otherwise

noted, the hydrolysis was carried outin a 250-mL Erlenmeyer flask

with the enzyme dosage of 0.06g g−1 biomass at pH 5.0–5.5, in a

shaker controlled at 50 ◦C and 150 rpm, for 2–3 days. The cellu-

lose (glucan) and hemicellulose (xylan) hydrolysis yields (%) were

calculated as the percentages of obtained glucose and xylose in

the hydrolysate to the total glucose and xylose present in the

substrate, respectively. For complete hydrolysis, the theoretical

sugar yields from cellulose and xylan are 1.11g glucose g−1 glucan

and 1.14g xyloseg−1 xylan, respectively [17]. Allexperiments were

duplicated, and the average values are reported. Before use as sub-

strate in fermentation, the enzymatic hydrolysate was centrifuged

and filtered to remove solid residues, and then supplemented with

minerals as follows (g L −1): 1 (NH4)2SO4, 0.38 MgSO4·7H2O, 0.10

ZnSO4·7H2O, 0.15 KH2PO4.

The fermentation was thenstudied in 250-mL Erlenmeyerflasks

each containing 100mL of the enzymatic hydrolysate of XOS waste

residue. CaCO3 was added at 50% of the theoretical amount of glu-cosederivedfrom thedried material(w/w) to maintainthe medium

pH at >6.0 for good cell growth and l -lactic acid production in the

fermentation. After autoclaving at 121◦C for 30min, eachflaskwas

inoculated with the preculture at an inoculation size of 10% (v/v).

Unless otherwise noted, the fermentation was performed at 40 ◦C

in a rotary shaker at 170 rpm for 2–3 days or until glucose was

depleted or lactic acid production ceased. Unless otherwise noted,

each fermentation condition was studied in duplicate.

2.4. SSF for lactic acid production

The SSF process was studied in 250-mL Erlenmeyer flasks with

a 100 mL working volume in a rotary shaker at 170 rpm and 40◦C,

unless otherwise noted. The fermentation medium containing thesame inorganic salts as in the SHF medium and XOS waste residue

(5%, 10%, 15%, or 20% w/v), with pH of ∼5.5, was sterilized by auto-

claving at 121 ◦C for 30 min. After cooling, enzymes were added at

the loading rateof 0.06g g−1 biomass,and each flask was then inoc-

ulated with the preculture at 10% (v/v). CaCO3 was added at 50% of

the theoretical amount of glucose derived from the dried material

(w/w) after 12 h of fermentation to keep the medium pH at >6.0.

Unless otherwise noted, all batch fermentations were duplicated.

2.5. l-lactic acid production in bioreactor

The SSF process was also studied in a 5-L stirred tank biore-

actor (Biostat B, B. Braun) with a rotating fibrous matrix, made

of a cotton cloth (9×15×0.2cm) fixed on the outer surface of aperforated stainless steel cup mounted on the impeller shaft, for

cell immobilization [6]. The bioreactor with 3 L of the medium was

sterilized at 121 ◦C for 30 min. After cooling, the bioreactor was

inoculated with 10% (v/v) preculture and operated at 40 ◦C, with

agitation at 200rpm andaerationat 1.0vvm. After 12h, the reactor

pH was maintained at >6.0 by adding CaCO3 solution periodically.

Antifoam 204 from Sigma (0.5 mL per L medium) was added to

prevent foaming during fermentation.

2.6. Analytical methods

The sporeconcentration was determined by countingthe spores

on a haemocytometer under a microscope. Analysis of chemical

composition in XOSwaste residues was carried out according to the


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94 L. Zhang et al. / Biochemical Engineering Journal 94 (2015) 92–99

National Renewable Energy Laboratory standard methods for the

determination of structural carbohydrates and lignin in biomass

[18]. For the analysis of fermentation broth samples, 72% (w/w)

sulfuric acid was first added to neutralize excessive CaCO3 and

acidify calcium lactate. The samples were then heated in boil-

ing water for 5 min to increase the solubility of lactic acid and

deactivate the hydrolytic enzyme and fungus. After centrifuga-

tion, the supernatant was analyzed for glucose, xylose, ethanol,

and l-lactic acid with a high performance liquid chromatograph

(HPLC)equippedwithan AminexBioRad HPX-87H columnat 45◦C,

using 5mM H2SO4 as the mobile phase at 0.6 mL min−1. To sepa-

rate and determine l(+)-lactic acid and d(+)-lactic acid by HPLC,

the SCAS Sumichiral OA-5000 column at 35 ◦C and 1mM CuSO4in 5% (v/v) isopropanol at 1.0 mL/min as the mobile phase were

used. The detectionwas at UV 254nm. However, no d(+)-lactic acid

was detected,indicating thatthe R. oryzae strain producedoptically

pure l(+)-lactic acid.

3. Results

3.1. SHF of XOS waste residue

3.1.1. Enzymatic hydrolysis of XOS waste residueFig. 1 shows the profiles of glucose and xylose released from

enzymatic hydrolysis of the waste residue at 5%, 10%, 15%, and 20%

(w/v) substrate loadings. The glucose and xylose titers increased

over time and reached maximum levels in 24–60h, depending on

the solid loading. As expected, more glucose and xylose were pro-

duced at higher substrate loadings. For example, increasing the

substrate loading from 5% to 20% also increased the final glucose

concentrations in the hydrolysates from 31.68 g L −1 to104.97 g L −1.

However, the glucose yield decreased from 87.13% to 72.17% (see

Table 1), probably due to increased solution viscosity, mass trans-

fer limitation, and end-product inhibition [19]. At 5% solid loading,

the enzymatic hydrolysis process was completed in 48h, with over

30g L −1 glucose and 8 g L −1 xylose released from the XOS waste

residue. The conversion yield of cellulose to glucose was 87.1%,whereas theconversionyield of hemicellulose to xylose was 64.2%,

which could be improved if an endoxylanase with a higher speci-

ficity (e.g., Cellic HTec2) was used. No cellobiose accumulation was

observed during the enzymatic hydrolysis process, indicating a

rapid conversion of cellobiose to glucose under the synergism of

cellulase complex. The high enzymatic conversion of XOS waste

residue makes this raw material a promising feedstock for sugar-

platform biorefinery.

3.1.2. Effect of temperature on l-lactic acid production from

hydrolysates

Usually, a moderate temperature, around 30 ◦C, is required

for fungal cell growth [20], but a higher temperature (>40◦

C) isfavorable for enzymatic hydrolysis. R. oryzae usually grows at a

temperature between 27 and35 ◦C [4] and pH 5.0–6.5 [6,21]. How-

ever, it is desirable to perform the fermentation at a temperature

closer to the temperature for enzymatic hydrolysis. Therefore, we

first studied the effect of temperature on l-lactic acid production

from XOS enzymatic hydrolysates. As shown in Fig. 2, 13.7 g L −1 of

l-lactic acidwitha yield of 0.274 g g−1 waste residue was produced

at 25 ◦C. The l-lactic acid production increased to 15.9g L −1 when

temperature increased to 30 ◦C. However, from 30 ◦C to 40 ◦C, the

l-lactic acid titer and yield did not show any significant change.

Further increasing the temperature to 45 ◦C resulted in no lactic

acid production, as R. oryzae could not be adapted to grow at the

higher temperature. Thus, 40 ◦C was chosen for the subsequent

fermentation studies.

B

A

Fig. 1. Time course profiles of sugars released in enzymatic hydrolysis of xylo-

oligosaccharides waste residues at 5%, 10%, 15%, and 20% (w/v) substrate loading.

A: glucose; B: xylose. Data shown are the average with error bar representing the

standard deviationfromduplicateruns.Error baris notvisible forsomedata points

because the standard deviation is smaller than the size of the symbol.

Fig. 2. Effect of temperature on l-lactic acid production from enzymatic

hydrolysates of XOS waste residues by R. oryzae in shake-flasks at 5% (w/v) sub-

strateloading.Data shown arethe average with error barrepresenting thestandard

deviation from duplicated runs.


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Table 1

Effects of substrate loading on enzymatic hydrolysis of XOS waste residue.

Substrate loading (%) Glucose Xylose

Titer (g L −1) Yield (%) Productivity (g L −1 h−1) Titer (g L −1 ) Yield (%) Productivity (g L −1 h−1)

5 31.68 ± 0.00 87.13 ± 0.01 0.66 ± 0.00 8.10 ± 0.02 64.24 ± 0.16 0.17 ± 0.00

10 61.32 ± 0.35 84.32 ± 0.49 1.28 ± 0.01 15.90 ± 0.05 63.06 ± 0.18 0.33 ± 0.00

15 88.89 ± 0.07 81.49 ± 0.06 1.48 ± 0.00 23.46 ± 0.39 62.02 ± 1.04 0.39 ± 0.01

20 104.97 ± 1.12 72.17 ± 0.77 1.75 ± 0.02 29.38 ± 0.12 58.26 ± 0.23 0.49 ± 0.00

Data shown are the average± standard deviation from duplicated runs.

B

DC

A

Fig. 3. l-lactic acid fermentation profiles of R. oryzae using XOSwasteresiduehydrolysates as substratein shake-flasks at 40 ◦C andvarious substrateloadings. A: 5%;B: 10%;

C: 15%; and D: 20% (w/v) loading. Data shown are the average with error bar representing the standard deviation from duplicated runs. Error bar is not visible for some data

points because the standard deviation is smaller than the size of the symbol.

3.1.3. l-lactic acid fermentation of enzymatic hydrolysates

Fig.3 shows the fermentation profiles of R. oryzae grownon XOS

waste residue hydrolysate at various substrate loadings. The fer-

mentation pH was maintained at >6.0by adding calcium carbonate.

There was not much difference in the pH profile among the differ-

ent substrate loadings, except for the 20%loading that had a higher

initial pH of ∼7.5 due to more CaCO3 were added. Nevertheless,

the pH in the range of 5.5–7.5 should have a minimal effect on the

fermentation [6]. In general, little or no lactic acid was produced

in the first 12 h, indicating that glucose was mainly used to gen-erate fungal biomass. Thereafter, a rapid consumption of glucose

with fast accumulation of l-lactic acid and ethanol occurred until

allglucosehad been consumed,exceptfor the 20%loading, inwhich

lactic acid production ceased before glucose depletion. During the

fermentation, xylose was also consumed simultaneously with glu-

cose, but at a much slower rate. As can be seen in Fig. 3A, xylose

consumption rate increased significantly 12h after glucose deple-

tion, suggesting that xylose uptake by the cells was inhibited or

repressed by glucose [9]. The slower xylose uptake by cells might

also be because xylose transport was much slower than glucose or

was strongly inhibited by glucose. Increasing the substrate loading

from 5% to 15% also increased lactic acid production titer (from

16.5gL −1 to 36.5gL −1) and productivity (from 0.34 g L −1 h−1 to

0.76gL −

1 h−

1) (see Table 2). Further increasing the substrate load-ing to 20% significantly decreased lactic acid production, which

might be attributed to the limitation in oxygen transfer at the

higher solid loading. The lactic acid yield was 0.33–0.34 g g−1

dry waste residue when the substrate loading was 5–10%, but

Table 2

Effects of substrate loading on l-lactic acid fermentation by R. oryzae with the enzymatic hydrolysates of XOS waste residues.

Substrate loading (%) Lactic acid (gL −1) Ethanol (g L −1 ) Lactic acid/ethanol ratio (gg−1) Lactic acid yield (gg−1 ) Lactic acid productivity (gL −1 h−1)

5 16.53 ± 0.38 5.04 ± 0.45 3.28 0.33 ± 0.01 0.34 ± 0.01

10 34.00 ± 0.04 9.50 ± 0.25 3.58 0.34 ± 0.00 0.71 ± 0.00

15 36.50 ± 0.15 12.00 ± 0.19 3.04 0.24 ± 0.00 0.76 ± 0.00

20 24.44 ± 0.01 15.85 ± 0.71 1.54 0.12 ± 0.00 0.51 ± 0.00

Data shown are the average±

standard deviation from duplicated runs.


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decreased significantlyto 0.24g g−1 at15%loadingand0.12gg−1 at

20% loading. Product (lactic acid) inhibition could adversely affect

microbial accessibility and fungal cell growth, as well as lactic

acid biosynthesis, which might limit lactic acid accumulation to

a higher concentration in the fermentation with higher substrate

loadings. The lower lactic acid production at the higher substrate

loading might also be attributed to the xylose inhibiting lactic

acid production [22]. The fermentation could also be inhibited by

unknown inhibitors produced from the degradation of ligninin the

hydrolysate[23]. The observed diauxic growth withthe accelerated

xylose consumption after glucose depletion suggests that xylose

utilization by R. oryzae is regulated by the carbon catabolite repres-

sion [24]. The slower xylose uptake by cells might also be because

xylose transport was much slower than glucose or was strongly

inhibitedby glucose.Moreover, Maaset al.[25] suggestedthatmore

xylose might be respired to carbon dioxide rather than lactic acid,

and the nutrients were limiting the conversion of xylose.

It is noted that there were significant amounts of ethanol

co-produced in the fermentation, with the final titer increased

from 5.0 g L −1 at 5% loading to 15.9 g L −1 at 20% loading. Ethanol

production suggested insufficient oxygen supply in these shake-

flask fermentations [26,27]. The lactic acid/ethanol ratio was ∼3.3

(g g−1) for 5–15% substrate loading and 1.5 (g g−1) at 20% load-

ing. Apparently, 20% loading was more favorable for ethanolproduction.

Overall, 10% substrate loading appeared to give the best fer-

mentation results, comparable to previously reported studies on

lactic acid production by R. oryzae from lignocellulosic biomass

in similar SHF processes [12,22–25,27–30]. Nevertheless, the l-

lactic acid production from enzymatic hydrolysates is a complex

and labor- and time-consuming process. Even the best runs at 10%

loading did not give sufficiently high l-lactic acid yield and produc-

tivity from the XOS waste residue hydrolysate to be economically

competitive.

Fig. 4. Effect of temperature on l -lactic acid production from XOS waste residues

at 5% (w/v) substrate loading in simultaneous saccharification and fermentation in

shake-flasks. Data shown are the average with error bar representing the standard

deviation from duplicated runs.

3.2. SSF of XOS waste residue

3.2.1. Effect of temperature on l-lactic acid production

The SSF process can potentially overcome problems in SHF and

increase l-lactic acid production from XOS waste residues. One

challenge associated with SSF is that the optimal temperatures

and pHs for the saccharification and fermentation processes are

different [31]. It is thus, necessary to determine the optimal tem-

perature for l-lactic acid production in the SSF process. As shown

in Fig. 4, l-lactic acid production increased significantly in both the

final titer (from ∼8.0gL −1 to ∼25.0gL −1) and product yield (from

0.16gg−1 to0.50 gg−1) as the temperature increased from 25◦C to

A B

DC

Fig. 5. Profiles of simultaneous saccharificationand fermentation of XOS waste residues by R. oryzae in shake-flasks at 40 ◦C andvarious substrateloadings. A: 5%;B: 10%;C:

15%; and D: 20% (w/v) loading. CaCO3 was added at 12 h, which caused an increase in the pH. Data shown are the average with error bar representing the standard deviation

from duplicated runs. Error bar is not visible for some data points because the standard deviation is smaller than the size of the symbol.


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40 ◦C, which might be attributed to the fact that more glucose was

producedfrom celluloseat thehighertemperature in theenzymatic

hydrolysis by cellulase and available for cells to use in the fer-

mentation. However, when the temperature increased to 45 ◦C, no

l-lactic acid was produced, indicating that R. oryzae cells could not

be adapted to survive under thisstressful condition. This study con-

firmed thathigh-titerlactic acidproductioncan be achievedin a SSF

process at 40 ◦C, a temperature that can improve substrate utiliza-

tion efficiency and reduce production cost by efficiently balancing

enzymatic hydrolysis and sugar fermentation processes [20].

3.2.2. l-lactic acid production in SSF process

Fig. 5 shows the fermentation time course data, including glu-

cose, xylose, lactic acid, and ethanol concentration profiles, during

the SSF process at differentsubstrate loadings. At 5% substrateload-

ing (Fig. 5A), there was initially a fast accumulation of glucose,

which reached ∼16g L −1 by 12 h, indicating that the rate of enzy-

matic hydrolysis was significantly higher than sugar fermentation

in this period. Thereafter, a rapid decrease in the glucose con-

centration coupled with a sharp accumulation of l-lactic acid was

observed, indicating that acid production from glucose was faster

than glucose released from cellulose hydrolysis [14]. In fact, the

glucose concentration was kept at near zero since 24 h, suggestingthatall glucose released fromcellulosewas immediately consumed

in the SSF process. The lactic acid titer reached the maximum

level of24.9g L −1 at 36h, achieving a product yield of 0.497 g lactic

acidg−1 dry waste residue. Thus, a continuous and simultane-

ous saccharification and fermentation for l-lactic acid production

from XOS waste residues was realized. Meanwhile, ethanol was

also produced in the SSF, reaching 4.64 g L −1 by 24 h. Interestingly,

ethanol production ceased after 24 h, even though lactic acid pro-

duction continued until 36 h. This was probably because the low

glucose concentration limited cell growth and oxygen uptake by

cells, resulting in a higher oxygen tension that favored lactic acid

biosynthesis. Clearly, the SSF process favored lactic acid produc-

tion, resulting in a higher lactic acid/ethanol ratio of 5.4 (g g−1), as

compared to the SHF process.R. oryzae was also able to ferment pentose for l -lactic acid pro-

duction [9]. As can be seen in Fig. 5A, the xylose concentration in

the fermentation broth increased to ∼5 g L −1 and remained rela-

tively constant until a significant decrease was observed after 36h.

The increased utilization of xylose as carbonsource coincided with

the depletion of glucose and cellulose (data not shown). Neverthe-

less, the utilization of xylose by R. oryzae was not as efficient as

glucose due to catabolite repression and other reasons discussed

earlier [9,22,25].

Since substrate loading plays an important role in the SSF pro-

cess, we also investigated the fermentation at higher substrate

loadings of 10% (Fig. 5B), 15% (Fig. 5C), and 20% (Fig. 5D). In gen-

eral, the fermentation profiles at 10% loading were similar to those

at 5% loading, but reached a higher lactic acid titer of 60.3 g L −1

and yield of 0.60g g−1 dry waste residue with a significantlyhigher

productivity(1.0 g L −1 h−1 vs.0.69gL −1 h−1). Further increasing the

substrate loading resulted in a significant reduction in lactic acid

production, with the final lacticacid titer dropped to 47.7 g L −1 and

yield to 0.32g g−1 waste residue at 15% loading and 33.8g L −1 and

0.17gg−1 waste residue at 20% loading, respectively. At the higher

solid loadings of 15% and 20%, sugar consumption seemed to be

greatly reduced, resulting in high concentration levels of glucose

and xylose that were not completely consumed in the fermenta-

tion. In fact, xylose consumption was inhibited in the presence of

large amounts of glucose. The poor fermentation performance at

the higher solid loadings can be attributed to increased broth vis-

cosity and reduced oxygen transfer, which not only decreased the

sugar consumption rate but also increased ethanol production, as

indicated by the decreased lactic acid/ethanol ratio at 20% solid

loading (see Table 3). A decrease in oxygen transfer was usually

observed at a higher substrate loading, which was not favorable

for lactic acid production by R. oryzae [6]. It has also been reported

that the fermentation kinetics in the SSF process could be affected

by the reducing sugar concentration in the hydrolysate, as well as

substrate loading, because of increased viscosity and substrate and

product inhibition [11]. In addition, imbalanced osmotic pressure

inducedby highsugarconcentration wasanothercritical factor that

might havealso inhibitedcell growth andreducedsubstrateutiliza-

tion efficiency [32]. Clearly, poor mixing and mass transfer at the

elevated solid loading in shake-flasks inhibited the fermentation

with R. oryzae in the SSF process.

Table 3 summarizes and compares the results of SSF at vari-

ous solid loadings. It is clear that 10% loading was optimal, withhigh-titer and high-yield l -lactic acid production from XOS waste

residues in the SSF. Previous studies also found that lactic acid

production from cellulose decreased with increasing the substrate

loading, resultingin a significant reductionin the product yield and

conversion efficiency, even with a prolonged fermentation period

[33,34]. Ruengruglikit and Hang [35] reported that 5% (w/v) was

the optimal loading for corncob as substrate, and a substrate load-

ing below 10% (w/v) was also suggested by Moritz and Duff [36].

Compared to SHFat the same substrateloading of 10%, higherlactic

acid titer (60.3 g L −1 vs. 34.0g L −1) and yield (0.60 vs. 0.34g g−1 dry

waste residue) were achieved in the SSF process. This can be par-

tiallyattributed to the fact that lacticacid production becamemore

favorable than ethanol production in the SSF process, as evidenced

by the much higherlactic acid/ethanolratio inSSF (10.6g g−1) com-pared to the SHF process (3.6g g−1). It is noted that the glucose

inhibition effect on cellulose hydrolysis was alleviated in the SSF

process, whichmight have also contributed to thehigher lacticacid

yield.

3.3. SSF for l-lactic acid production in bioreactor

In order to investigate the scale-up feasibility of the SSF pro-

cess, fermentation with 10% substrate loading was carried out in

a 5-L stirred tank bioreactor, and the results are shown in Fig. 6.

In general, the fermentation performed well, producing 41.9g L −1

lactic acid with a product yield of 0.42g g−1 dry biomass, which

were lower than those obtained in shake-flask fermentation. The

lower lactic acid production in the stirred-tank bioreactor could be

attributed to the differences in cellmorphology,which would affect

mass transfer and lactic acid production [7], andthe dissolved oxy-

gen (DO) concentration level. Too much oxygen (high DO) would

promote cell growth and led more substrate into the TCA cycle for

Table 3

Effects of substrate loading on simultaneous saccharification and fermentation for l-lactic acid production from XOS waste residue by R. oryzae.

Substrate loading (%) Lactic acid (gL −1) Ethanol (g L −1) Lactic acid/ethanol ratio (gg−1) Lactic acid yield (gg−1) Lactic acid productivity (gL −1 h−1)

5 24.87 ± 0.10 4.64 ± 0.17 5.36 0.50 ± 0.00 0.69 ± 0.00

10 60.29 ± 0.26 5.71 ± 0.32 10.56 0.60 ± 0.00 1.00 ± 0.00

15 47.72 ± 0.19 7.28 ± 0.21 6.55 0.32 ± 0.00 0.66 ± 0.00

20 33.75 ± 1.01 8.13 ± 1.03 4.15 0.17 ± 0.01 0.35 ± 0.01

Data shown are the average±

standard deviation from duplicated runs.


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Table 4

Comparison of l-lactic acid production from lignocellulosic biomass by R. oryzae.

Strain Substrate Titer (g L −1) Yield (g g−1) Productivity (g L −1 h−1) References

SHF

R. oryzae HZS6 Corncob 77.2 – 0.99 [12]

R. oryzae UMIP 4.77 Wheat straw 10 0.26 0.27 [22]

R. oryzae NRRL 395 Waste office paper 49.1 – 0.51 [23]

R. oryzae NBRC 5378 Wheat straw 2 – – [24]

R. oryzae CBS 112.07 Wheat straw 6.8 0.23 – [25]

R. oryzae NRRL 395 Cassava pulp 16.7 0.24 0.29 [27]R. sp. MK-96-1196 Corncob 26 0.26 – [28]

R. oryzae NRRL 395 Wheat bran 6 – – [29]

R. oryzae GY 18 Corncob – 0.36 – [30]

R. oryzae NLX-M-1 XOS waste residue 34.0 0.34 0.71 This study

SSF

R. oryzae UMIP 4.77 Paper pulp 24.1 – 0.5 [22]

R. oryzae NBRC 5378 Wheat straw 6 0.23 – [24]

R. sp. MK-96-1196 Corncob 24 0.24 – [28]

R. oryzae NRRL 395 Corncob – 0.30 – [35]

R. oryzae NLX-M-1 XOS waste residue 60.3 0.60 1.0 This study

SHF, separate hydrolysis and fermentation; SSF, simultaneous saccharification and fermentation.

Fig. 6. Profiles of simultaneous saccharification and fermentation of XOS waste

residues by R. oryzae at 40 ◦C at 10% (w/v) substrate loading in a 5-L stirred tank

bioreactor. CaCO3 was added at 12 h and periodically thereafter to maintain the pH

above 6.0.

ATP generation andbiomassformation, thereby loweringlacticacid

yield. In addition, continuous aeration at a high rate (1vvm) would

strip away CO2 and ethanol, which could promote more pyruvate

conversion to acetaldehyde and ethanol, thus reducing lactic acid

production. Compared to shake-flasks, a relatively high DO and

low pCO2 in the bioreactor with continuous aeration could have

a negative effect on lactic acid production [37]. Further optimiza-

tion in agitation and aeration rates would be necessary to improve

the fermentation performance. It is noted that the ethanol titer inthe stirred-tank bioreactor was much less than that in flasks, only

2.26gL −1, indicating that some of theproduced ethanol was lost as

vapor due to continuous aeration.

4. Discussion

Several studies on l-lactic acid fermentation with lignocellu-

losic biomass have been reported and are summarized in Table 4

for comparison. For SHF, Park et al. [23] reported the highest lac-

tic acid production of 49.1g L −1 from the enzymatic hydrolysate of

waste office paperin a 4-day culture,whereasa moderatelacticacid

yield of 0.36g g−1 corncob was obtained from corncob hydrolysate

[30]. Compared to these SHF studies, comparable lactic acid titer

andyield, buta higher productivityof 0.71 g L −1

h−1

, were obtained

from XOS waste residues in our study. For SSF, most of the previ-

ous studies with R. oryzae producedonly upto 24g L −1 of lacticacid

with a moderate yield of 0.23–0.30g g−1

substrate and a productiv-ity of ∼0.5gL −1 h−1. On the other hand, our SSF process produced

a high lactic acid titer of 60.3 g L −1 with a yield of 0.6gg−1 and

productivity of 1.0g L −1 h−1 from XOS waste residues, which were

among the highest ever reported with R. oryzae using lignocellu-

losic biomass as raw material. It is noted that the lactic acid yield

from XOS waste residues by R. oryzae was comparable to those

obtained from similar lignocellulosic biomass with lactic acid bac-

teria. Sreenath et al. [15] used Lactobacillus plantarum to produce

lactic acid from alfalfa fiber in a SSF process, achieving a yield of

0.61gg−1 drymatter of fiber. A coculture of Lactobacillus rhamnosus

and Lactobacillus brevis was used in a SSF process, which produced

lactic acid from corn stover with a yield of 0.70g g−1 cellulose and

hemicellulose [16].

For the same lignocellulosic materials, R. oryzae could produce

more l-lactic acid ata higher final titer in SSFthanin SHF. Forexam-

ple, only ∼2 g L −1 of lactic acid was produced from wheat straw in

SHF, while lactic acid production reached 6 g L −1 in SSF [24]. This is

because the inhibition of cellulase–catalyzed reaction by glucose

can be eliminated or greatly alleviated by simultaneously using

glucose in the fermentation, which prevents the accumulation of

glucose generatedfrom the hydrolysis of cellulosic materials in the

SSF process [38]. However, Miura et al. [28] reported that 26 g L −1

l-lactic acid was produced from the enzymatic hydrolysate of

corncob, whilein the SSFprocess usinga mixed culture of cellulase-

producing Acremonium thermophilus and Rhizopus sp. MK-96-1196

at 35 ◦C, 24gL −1 of l-lactic acid was produced from 100 g L −1 of

untreated raw corncob. It is thus, clear that the performance of SSF

will also be dependent on the raw materials used, and each SSF

process will have to be optimized for the conditions used in theprocess, including solid loading and agitation and aeration rates.

Further studies including process optimization, lactic acid conver-

sion from xylose and economic analysis will have to be performed

before the process canbe commercialized. Better understandingof

the metabolicpathways involvedin xylose utilization by R. oryzae is

also needed in order to maximize l-lactic acid production in terms

of final product titer, yield, and productivity [9].

5. Conclusion

The waste residues from corncobs used in XOS manufacturing

can be used as a low-cost substrate for l-lactic acid production

by R. oryzae. The solid waste residues can be efficiently treated


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and hydrolyzed with commercial cellulase enzymes to release fer-

mentable sugars, glucose, and xylose, for lactic acid production. A

high cellulose-to-lactic acid conversion and yield can be obtained

in a SSF process with R. oryzae at a relatively high temperature of

40 ◦C. Although lactic acid production in an SSF process has been

demonstrated with starchy materials and lactic acid bacteria [39],

this study is the first demonstration of SSF for high-titer and high-

yield l-lactic acid production from lignocellulosic materials by R.

oryzae. The utilization of cheap XOS waste residues for l-lactic acid

production can provide a cost-effective approach for organic acid

production as well as in waste management.

Acknowledgements

This work was supported by the International Advanced

Forestry Technology IntroductionProject Funding (Grant No. 2012-

4-18), Natural Science Foundation of Jiangsu Province (Grant No.

BK20131426), Excellent Youth Foundation of Jiangsu Province of

China (BK2012038), the Doctorate Fellowship Foundation of Nan-

jing Forestry University, Graduate Research Innovation Projects of

Jiangsu Province Ordinary University (CXZZ13 0544), and the Pri-

ority Academic Program Development of Jiangsu Higher Education

Institutions.

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