Lactic acid production from paper sludge by SSF with thermotolerant Rhizopus sp.
© The Author(s) 2016
Received: 4 January 2016
Accepted: 23 May 2016
Published: 2 June 2016
Rhizopus fungi is suitable for the production of lactic acid, which is the backbone material of polylactic acid used as green plastic from lignocellulosic biomass, since it can grow and ferment in simple medium with various carbon sources such as starch and cellulose. Although paper sludge (PS) contains a lot of cellulosic fibers and in general was incinerated for volume reduction and heat recovery, other efficient utilizations have hardly been developed. In effective production of lactic acid from PS, the research of the extraction of cellulosic fiber from raw PS to obtain effectively fermentable sugars by cellulase and the selection of lactic acid microorganism are necessary. In this study, the PS pretreatment method with NaOH and HCl and the optimization of cellulase reagent were achieved, and also a desirable thermotolerant Rhizopus was selected. Finally, the production of lactic acid from the treated PS at 40 °C by simultaneous saccharification and fermentation (SSF) with the strain and an optimized cellulase cocktail was investigated.
Rhizopus oryzae NBRC 5384 was selected for thermotolerant lactic acid production from Rhizopus library because of its heat tolerance up to 40 °C and high lactic acid production of 80 g/L. The strain can ferment to lactic acid from hexose, pentose, sugar alcohol, disaccharide and starch. The soaking of raw PS in NaOH and HCl was able to reduce effectively inorganic materials and other reagents for repulping, and the content of Al and Ca per PS dry matter was mainly decreased from 32.9 and 30.8 to 14.1 and 1.66 %, respectively. SSF of the treated PS of 50 g/L with optimized cellulase cocktail and 5384 at 40 °C resulted in lactic acid production of 9.33 g/L for 96 h.
The thermotolerant Rhizopus fungus was found based on its high performance in lactic acid production at high temperature from not only glucose, but also other various carbon sources including polysaccharides and the secretion of amylases and cellulases. The treatment of raw PS by NaOH and subsequent HCl was able to remove a large amount of inorganic materials with decrease of hydrophobicity. In SSF of the treated PS with the strain and the optimized cellulase cocktail, lactic acid was able to be produced. However, the increase of initial PS concentration in SSF led to the decrease of the yield with ethanol production, because of limited aeration due to increase of density. An appropriate oxygen supply to the strain is necessary to improve lactic acid production.
Petrochemical production has caused worries amid concern about depletion of fossil fuel. Additionally, disposal treatment of a lot of the waste plastic is also a major consideration. As one of the sustainable green plastic materials, polylactic acid synthesized from lactic acid has been used increasingly as an alternative to petrochemical plastics. It can be discarded into the soil because of its biodegradability; the matter may be decomposed by microorganisms and used as carbon sources for plants and microorganisms. Lactic acid and its derivatives are also important chemicals that have many applications in food, chemical and pharmaceutical products (Passos et al. 1994; Datta et al. 1995; Lunt 1998; Grade et al. 2002). While it is produced by chemical synthesis, this manufacturing method results in a racemic mixture of d- and l-lactic acids. As enantiopure lactic acid is required to produce polylactic acid which is stable structurally and does not contain impurities, lactic acid production by bioconversion has attracted attention because of its economical procedure compared with chemical synthesis. Moreover, the biological production method of lactic acid is valid because of the possibility of using lignocellulosic materials as feedstock.
Rhizopus fungi belonging to Zygomycota are well known microbes of enantiopure l-lactic acid from glucose and other carbon sources. The fungi are also able to ferment starch to lactic acid (Ping Huang et al. 2005; Yin et al. 1997; Zhou et al. 1999). Furthermore, several researches have been reported about the fungus, by which cellulolytic and xylanolytic enzymes were secreted (Bakir et al. 2001; Murashima et al. 2002). The saprophytic fungus has high potential for lactic acid production from various abundant cellulosic biomasses, such as agricultural (rice straw, wheat straw), forestry (wood) and industrial waste (old paper, paper sludge) (Lynd et al. 1991, 1999; Lynd et al. 2001; Wyman 2003; Gao et al. 2008; Marques et al. 2008; Hama et al. 2015). Rhizopus fungi can produce l-lactic acid from simple media with not only several monosaccharides, but also starch and lignocelluloses with lower production cost than that by other lactic acid bacteria though many kinds of microorganisms such as Lactobacillus, Lactococcus and Enterococcus (Woiciechowski et al. 1999; Thongchul and Yang 2003; Wang et al. 2010). Additionally, Rhizopus can produce three major end products of l-lactic acid, fumaric acid and ethanol, unlike homofermentative lactic acid bacteria. These products are affected by culture condition, and sufficient aeration induces efficient lactic acid (Cui et al. 1998; Skory et al. 1998).
Paper sludge (PS) is a solid residue arising from paper manufacture and the generated amount is more than 3.5 million ton per year in Japan, according to statistic data on industrial waste from paper manufacturing by the Japan Paper Association. Since it is mainly incinerated to reduce the volume and regenerate thermal energy, the use of a lot of fossil fuel for the discard treatment has been viewed with suspicion, because PS has high moisture content up to 50 % and incineration discharges a large amount of CO2. Also, the utilization of PS ash derived from the incineration is limited and is used as filling materials for cement, asphalt and rubber, or as a material for manufacturing cement (Ishimoto et al. 2000). Though a small part of PS is used as a soil conditioner and a fertilizer in agriculture, a large part of ash is disposed of in sanitary landfills (Henry 1991; Tripepi et al. 1996). Therefore, new technology of effective utilization of PS should be developed for not only the production of sustainable materials as alternative to petrochemicals, but also the reduction of paper waste. PS is a promising material as one of a lignocellulosic feedstock for lactic acid production from the content of a large amount of short cellulosic fibers already treated by delignification of paper making. While PS has already been transmuted to cellulosic fiber in the pulping process by digestion with alkali and steam, general pretreatment of lignocellulosic biomasses needs high energy to delignify and expose the cellulose fiber. However, the PS contains a large amount of paper filler materials, such as CaCO3, kaolin (Al2Si2O5(OH)4), talc (Mg3Si4O10(OH)2) and other inorganic materials, and it is possible to be an inhibitor of enzymatic reaction and metabolism of microorganism for the bioconversion of PS. The fillers and the other additives in PS envelop the cellulose fibers and obstruct the access of microbes and enzymes to the cellulose substrate (Nikolov et al. 2000). Cellulase hydrolysis is especially inhibited by CaCO3, because the pH of this reaction suspension (pH 6–8) is relatively higher than the optimum pH of cellulases. (Kang et al. 2010). Although CaCO3 is essential for pH control for lactic acid production to suppress pH decrease by the acid, removal of ash in PS leads to effective hydrolysis of PS and lactic acid production. CaCO3 can be removed by acid dissolution or CO2 treatment (Kang et al. 2011).
In this study, lactic acid production by an appropriate Rhizopus fungus from PS that removed the inorganic materials was investigated using simultaneous saccharification and fermentation (SSF) method. In SSF, the enzymatic hydrolysis step of cellulose and the fermentative step by microorganisms are typically combined and the cultivation is operated on a one-batch system (Abe and Takagi 1991; Yanez et al. 2003). Unfortunately, appropriate temperatures for the enzymes and microbes are in general very different. As optimum temperature of cellulases are generally 40–60 °C and the growth temperature of the common lactic acid bacteria is around 30 °C, SSF must be operated at the growth temperature which causes the cellulase activities to decrease. To achieve effective SSF, thermotolerant Rhizopus strain was selected. Several strains can grow and produce lactic acid up to 40 °C (Kitpreechavanich et al. 2008). Conversion of PS to lactic acid by SSF was studied with thermotolerant Bacillus; however, it was not able to secrete cellulases (Marques et al. 2008; Naresh and Zhiliang 2009). Since Rhizopus sp. is able to secrete cellulases and hemicellulases, SSF will be able to perform with addition of relatively low-dose cellulase economically. Therefore, the selection of thermotolerant Rhizopus strain, removal of CaCO3 from raw PS and lactic acid production from the treated PS by SSF with the strain and cellulase cocktail were investigated.
To evaluate lactic acid production, 55 kinds of Rhizopus sp. was obtained from NBRC (NITE Biological Resource Center, Chiba, Japan). These strains were maintained on Difco™ potato dextrose agar (PDA) slants at 4 °C.
The paper sludge (PS) used in this study was supplied from the final waste sludge discharge spout of deinked pulp manufacturing process in Chuetsu Pulp & Paper Co., Ltd. (Toyama, Japan). The moisture content of PS was around 50 % by squeezing of the filler-pressed machine. It was sealed in plastic Ziploc bags and stored at −30 °C until experimental use. PS was analyzed after oven drying at 50 °C to constant weight. The PS components of cellulose, hemicellulose, lignin, acid-soluble lignin and ash were assayed by Klason lignin method (Lopez et al. 2010).
Cellulases in this study used 15 kinds of commercial reagents as follows: Meicelase (Meiji Seika Pharma, Japan), Cellulase T “Amano” 4, Cellulase A “Amano” 3, Pectinase G “Amano”, Hemicellulase “Amano” 90(Amano Enzyme, Japan), Cellulase Y-NG, Cellulase “Onozuka” 3S, Cellulase “Onozuka” R-10 (Yakult Pharmaceutical Industry, Japan), Sumizyme AC, Sumizyme X, Sumizyme SNX (Shin-Nihon Kagakukogyo, Japan), Cellulase Trichoderma viride, Novozyme 188 (Sigma-Aldrich Co., St Louis, MO), Accellerase® (DuPont, DE, USA) and Vecelex (Godo-Shusei, Japan).
Removal of inorganic materials from raw PS by alkali and acid solution
Raw PS was suspended in 1 M NaOH solution with 200 g/L consistency and soaked overnight. After the PS fiber was separated from the alkaline liquid, it was resuspended in appropriate HCl solution of the required concentration for neutralization of both residual NaON and CaCO3 contained in PS and soaked overnight. The PS was separated from the acid solution and thoroughly washed with water. The PS treated by NaOH and HCl was oven dried at 50 °C, completely dried and then powdered finely in a mill.
Selection and determination of optimum cellulase cocktail
The treated PS of 50 g/L was hydrolyzed by each 15 kinds of commercial cellulase at the protein concentration of 1 g/L in 100 mL bottles containing 25 mL acetic acid buffer (pH 5.0). These were shaken in a rotary shaker at 120 rpm at 40 °C for 48 h and then hydrolysates were separated by filtration (Filter paper No.131, Advantec Co., Tokyo, Japan) and analyzed by HPLC. The cellulase reagents that were able to produce a lot of fermentable sugar (glucose and xylose) from the PS were selected, and their blending ratio was determined by the design of experiment method using Design Expert 8 (Stat-Ease, Inc. MN).
Cultivation of Rhizopus fungi
Fungi were precultured on PDA plate at 40 °C until they covered the overall surface. The mycelia on a plate were milled using a blender (Milser LM-Plus, Osaka chemical Co., Osaka, Japan) in 100 mL saline per plate and it was used as inoculums. The composition of the culture medium was 120 g/L glucose, 3 g/L (NH4)2SO4, 0.3 g/L KH2PO4, 0.25 g/L MgSO4·7H2O, 0.04 g/L ZnSO4·7H2O, and 50 g/L CaCO3. All reagents used were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). The medium of 25 mL was put in a 100 ml medium bottle and then sterilized at 121 °C for 15 min. After getting cold, the cap of the medium bottle was exchanged with a silicone plug (Type C: Asone Co. Tokyo, Japan). Cultivation of fungi was started by inoculation of 1 mL hypha suspension into the medium and then incubated in a rotary shaker (120 rpm) at 40 °C. After the cultivation, 5 ml of 5 M HCl was added to each bottle for dissolution of CaCO3, and the broth was separated by filtration. Residual sugar and lactic acid in the culture were analyzed by HPLC.
Enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) of the treated PS
For lactic acid production from treated PS, 50 or 100 g/L of the treated PS was used as substrate instead of glucose. Medium components and the PS in 500 mL baffle flasks were sterilized at 121 °C for 15 min. Enzymatic hydrolysis was started by the addition of filter-sterilized cellulase cocktail, which consisted of three enzyme reagents at a calculated rate and protein with a concentration of 3.5 g protein/L (containing endo-β-glucanase, endo-β-xylanase, cellobiohydrolase, β-glucosidase, β-xylosidase and FPase whose activities were 10.4, 30.4, 0.214, 2.44, 0.0429 and 0.473 U/mL, respectively), into the medium and then shaking at 130 rpm for 144 h at 40 °C. SSF was started by the addition of 8 mL of homogenized mycelium suspension and the cellulase cocktail, and then they were cultured at the same condition of hydrolysis. Hydrolysates and broths sampled from cultures were analyzed by HPLC for determination of sugars and lactic acid.
In the case of Klason lignin method, glucose was measured by Glucose C II-test Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and total sugar was measured by the phenol–sulfuric acid method. Elemental composition in the ash of PS was determined by X-ray fluorescence analysis (XRF) using wave length-dispersive X-ray spectroscopy (PW2404, Spectris Co., Ltd., Tokyo, Japan). The amount of glucose, xylose, and lactic acid from each cultivation was determined by HPLC (LC-10ADvp, Shimadzu Co., Kyoto, Japan) equipped with a refractive index detector (RID-10A, Shimadzu Co.) and ICSep WA-1 Wine Analysis column (Transgenomic, NE, USA) operated at 40 °C with 1.25 mM sulfuric acid as mobile phase at flow rate of 0.6 mL/min.
Result and discussion
Removal of inorganic materials from PS
Since raw PS is the final residue of recycled paper manufacturing, PS containing many components with the exception of cellulose and hemicelluloses fibers such as CaCO3 from loading materials, reagents and surfactants by the deinking and the repulping. These materials have the possibility of becoming inhibitors of hydrolysis of enzymes and fermentation of microorganism, especially, a large amount of CaCO3 covering the cellulose fiber might obstruct the attack of enzymes and microbes. To remove these inhibitors for the effective hydrolysis of PS and subsequent fermentation, their removal process by dissolution with alkali and acid solution was investigated.
Components ratio in raw and each treated PS
Elemental components ratio of ash in raw and treated PS
Ratio of ash
Elemental components (%)a
Selection of thermotolerant lactic acid-producing strain
Comparison of lactic acid and fumaric acid produced by three strains of Rhizopus
Lactic acid (g/L)
Fumaric acid (g/L)
Lactic acid production by R. oryzae NBRC 5384 by difference of incubation temperatures
Characterization of lactic acid production from several carbohydrates by R. oryzae NBRC 5384
Ability of R. oryzae NBRC 5384 to produce lactic acid using various carbon sources
Lactic acid (g/L)
Lactic acid production from PS treated by SSF with thermotolerant fungus
Moreover, the hydrolysis rate of the cellulosic fiber might be greatly controlled by the compounding ratio of these enzymatic activities depending on the type of substrate. Although the commercial cellulase reagent, which has various cellulase activities, was optimized to hydrolyze effectively targeting cellulosic materials, the cellulase reagent for hydrolysis of PS is not available and effective hydrolysis is difficult by only a commercial reagent. Therefore, the blending of several commercial reagents is a promising strategy to obtain fermentable sugars from treated PS. First, the hydrolysis reaction of treated PS by 15 kinds of commercial reagents was carried out for selection at each concentration of 1 g protein/L and 50 g PS/L for 24 h to compare the initial hydrolysis rates. Among all the reagents, Cellulase Onozuka 3S (O) and Accellelase (A) were produced from most fermentable sugars (total amount of glucose and xylose) from the PS. Next, the optimization of commercial cellulase reagents was performed by Design of Experiment (DOE) method with Design Expert 8.0. for effective hydrolysis of treated PS. The ratio calculated using DOE was O:A = 0.157:0.843. Additionally, 0.5 g protein/L Pectinase G (P) was used as a supplement of β-xylosidase activity for xylose production from hemicellulose in treated PS, because the activities of two selected reagents were weak. From these results, a desirable cellulase cocktail was constructed at the ratio of O:A:P = 0.47:2.53:0.50 (total concentration was 3.5 g-protein/L).
On the bioconversion from lignocellulosic biomass, which is applied not only to lactic acid but also to the other fermentation products, SSF is a more valid process than separate hydrolysis and fermentation (SHF) because the process can be carried out in one tank batch, though higher yields of fermentation products are achieved by SHF than SSF since it is possible to start high initial fermentable-sugars by the concentration of the hydrolysate (Hetényi et al. 2011). The reason why SSF progressed the production of lactic acid effectively is that it can avoid product inhibition against hydrolases by fermentable sugars, as the lactic acid fermentation microorganisms consume sugars formed by hydrolases at once. Although the SSF is commonly adopted for fermentation, it has a problem about difference of appropriate temperatures between cellulases and fermenting-microorganisms, and therefore the process is in general forced to execute at the low temperature agreed with an optimum temperature for fermenting microbes despite cellulases cannot keep maximum potential. To overcome the temperature gap, the application of thermotolerant fermenting-microorganisms for high-temperature SSF is effectual. It will be able to achieve the production of a large amount of fermentable sugars immediately by high cellulase activity and the subsequent fermentation. Enhancement of cellulases activity by increasing the temperature can lead to a reduction of cellulase usage in SSF and therefore the reduction in production cost will be accomplished. Moreover, though for low-temperature SSF the biomass has to be sterilized, since lignocellulosic biomasses includes a large amount of environmental microbes such as bacteria and fungi, the high-temperature SSF has the advantage that the contamination from biomass can be avoided easily. Therefore, there are many possibilities for the use of thermotolerant zymogenic microorganisms as biocatalyst for bioconversion of lignocellulosic feedstock to fermentation products.
With the slow depletion of fossil resource such as crude petroleum, the utilization of untapped natural resources has been received worldwide attention. The economical production of green plastic from lignocellulosic materials by bioconversion has recently been of interest in terms of carbon-neutral. Polylactic acid is one of the green plastic and lactic acid that is a raw material widely used in the field of food, chemical and pharmaceuticals, etc. In this research, paper sludge (PS) excreted as a waste product from the paper-making plant was focused on as an unused cellulosic material, because in the circumstances the treatment is in general carried out by incineration with a large amount of fossil fuel and therefore carbon dioxide has been generated. In addition, the PS ash is industrial waste and stable supply is more possible all the year round.
The production of lactic acid from PS containing a great amount of cellulose material was investigated using simultaneous saccharification and fermentation (SSF) with a new-found thermotolerant Rhizopus fungus and optimized cellulase cocktail. The screening of the fungus that can not only grow, but also ferment glucose to lactic acid at 40 °C was performed among the Rhizopus library in our laboratory, and some fungi belonging to R. oryzae and R. microsporus were able to grow at more than 40 °C and produce lactic acid. Taking the tolerance to high temperature and the ability of lactic acid production into consideration, R. oryzae NBRC 5384 was selected as the fungus for the production of lactic acid by SSF of PS. The strain was able to produce lactic acid from glucose in high yield under the aerobic condition at 40 °C. Moreover, various sugars such as hexose, pentose, disaccharide and starch were directly bioconverted to lactic acid by the fungus. These results suggested that the strain can secrete α- and β-glucosidases, α-amylase and glucoamylase.
Since in common SSF, the hydrolysis process was a rate-limiting step, and the improvement of its hydrolysis rate was the most important task for the effective production of lactic acid. To remove the unnecessary inclusion such as inorganic materials and deinked reagents used for paper manufacturing which inhibit cellulases activity and/or fermentation by thermotolerant fungus, the pretreatment of raw PS was investigated. PS treatment of soaking in NaOH and subsequently HCl was very efficient in the exposure of cellulosic fiber from PS, though its hydrophilicity was increased. Ca from CaCO3 in the loading material and Al from Al2Si2O5(OH)4 in kaolin were mainly decreased by the treatment. Removal of inhibitors against enzymatic reaction by cellulases and fermentation by the fungus were efficiently executed with recovery of the cellulosic fiber in PS.
The optimization of cellulase reagent was performed by the DOE method based on the total amount of fermentable sugar (glucose plus xylose) produced from the treated PS with 14 kinds of commercial cellulase reagents. The total amount of fermentable-sugars formed from the treated PS by using the cocktail at 40 °C was vastly superior to that at 28 °C. Moreover, lactic acid was able to be produced by SSF of the treated PS at 40 °C with a new thermotolerant fungus, Rhizopus oryzae NBRC 5483, combined with the desirable cellulase cocktail. In the culture condition with high substrate concentration, the stagnation of oxygen supply to the strain might be caused the change of metabolic pathway. In particular, limitation of oxygen supply led to not only lactic acid, but also ethanol production.
As it now stands, although the amount of lactic acid produced from the treated PS by the SSF at 40 °C was low and the bioconversion has a problem relating to oxygen supply, the SSF might be one of promising methods for the utilization of PS as an unused cellulosic waste without CO2 emission, pushing ahead with the research as quickly as possible. The SSF process from PS will be able to achieve the production of various biomaterials such as not only lactic acid, but also other organic acids or ethanol in the near future.
TM carried out all PS treatments, enzymatic reactions, cultivations and analyses. TM has been involved in drafting the manuscript. HK has been involved in revising it critically for important intellectual content. HK has given final approval of the version to be published. Both authors read and approved the final manuscript.
This research was supported by A-STEP program (Industrial needs response type) of the Japan Science and Technology Agency (JST).
This study was presented at 12th Asian Congress on Biotechnology (ACB2015). Topic: Bioenergy and Biorefinery.
The authors declare that they have no competing interests.
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