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Different Strategies to Improve Lactic Acid Productivity based on Microorganism Physiology and Optimum Operating Conditions REGIANE ALVES DE OLIVEIRA, Carlos Eduardo Vaz Rossell, Bêtania Hoss Lunelli, Pedro Otávio Marques Schichi, Joachim Venus, and Rubens Maciel Filho Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.8b01655 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 15, 2018

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Industrial & Engineering Chemistry Research

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Different Strategies to Improve Lactic Acid Productivity based on Microorganism Physiology and Optimum Operating Conditions Regiane Alves de Oliveira * a,b; Carlos E. Vaz Rossell c; Betânia H. Lunelli d; Pedro O. M. Schichi b; Joachim Venus e; Rubens Maciel Filho a a

Laboratory of Optimization, Design and Advanced Process Control, School of Chemical

Engineering, University of Campinas (UNICAMP). Av. Albert Einstein, 500, Campinas – SP. 13083-852. Brazil. b

Brazilian Bioethanol Science and Technology Laboratory (CTBE), Brazilian Center for Research in Energy and Materials (CNPEM). Rua Giuseppe Máximo Scolfaro, 10000, Campinas – SP. 13083-100. Brazil.

c

Interdisciplinary Center of Energy Planning, University of Campinas (UNICAMP), Rua Cora Coralina 330, 13083-896, Campinas – SP, Brazil.

d

Faculdade de Química, Pontifícia Universidade Católica de Campinas (PUC). Rod. D. Pedro I, km 136. Campinas – SP. 13086-900. Brazil.

e

Leibniz Institute of Agricultural Engineering and Bio-economy e.V. (ATB). Max-Eyth-Allee 100, Potsdam. 14469. Germany.

* [email protected]

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Conflict of interest: The authors declare no conflict of interest

Abstract

Despite the broad variety of substrate tested for the lactic acid production, most of them have problems, even those with promising results. The use of sugarcane molasses appears as a viable alternative, allowing the development of high-performance lactic acid production with yeast extract. In this study, different strategies are proposed and evaluated to achieve high yield, productivity and lactic acid concentrations considering the suitable association among strain physiology, osmotic stress, substrates, pH control, and fermentation operational parameters. With these strategies and using molasses as a substrate, it was possible to obtain a yield of 95%, with a productivity of 5.38 g⋅L-1⋅h-1, and a titer of 157.95 g/L of lactic acid.

Keywords: Lactic acid; Fermentation; Yeast extract; Alkali dosing; Osmotic stress; Biorefinery.

1. Introduction Lactic acid is an organic acid with an α-hydroxyl and an acid functional group

1

that

provide a special characteristic to be a building block for many chemicals. Its production by fermentation provides the product with desired properties related to each lactic acid isomer: D and L-lactic acid. Industrial applications of lactic acid are vast, ranging from commodity products to more sophisticated ones that have been gaining market share every year 2. Lactic acid has recently been emphasized in many areas, such as medical, chemical, environmental, and ecological as a building-block molecule and polymers such as poly (lactic acid) (PLA) 3,4. In addition, its use is already known for a long time in the food industry.


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Research attempts have been targeted to find new and efficient nutritional sources always associated with either new fermentation techniques or improvements in the existing ones 5. Regardless the extensive variation of supplies tested for the lactic acid production, even those presenting favorable results have problems with price, seasonality, continuous availability for large-scale production, fermentation rate, number of contaminants present, yields of lactic acid production, and by-products formation

6,7

. Further, the substrate used for fermentation

depends on the geographic location of the lactic acid industry, in reason of the availability and logistics of transportation and use 8. Sugarcane molasses is a rich nutritional source with a significant amount of mineral salts and vitamins, including sucrose, glucose and fructose 9,10. Its association to yeast extract is able to provide to lactic acid bacteria all the required compounds for a fast growth and a high lactic acid production. Sugar-based feedstocks, including Total Sugar after Inversion (TSAI) from molasses, are conventional feedstocks applied to biotechnological production processes

11

. They make molasses a very desirable

substrate for lactic acid production, since their production is already widespread in many countries and their composition is well-known. They also have relatively low inhibitor concentrations compared with other agribusiness products and allows them to be stored and used all year as feedstock. Another important issue to be considered about lactic acid bacteria is their demand for an ample range of vitamins and minerals for maintenance of cellular growth and lactic acid production, besides their nitrogen requirement to synthesize molecules for their own growth. As a consequence, the final product becomes substantially more expensive. The proportion of carbon and nitrogen sources is a fundamental aspect affecting the sugar conversion to lactic acid. The inclusion of complex nitrogen sources such as yeast extract can overcome this situation 5, since it may be imperative to achieve a fast cellular growth and lactic acid production, since lactic acid bacteria have a partial growth-associated metabolism


12

. These


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good results are believed to be a consequence of the presence of amino acids, B vitamins, soluble proteins and additional nutrients available to the microorganism. The prices of all the inputs for a biotechnological process are region dependent 13. In this sense, this study presents a possibility in which yeast extract can be an important alternative if the lactic acid production process is integrated to an ethanol industrial plant under a biorefinery concept, as shown in Figure 1. In general, this design presents economic, environmental, and social advantages over individual processes. In this case, a first-generation sugarcane biorefinery will produce sugar, ethanol, yeast (as a byproduct) and lactic acid. The bagasse produced during juice extraction is also used to provide steam and electricity to supply demand processes. The surplus electricity can be then exported to the grid. Ethanol fermentation, as it is done in Brazilian Distilleries, generates an excess of 20 kg of dry yeast per 1000 L of produced ethanol 14. This yeast surplus can be recovered and dried to “Fodder Yeast” or processed to specialized products. In this way, yeast extract can be obtained through an autolysis process done by lytic enzymes and provided by the yeast itself, with the addition of sodium chloride to a final concentration of 2%, and ethanol to attain 7% v/v, at 55ºC

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(Figure 2). This yeast extract as a concentrated solution, or a dried final product, can be produced at a comparatively low production cost and used as amino acids and growth factors for lactic acid fermentation as well as other bio-processes requiring yeast extract. This process is already cost-effective in Brazil, and the products are commercialized by Biorigin 15, Grupo São Martinho 16 and Biomin 17. Considering this scenario, our study offers a contribution to lactic acid production in developing countries, such as Brazil, India and China, which have a large availability of sugarcane to produce molasses, ethanol, yeast extract and, consequentially, lactic acid without worrying about yeast extract costs.


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Bearing all this in mind and recognizing the fact that molasses has been widely studied as an efficient substrate for lactic acid production, this study investigates the association between a Lactobacillus plantarum strain and molasses to produce lactic acid. To find a feasible process for lactic acid production in Brazil, sugarcane molasses was associated with yeast extract and different alkali solutions in order to achieve high titer, productivity and yields, and understand how each component affects the microorganism physiology. 2. Experimental 2.1. Propagation of microorganisms and inoculum preparation Lactobacillus plantarum CCT 3751 (Fundação André Tosello – Coleção de Culturas Tropical, Campinas, Brazil) was reactivated in de Man, Rogosa and Sharpe broth (MRS)

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and incubated for 24 hours at 37°C. The inoculum was prepared in a 250 mL Erlenmeyer flask containing 100 mL of MRS broth and incubated for 18 hours, at 37°C and 120 rpm. Subsequently the inoculum was centrifuged for 10 minutes at 4°C and 6,000 rpm. The cell pellet was suspended in 100 mL of sterile water before been used as inoculum. 2.2. Analytic procedures Samples withdraw were performed to evaluate lactic acid production, TSAI consumption and dry cell weight. For dry cell weight measurement, an aliquot of fermented broth was centrifuged for 10 minutes at 4°C and 6,000 rpm. The cells’ mass was placed in a weighed bowl. The bowl was dried in an incubator for 24 hours at 80°C. The bowl was cooled to room temperature and weighed again. The dry cell weight was calculated by the weight difference. Measurements of TSAI consumption and lactic acid production were performed using Highperformance liquid chromatography, as described in Table 1. 2.3. Preliminary tests for optimizing the concentration of yeast extract Preliminary tests were conducted in Erlenmeyer flasks equipped with airtight lids. The working volume was 200 mL (inoculum: 10% v/v). The medium was composed of 100 g/L of



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TSAI from sugarcane molasses, 5 g/L of sodium acetate to inhibit fungal contamination

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,

and yeast extract (tested concentrations: 0, 20, 40 and 60 g/L). In this case, the pH was adjusted up to 6 with 30 g/L of CaCO3 added before the sterilization. Fermentations were conducted in an orbital shaker at 37°C and 120 rpm for 24 hours. All the tests were carried out in duplicate. 2.4. Preparation of the bioreactor and fermentation broth Batch fermentation tests were performed using bioreactor New Brunswick Bioflo®/ Celligen® 115 with a working volume of 1 L. The bioreactor filled with the fermentation broth was sterilized in a vertical autoclave at 121°C for 30 minutes. The fermentation broth was composed of 200 g/L of TSAI from sugarcane molasses, 5 g/L of sodium acetate and yeast extract (concentration from item 2.3). The bioreactor was adjusted at 37°C, stirring at 200 rpm and oxygen removed by adding nitrogen gas for 30 minutes before inoculation. The pH was kept at 6.0 ± 0.1 through real-time monitoring and an automatic dosing of a sterile base solution. All the tests were carried out in duplicate. The first test round in the bioreactors was used to determine the most suitable alkali solution for the fermentation process: NaOH 1M; NaOH 6M; Ca(OH)2 4M. Once the Ca(OH)2 has low solubility in water, the concentration of 4M was the maximum possible concentration to keep in suspension using a magnetic stirrer. After determining the most suitable alkali solution to be used, the requirement of yeast extract was optimized. The yeast extract concentrations tested ranged from 5 to 20 g/L. In all the cases, yield (Y), productivity (P) and residual TSAI (RTSAI) were calculated to evaluate the processes, as shown in Equations 1, 2 and 3: Y(g/g) = Lactic acid produced (g) / TSAI consumed (g)

(1)

P(gL-1h-1) = Lactic acid (g/L) / Time (hour)

(2)

RTSAI (%) = (TSAI final (g) / TSAI initial (g)) x 100

(3)


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2.5. Material and energy balance A preliminary material and energy balance was developed based on the data obtained and the literature available. All the calculations were done using Microsoft Excel®. 3. Results Preliminary experiments were used to determine an ideal range of yeast extract concentration. Figure 3 shows the results of lactic acid concentration (g/L), residual TSAI (%) and productivity (gL-1h-1) after 24 hours of fermentation. The maximum standard deviation was 5% and the lactic acid yield was 0.96 g/g. The residual sugar after 24 hours of the process without yeast extract addition is approximately double, compared with 20 g/L of this compound. However, the increase of yeast extract from 20 to 60 g/L resulted in an insignificant increase in acid lactic production. This is probably a result of the excess of yeast extract when the concentrations are higher than 20 g/L. Therefore, it offers more nutrients than the microorganism can use. Consequentially, this concentration of yeast extract associated with molasses from sugarcane is enough to sustain the lactic acid production. Other tests were carried out in the bioreactor using 20 g/L of yeast extract (as previous determined) and different types of alkalis to neutralize the lactic acid produced. The first test was conducted using NaOH 1M, since this compound is widely used as a good neutralizing agent. However, the NaOH 1M injection in this process resulted in a large increase in the volume of the fermentation medium, which is quite undesirable. The injection prevented reaching the total sugar consumption. After 36 hours of the process, when the alkali injection had stopped, 49% of TSAI was still available. As a result, the pH of the fermentation dropped to 3.5, inhibiting the microorganism growth and lactic acid production. Considering the end of the log phase of Fermentation 1, the yield achieved was 0.87 g/g, with an overall productivity of 2.33 gL-1h-1. However, more than 50% of the TSAI supplied initially was not consumed,



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and the log phase was ended after 20 hours with a lactic acid concentration of 50.07 g/L, as shown in Figure 4. To reduce the volume of alkali injected, the solution NaOH 6M was used in Fermentation 2 aiming to improve the results of lactic acid production and productivity. Thus, it was possible to conduct the fermentation for 46 hours, when the maximum useful volume of the reactor was achieved. Although the process time was increased, the log phase also finished in 20 hours, as well as Fermentation 1. However, in this case, there was an increase in the yield (0.97 g/g), productivity (3.96 gL-1h-1), and lactic acid production (72.69 g/L), as shown in Figure 4. However, 65% of the TSAI supplied at the beginning remained, which is undesirable. As an alternative to alleviate this issue, NaOH was substituted by Ca(OH)2 4M, and this test was performed in triplicate. During the first 24 hours (end of the log phase), the yield was 0.95 ± 0.06 g/g. The productivity was 1.4 times higher than the productivity of Fermentation 2, reaching 5.38 ± 0.89 gL-1h-1, higher than those obtained by Wang et al. (4.40 gL-1h-1) 20 and Wang et al. (2.56 gL-1h-1)

21

using calcium alkalis. The lactic acid concentration in the

end of the log phase reached 143.95 ± 28.49 g/L, as shown in Figure 4, and the residual TSAI at the end of the log phase was 21% of the initial value, whereas it was more than 50% in the previous tests. In this case, the fermentation was carried out until the end of the TSAI consumption with only 3% of the initial concentration, while the total lactic acid production reached an average of 157.95 g/L. Bearing all this in mind, Ca(OH)2 4M was the most suitable alkali to be used in the process. The last tests were performed using Ca(OH)2 4M as alkali to achieve a final optimal yeast extract concentration. Fermentations performed in absence of yeast extract, and with concentrations above 20 g/L did not present a performance improvement, as shown in the


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preliminary tests (Figure 3). Therefore, fermentations with 5, 10 and 15 g/L of yeast extract were performed. The obtained results were compared with those from the previous case (20 g/L of yeast extract and Ca(OH)2 4M), as shown in Figure 5. The results confirmed that 20 g/L of yeast extract is the suitable requirement to obtain high lactic acid productivity and titer. In addition, the residual TSAI at the end of the log phase had reduced in comparison with the other yeast extract concentrations (Figure 5). The results obtained for the concentrations of 10 and 15 g/L yeast extract shows no great difference between them (Figure 5), but when the results are compared with those obtained with 20 g/L of yeast extract, there was an increase of 39% and 62% in lactic acid concentration and productivity, respectively. Besides, the reduction of the residual TSAI reached 62%. 4. Discussion 4.1. Experimental process Yeast extract is a key factor in the lactic acid production. According to the data obtained, the absence of yeast extract reduces lactic acid productivity and production by approximately half. Furthermore, comparing the absence of yeast extract with 20 g/L of this compound, the residual sugar after 24 hours of the process is approximately double. This is a result of Lactobacillus sp. being organisms with a poor physiological mechanism to produce their own amino acids. In addition, they also require B vitamins to grow. Both nutrients are available in yeast extract. However, higher yeast extract concentrations (higher than 20 g/L) do not increase the microorganism performance, meaning that yeast extract is important, but it is not the only factor to be considered. Previous tests had shown that the optimum pH for L. plantarum is 6.0, which is an important factor for lactic acid production and cell growth. Furthermore, the alkali solution used to keep the pH at 6.0 has an influence on results such as lactic acid concentration and productivity.



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Stopping the alkali addition in Fermentation 1 resulted in a pH drop to an inhibitory level (3.5), which prevented lactic acid production. In this case, lactic acid causes an acid stress condition in the cells. This is actually generated by the lactic acid production of the cell itself, which is why the process requires a mechanism to control the medium acidity. The process of acid stress directly affects the energy applicable for the cell production and for the catabolic flux of the microorganism

22

. Even though the system is not completely explained, acidic

environment results in a considerable boost in glycolytic enzymes, preparing the cell for a rapid growth restoration and normalization of metabolism once the pH stress is finished

22

.

The effects of acid stress to lactic acid bacteria were demonstrated using L. delbrueckii bulgaricus

23

. In this case, the authors showed that the pyruvate from sugars catabolism is

relocated to the acetyl-CoA production instead of lactate production. This change moves the acetyl-CoA to fatty acids production, modifying the cell membrane fluidity

23,24

. As a result,

the membrane becomes more resistant to exchanges with the environment, in order to save energy for the cell in the transfer of protons

23

. At the same time, ATP is used in the

maintenance of proton motive force and no more to microorganism growth and lactic acid production. L. casei Other studies

22,23,27

25

and L. rhamnosus

26

also presented the same metabolic processes.

corroborate this fact since they revealed that exposition to acid stress

leads the microorganism to prevent the protons inflow by turning the membrane more condensed and inflexible. This assumption is extensively accepted for different species of Lactobacillus. Changes in the environment are noticed by the microorganism’s translational signal system that is in charge for the cell adaptive changes of physiology, metabolism and cell behavior

28

. Numerous cellular mechanisms get signals from this system each time a

change in the environment is sensed. Thus, the system presents a membrane sensor and a cytoplasmic response regulator. Based on this theory, the authors


23

noticed changes in L.

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delbrueckii bulgaricus at protein and transcriptional levels with a fast cell response to the deleterious effects of acid stress, repairing damages and allowing the survival. To prevent acid stress to the microorganisms, NaOH has been widely used as a neutralizing agent in lactic acid production

29,30

. However, high NaOH concentrations negatively affect

lactic acid bacteria growth, and thus, the production of lactic acid (growth-associated metabolism), due to the high environmental osmotic pressure 31. Even keeping the pH at 6.0, the result did not reach the expected values in Fermentation 2, and a high amount of TSAI was not consumed. This can be strongly associated with the environmental osmotic stress caused by the addition of NaOH and the formation of sodium lactate. These compounds seem to adversely affect the microorganism that, besides growing and producing lactic acid, has to spend some energy to maintain cell stability 31. Bearing this in mind, the alternative chosen to reduce this problem was to change the NaOH by an alkali solution containing calcium. Using NaOH, one mol neutralizes one lactic acid mol, forming sodium lactate and water (Equation 4). On the other hand, one mol of calcium hydroxide neutralizes two lactic acid mol, resulting in calcium lactate and water (Equation 5). 1M glucose 2M lactic acid + 2M NaOH 2M sodium lactate + 2M H2O

(4)

1M glucose 2M lactic acid + 1M Ca(OH)2 1M calcium lactate + 2M H2O

(5)

The lower solubility of calcium lactate may lead to the higher lactic acid yield and productivity

32

, since high lactate concentrations induce calcium lactate precipitation. It

reduces the quantity of soluble lactate and alleviates the lactate inhibition. As a result, the lactate inhibition can be controlled regardless of the amount of total lactate produced. The opposite happens with the presence of sodium lactate, which is miscible in high concentrations 32. The lower lactate molarity is also positive reducing product inhibition, since the lactate molarity can affects lactic acid bacteria growth and productivity 33. For that reason, Ca(OH)2 is a more suitable neutralizing agent than NaOH concerning the obtainment of



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higher lactic acid concentrations and productivities in the same conditions

32

. Lactic acid

productivity using Ca(OH)2 was around 1.4 times higher than the productivity using NaOH, and the titer was almost the double. Similar results of production and productivity were obtained using commercial xylose and Ca(OH)2

32

, sugarcane bagasse and Ca(OH)2

34

,

bagasse sulfite pulp and CaCO3 35, and palm oil lignocellulose and Ca(OH)2 36. All the data exposed emphasize that, although both alkalis act as neutralizing agents widespread in lactic acid production, fermentation parameters such as lactic acid productivity and total production are highly influenced by the presence and concentration of yeast extract, pH and the chosen alkaline solution. This proves the need to monitor the process as a whole, as the microorganism physiology influences the economic bias of the process, maximizing gains and minimizing the optimization time. Therefore, it is proposed the production of lactic acid using yeast extract to provide amino acids to the microorganism, which has no need to recycle misfolded proteins. Thus, the ATP molecules can be driven to the microorganism grow and lead to the consequent production of lactic acid. Also, the use of alkalis containing Ca++ is proposed to prevent both acid and osmotic stress to the microorganism. 4.2. Large-scale industrial production The biorefinery concept can be applied to lactic acid production when molasses and yeast extract are available, as it has been done for a long time on sugar and ethanol industries. Furthermore, large sugarcane producers, such as Brazil, India and China

37

may become

viable alternatives to produce lactic acid on an industrial scale. One of the advantages of using molasses – a byproduct of sugar production – for lactic acid production is that its industrial plant can be easily integrated to a sugarcane mill and follow the biorefinery concept, producing sugar, ethanol, bioelectricity and lactic acid. Industrial utilities such as process steam and electricity can be provided by the cogeneration system


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already installed to supply the energy demand, which makes it self-sufficient. A preliminary material and energy balances were performed to evaluate a proposed integrated scenario, as shown in Figure 6. In the design proposed, ethanol and sugar are produced from sugarcane juice. Sugarcane molasses is used on the production of ethanol and lactic acid. Process utilities (e.g. steam and electricity) are provided by the cogeneration system. Sugarcane bagasse, which is obtained after the juice extraction, is diverted to the cogeneration to be burnt in the boilers. The data used in the material and energy balances were obtained from the literature

38–41

.

Table 2 shows a summary of the main efficiencies and process yields. The sugarcane mill capacity was defined according to the average capacity of producing plants in the Brazilian central-south region. For this study, a medium-sized plant with a milling capacity of four million metric tons of sugarcane per season (≈ 200 days of operation) was adopted. This plant has the potential to produce around 175 million liters of anhydride ethanol and 223,000 metric tons of sugar per year. As a byproduct, 20 metric tons of molasses are produced per hour. A fraction of this molasses (≈ 35%) is diverted to the lactic acid plant, which produces 12,000 metric tons of lactic acid per year. Our assumption is that 90% of TSAI is converted into the fermentation process with a theoretical yield of 100% (Equation 4). The produced lactic acid is then recovered by means of a liquid-liquid extraction process and a stripping separation, as presented by Udachan and Sahoo 39. 5. Conclusions Lactic acid using sugarcane molasses has high yield, productivity, and can reach high lactic acid

concentrations

as

long

as

the

correct

association

between

organism/substrates/alkali/parameters of fermentation is made. Sugarcane molasses is a rich nutritional source that does not require the addition of minerals and synthetic vitamins when associated with a complex nitrogen source. Its use for the production of lactic acid opens an



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opportunity for the implementation of a biorefinery that produces sugar, ethanol, yeasts, electricity and lactic acid in a single site, as it has been done in the sugar and alcohol sectors, the exception of lactic acid production. This fact is further emphasized due to the importance of the yeast extract in the lactic acid fermentation, which can be easily obtained from the biorefinery concept. Also, to attain an efficient production of lactic acid it is necessary to use a neutralizing agent for the acid produced, in an attempt to maintain a stable internal pH of the cells. In this case, the best benefit was found with the use of Ca(OH)2 largely due to factors related to osmotic pressure on the cell. Besides, it is easier to find solutions for physical and mechanical problems in the system, rather than control the adverse effects caused by alkalis with high solubility without using genetically modified organisms.

Funding: This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo – Fapesp (2013/26290-5).

Acknowledgments: The authors thank Espaço da Escrita – Pró-Reitoria de Pesquisa – UNICAMP - for the language services provided.

References (1)

Auras, R.; Lim, L. T.; Selke, S. E. M.; Tsuji, H. Poly(Lactic Acid): Synthesis, Structures, Properties, Processing, and Applications; Grossman, R. F., Nwabunma, D., Eds.; John Wiley & Sons, Inc.: New Jersey, 2010.

(2)

Tan, J.; Abdel-Rahman, M. A.; Sonomoto, K. Biorefinery-Based Lactic Acid Fermentation: Microbial Production of Pure Monomer Product. In Romanian Reports of Physics; 2017; Vol. 54, pp 27–66.


Page 15 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


15

(3)

Castro-Aguirre, E.; Iñiguez-Franco, F.; Samsudin, H.; Fang, X.; Auras, R. Poly(Lactic Acid)—Mass Production, Processing, Industrial Applications, and End of Life. Adv. Drug Deliv. Rev. 2016, 107, 333–366.

(4)

Tajitsu, Y. Poly(Lactic Acid) for Sensing Applications. In Romanian Reports of Physics; 2017; Vol. 54, pp 349–359.

(5)

Wang, Y.; Tashiro, Y.; Sonomoto, K. Fermentative Production of Lactic Acid from Renewable Materials: Recent Achievements, Prospects, and Limits. J. Biosci. Bioeng. 2015, 119 (1), 10–18.

(6)

Farooq, U.; Anjum, F. M.; Zahoor, T.; Sajjad-Ur-Rahman; Randhawa, M. A.; Ahmed, A.; Akram, K. Optimization of Lactic Acid Production from Cheap Raw Material: Sugarcane Molasses. Pakistan J. Bot. 2012, 44 (1), 333–3338.

(7)

Ghaffar, T.; Irshad, M.; Anwar, Z.; Aqil, T.; Zulifqar, Z.; Tariq, A.; Kamran, M.; Ehsan, N.; Mehmood, S. Recent Trends in Lactic Acid Biotechnology: A Brief Review on Production to Purification. J. Radiat. Res. Appl. Sci. 2014, 7 (2), 222–229.

(8)

Abdel-Rahman, M. A.; Tashiro, Y.; Sonomoto, K. Lactic Acid Production from Lignocellulose-Derived Sugars Using Lactic Acid Bacteria: Overview and Limits. J. Biotechnol. 2011, 156 (4), 286–301.

(9)

Hashizume, T.; Higa, S.; Sasaki, Y.; Yamazaki, H.; Iwamura, H.; Matsuda, H. Constituents of Cane Molasses. Agric. Biol. Chem. 1966, 30 (4), 319–329.

(10)

Olbrich H. The Molasses. Biotechnol. Kempe GmbH 2006, 131.

(11)

Buschke, N.; Schäfer, R.; Becker, J.; Wittmann, C. Metabolic Engineering of Industrial Platform Microorganisms for Biorefinery Applications – Optimization of Substrate Spectrum and Process Robustness by Rational and Evolutive Strategies. Bioresour. Technol. 2013, 135, 544–554.

(12)

Selmer-Olsen, E.; Sorhaug, T. Comparative Studies of the Growth of Lactobacillus



Page 16 of 30

16

Plantarum in Whey Supplemented with Autolysate from Brewery Yeast Biomass or Commercial Yeast Extract. Milchwissenschaft. 1998, 53 (7), 367–370. (13)

Pleissner, D.; Dietz, D.; van Duuren, J. B. J. H.; Wittmann, C.; Yang, X.; Lin, C. S. K.; Venus, J. Biotechnological Production of Organic Acids from Renewable Resources. In Advances in Biochemical Engineering/Biotechnology; Springer Berlin Heidelberg: Heidelberg, 2017.

(14)

Sgarbieri, V. C.; Alvim, I. D.; Vilela, E. S. D.; Baldini, V. L. S.; Bragagnolo, N. Pilot Plant Production of Yeast (Saccharomyces Sp.) Derivatives for Use as Ingredients in Food Formulations. Brazilian J. Food Technol. 1999, 2, 119–125.

(15)

Biorigin

-

Arte

em

Ingredientes

Naturais

http://www.biorigin.net/biorigin/index.php/br/solucoes/fermentacao-industrial (accessed Jun 11, 2018). (16)

Grupo São Martinho - Produtos e Subprodutos http://www.saomartinho.com.br/ (accessed Jun 11, 2018).

(17)

Biomin http://www.biomin.net/pt/produtos/ (accessed Jun 11, 2018).

(18)

De Man, J. C.; Rogosa, M.; Sharpe, M. E. A Medium for the Cultivation of Lactobacilli. J. Appl. Bacteriol. 1960, 23 (1), 130–135.

(19)

Cortés-Zavaleta, O.; López-Malo, A.; Hernández-Mendoza, A.; García, H. S. Antifungal Activity of Lactobacilli and Its Relationship with 3-Phenyllactic Acid Production. Int. J. Food Microbiol. 2014, 173 (7), 30–35.

(20)

Wang, L.; Zhao, B.; Li, F.; Xu, K.; Ma, C.; Tao, F.; Li, Q.; Xu, P. Highly Efficient Production of D-Lactate by Sporolactobacillus Sp. CASD with Simultaneous Enzymatic Hydrolysis of Peanut Meal. Appl. Microbiol. Biotechnol. 2011, 89 (4), 1009–1017.

(21)

Wang, Y.; Yang, Z.; Qin, P.; Tan, T. Fermentative L-(+)-Lactic Acid Production from


Page 17 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


17

Defatted Rice Bran. RSC Adv. 2014, 4 (17), 8907. (22)

Cotter, P. D.; Hill, C. Surviving the Acid Test: Responses of Gram-Positive Bacteria to Low PH. Microbiol. Mol. Biol. Rev. 2003, 67 (3), 429–453.

(23)

Zhai, Z.; Douillard, F. P.; An, H.; Wang, G.; Guo, X.; Luo, Y.; Hao, Y. Proteomic Characterization of the Acid Tolerance Response in Lactobacillus Delbrueckii Subsp. Bulgaricus CAUH1 and Functional Identification of a Novel Acid Stress-Related Transcriptional Regulator Ldb0677. Environ. Microbiol. 2014, 16 (6), 1524–1537.

(24)

Siragusa, S.; De Angelis, M.; Calasso, M.; Campanella, D.; Minervini, F.; Di Cagno, R.; Gobbetti, M. Fermentation and Proteome Profiles of Lactobacillus Plantarum Strains during Growth under Food-like Conditions. J. Proteomics 2014, 96 (0), 366– 380.

(25)

Broadbent, J. R.; Larsen, R. L.; Deibel, V.; Steele, J. L. Physiological and Transcriptional Response of Lactobacillus Casei ATCC 334 to Acid Stress. J. Bacteriol. 2010, 192 (9), 2445–2458.

(26)

Koponen, J.; Laakso, K.; Koskenniemi, K.; Kankainen, M.; Savijoki, K.; Nyman, T. a.; de Vos, W. M.; Tynkkynen, S.; Kalkkinen, N.; Varmanen, P. Effect of Acid Stress on Protein Expression and Phosphorylation in Lactobacillus Rhamnosus GG. J. Proteomics 2012, 75 (4), 1357–1374.

(27)

Zhang, Y.-M.; Rock, C. O. Membrane Lipid Homeostasis in Bacteria. Nat. Rev. Microbiol. 2008, 6 (3), 222–233.

(28)

Galperin, M. Y. Bacterial Signal Transduction Network in a Genomic Perspective. Environ. Microbiol. 2004, 6 (6), 552–567.

(29)

Meng, Y.; Xue, Y.; Yu, B.; Gao, C.; Ma, Y. Efficient Production of L-Lactic Acid with High Optical Purity by Alkaliphilic Bacillus Sp. WL-S20. Bioresour. Technol. 2012, 116 (0), 334–339.



Page 18 of 30

18

(30)

Ouyang, J.; Ma, R.; Zheng, Z.; Cai, C.; Zhang, M.; Jiang, T. Open Fermentative Production of L-Lactic Acid by Bacillus Sp. Strain NL01 Using Lignocellulosic Hydrolyzates as Low-Cost Raw Material. Bioresour. Technol. 2013, 135, 475–480.

(31)

Tian, X.; Wang, Y.; Chu, J.; Zhuang, Y.; Zhang, S. Oxygen Transfer Efficiency and Environmental Osmolarity Response to Neutralizing Agents on L-Lactic Acid Production Efficiency by Lactobacillus Paracasei. Process Biochem. 2014, 49 (12), 2049–2054.

(32)

Ye, L.; Zhou, X.; Hudari, M. S. Bin; Li, Z.; Wu, J. C. Highly Efficient Production of LLactic Acid from Xylose by Newly Isolated Bacillus Coagulans C106. Bioresour. Technol. 2013, 132 (0), 38–44.

(33)

Nakano, S.; Ugwu, C. U.; Tokiwa, Y. Efficient Production of D-(−)-Lactic Acid from Broken Rice by Lactobacillus Delbrueckii Using Ca(OH)2 as a Neutralizing Agent. Bioresour. Technol. 2012, 104 (0), 791–794.

(34)

van der Pol, E. C.; Eggink, G.; Weusthuis, R. A. Production of L(+)-Lactic Acid from Acid Pretreated Sugarcane Bagasse Using Bacillus Coagulans DSM2314 in a Simultaneous Saccharification and Fermentation Strategy. Biotechnol. Biofuels 2016, 9 (1), 248.

(35)

Zhou, J.; Ouyang, J.; Xu, Q.; Zheng, Z. Cost-Effective Simultaneous Saccharification and Fermentation of L-Lactic Acid from Bagasse Sulfite Pulp by Bacillus Coagulans CC17. Bioresour. Technol. 2016, 222, 431–438.

(36)

Juturu, V.; Wu, J. C. Production of High Concentration of L-Lactic Acid from Oil Palm Empty Fruit Bunch by Thermophilic Bacillus Coagulans JI12. Biotechnol. Appl. Biochem. 2018, 65 (2), 145–149.

(37)

Food And Agriculture Organization Of The United Nations: Statistics Division http://www.fao.org/faostat/en/#compare (accessed Aug 23, 2017).


Page 19 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


19

(38)

Dias, M. O. S.; Junqueira, T. L.; Cavalett, O.; Cunha, M. P.; Jesus, C. D. F.; Mantelatto, P. E.; Rossell, C. E. V.; Maciel Filho, R.; Bonomi, A. Cogeneration in Integrated First and Second Generation Ethanol from Sugarcane. Chem. Eng. Res. Des. 2013, 91 (8), 1411–1417.

(39)

Udachan, I. S.; Sahoo, A. K. A Study of Parameters Affecting the Solvent Extraction of Lactic Acid from Fermentation Broth. Brazilian J. Chem. Eng. 2014, 31 (3), 821–827.

(40)

Brazilian Sugarcane Industry Association http://www.unica.com.br/ (accessed Jan 6, 2018).

(41)

Brazilian Agricultural Research Corporation https://www.embrapa.br (accessed Jan 6, 2018).



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Table 1: Parameters used to analyze TSAI and lactic acid concentration on HPLC. Parameter Column Mobile phase Flow Column temperature Analysis time

Analyte TSAI Aminex ®HPX-87P 300 x 7.8 mm x 9 µm (Bio rad) Milli-Q water

Lactic acid Aminex ®HPX-87H 300 x 7.8 mm x 9 µm (Bio rad) Sulfuric acid (5mM)

0.5 mL/min

0.6 mL/min

55°C

35°C

30 min

30 min


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Table 2: Main parameters, efficiencies and process yields adopted on the material and energy balances. Parameters Sugarcane processed Days of operation Reducing sugar content in sugarcane (wet basis) Sugarcane fibers content (wet basis) Efficiency of juice extraction in the mills Fermentation efficiency Anhydrous ethanol purity 85 bar boiler efficiency 85 bar steam temperature Dried yeast per cubic meters of ethanol Efficiency of lactic acid purification unit Turbines isentropic efficiency Generator efficiency Steam pressure – process Steam pressure – molecular sieves

Value 833,333 kg/h 200 15.3% 13% 96% 90% 99.6% 87% 520°C 25 kg 85% 85% 98% 2.5 bar 6 bar


References 40 40 38 38 38 38 38 38 38 41 39 38 38 38 38


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Figure 1. Sugarcane Biorefinery: Production of sucrose, ethanol, yeast and “yeast products,” lactic acid, and electricity. Figure 2. Processing of yeast cell bled from ethanol fermentation to yeast extract. Figure 3. Results of lactic acid production, productivity, and residual TSAI in the preliminary tests using different yeast extract concentrations, after 24 hours of fermentation. Figure 4. Results of lactic acid production, productivity, and residual TSAI at the end of the log phase of the fermentations using different alkalis. Figure 5. Results of lactic acid production, productivity, and residual TSAI using different yeast extract concentrations for molasses fermentation at the end of the log phase with Ca(OH)2 as alkali. Figure 6. Integrated process diagram for sugar, ethanol, lactic acid and bioelectricity production.


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Figure 1



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Figure 2


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Figure 3



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Figure 4


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Figure 5


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Figure 6


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For Table of Contents Only


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287x258mm (96 x 96 DPI)


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Different Strategies To Improve Lactic Acid ... - ACS Publications

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