Date Published: September 28, 2012
Author(s): Cirilo Nolasco-Hipolito, Octavio Carvajal Zarrabal, Rubena Malfia Kamaldin, Ling Teck-Yee, Samuel Lihan, Kopli Bin Bujang, Youji Nitta.
Enterococcus faecium No. 78 (PNCM-BIOTECH 10375) isolated from puto, a type of fermented rice in the Philippines was used to produce lactic acid in repeated batch fermentation mode. Enzymatically liquefied sago starch was used as the sole carbon source, since sago (Metroxylon spp) is a sustainable crop for industrial exploitation. Liquefied sago starch was inoculated with E. faecium to perform the saccharification and fermentation processes simultaneously. Results demonstrated that E. faecium was reused for 11 fermentation cycles with an average lactic acid yield of 36.3 ± 4.71 g/l. The lactic acid production was superior to that of simple batch mode and continuous fermentation in terms of lactic acid concentration. An un-dissociated lactic acid concentration of 1.15 mM affected the productivity of the cells. Work is in progress to maintain and increase the usability of the cells over higher fermentation cycles.
Lactic acid is an important commodity because it is a multi-functional versatile organic acid having a wide range of applications. One of the most important factors that affect the overall production cost in Lactic Acid Fermentation (LAF) is the raw material. The consortium CSM (
) reported that the raw material costs for lactic acid (LA) production, as a percentage of sales, increased from 52.9% in 2009 to 53.2% in 2010. For a long time, it has been stated that lignocellulosic materials are very promising for bio-refinery applications, but this technology is still problematic (Zhou et al.
). Recently Ou et al. (
), reported a process using Bacillus coagullans to produce LA from non food carbohydrates, and interestingly, an indigenous Clostridium phytofermentans was found as a potential microorganism for the efficient use of lignocellulosic materials to produce ethanol, hydrogen and organic acids (
[Leschine and Warnick 2010]). Among starchy materials, sago starch is being considered as an attractive raw material for food and industrial exploitation due to the fact that it is produced abundantly in the agricultural plant Metroxylon spp (Karim et al.
). In 2008, Malaysia exported 37,365.3 metric tons of sago flour, thereby earning RM44, 091.0 million (Malaysia Dept
[Statistics 2011]). The sago palm efficiently fixes carbon dioxide to synthesize starch in large quantities in its trunk. Sago starch granules are generally bigger than those of rice, (3–10 μm), corn (5–20 μm), wheat (22–36 μm), or cassava (5–25 μm), but smaller than those of potato (15–85 μm) (Nor-
[Nadiha 2010]). Sago starch contains approximately 74-80% of amylopectine and 24-31% of amylose (Karim et al.
) and has a crystalline structure (Yetti et al.
). These properties of sago starch make difficult its hydrolysis. Uthumporn et al. (
) reported that the relative order in the hydrolysis of the starchy materials studied was as follows: corn starch > mung bean starch > cassava starch > sago starch. These findings demonstrated that sago is a difficult substrate for raw starch degrading enzymes (Yetti et al.
). On the other hand, an improvement in the industrial production and efficiency of enzymes has decreased their cost in the market (Novozymes and BBI
[International 2005]). Nevertheless, to improve the economics of LAF, the use of microorganisms with amylolytic activity could be preferred because it saves in terms of enzymes and energy in the liquefaction/saccharification process. Some strains of fungi and bacteria capable of producing LA directly from starchy materials by using different strategies have been reported in the literature (Lu et al.
[Petrova and Petrov 2012]; Shibata et al.
; Xiao et al.
). For instance, the amylolytic bacterium L. amylovorus NRRL B4542 is reported to be capable of fully converting liquefied corn starch to LA, with a productivity of 25 g/lh in continuous culture through the use of a yeast extract concentration as high as 30 g/l as nitrogen source (
[Zhang and Cheryan 1994]). On the other hand, Shibata et al. (
) reported the use of Enteroccus faecium 78 as a promising microorganism to produce L-(+)-LA directly from raw sago starch (RSS) in continuous culture by using a hollow fibre cartridge to recycle the cells. It was reported that E. faecium 78 performed well at 30°C and pH 6.5 with a productivity of 3.04 g/lh at a LA concentration as low as 16.6 g/l (Shibata et al.
). In their research the fermentation mode was of capital importance to enhance the productivity of the system. In this regard the repeated batch fermentation (RBF) process combines the advantage of batch and fed-batch fermentation processes mainly making possible to conduct the process by long periods and improving the productivity compared to batch process (Treichel et al.
). RBF reduces the cost of fermentation process and enhance the productivity through the use of high cell density (Yamakawa et al.
). Moreover, from an industrial point of view by using the RBF mode the production period can be shortened, compared to standard fed-batch or batch processes resulting in a significant increase of the final product yield (Russ et al.
). Therefore, in this study we used the RBF for LA production with the strain E. faecium 78 (PNCM-BIOTECH 10375) in liquefied sago starch (LSS) as the only carbon source. In general, the main objective was to improve the productivity of the system, which includes the use of LSS, recycling of the yeast to speed-up the fermentation process.
Data from two replicate experiments and three repetitions of the analysis for each sample were expressed as the mean ± standard deviation (± SD). All analyses were conducted with PASW 18.0 software.
It was thought that glucose and hydrolysed sago starch (HSS) could promote the growth of the cells better than raw sago starch (RSS) and gelatinized sago starch (GSS). However, the trends obtained in all cases were similar, although it was observed that in the log phase the uptake of the GSS and RSS forms was slightly faster than that of the glucose and HSS. Similar observation was reported by Shibata et al. (
). They found that the performance of E. faecium using RSS was better than with corn, potato and wheat starches. It could be desirable to have RSS as the most appropriate form of starch to be used as direct substrate for the fermentation process; however, it is problematic to manage the RSS slurry at high concentration. Although Shibata et al. (
) did not reported how they prepared the raw sago starch, and if they sterilized the sago slurry, then; the form of starch used perhaps was the gelatinised sago starch. The gelatinised sago starch can be used as substrate but a very low concentration because when the concentration of starch is increased the viscosity of the media is very high. The viscosity of the slurry increases proportionally with the concentration of the starch. In general, when the starch concentration increased, sedimentation occurred due to saturation or insolubility. This situation was visualized by
[Zhang and Cheryan (1994]); therefore, these researchers used liquefied starch to avoid these problems and they improved the process by using the amylolytic Lactobacillus amylovorus strain. Shibata et al. used E. faecium No. 78 cultivated in RSS at a concentration no higher than 20 g/l, which is very low for an industrial application. Therefore, it could be reasoned that using batch and its extension RBF might be advantageous to produce higher LA concentration at a similar production rate.
In conclusion, Enterococcus faecium No. 78 (PNCM-BIOTECH 10375) isolated from puto, a type of fermented rice in the Philippines, was able to produce LA in RBF mode. The cells retained their metabolic activity during 566 h of fermentation. However, the growth of this strain was affected strongly by non-dissociated LA at a concentration of around 1.15 mM, by lowering the productivity of the system.
The authors of this manuscript declare that do not have any financial or non-financial competing interest in the submission of this experimental results in the journal AMB EXPRESS.
The authors declare that we belong to a group of researcher with common interest on research. The next are our individual contribution for the manuscript ID 2135787809763144 titled Lactic acid production by Enteroccocus faecium in liquified sago starch. CNH: The performed fermentations in repeated batch fermentation mode and drafted the manuscript. OCZ: The preparation of the manuscript and worked in the hydrolysis of the sago starch as well drafted the manuscript. RMK: The fermentations testing all the forms of sago starch and did the analysis needed during the research. LTY: The statistical analysis with its corresponding interpretation of the results. KB: Contributed with the preparation of the manuscript. SL: Contribute with the redaction of manuscript. YN: The scanning electron microscopy pictures for the starch granules. All authors read and approved the final manuscript.