Production of Lactic Acid from Seaweed Hydrolysates Via Lactic Acid Bacteria Fermentation

: Biodegradable polylactic acid material is manufactured from lactic acid, mainly produced by microbial fermentation. The high production cost of lactic acid still remains the major limitation for its application, indicating that the cost of carbon sources for the production of lactic acid has to be minimized. In addition, a lack of source availability of food crop and lignocellulosic biomass has encouraged researchers and industries to explore new feedstocks for microbial lactic acid fermentation. Seaweeds have attracted considerable attention as a carbon source for microbial fermentation owing to their non-terrestrial origin, fast growth, and photoautotrophic nature. The proximate compositions study of red, brown, and green seaweeds indicated that Gracilaria sp. has the highest carbohydrate content. The conditions were optimized for the saccharification of the seaweeds, and the results indicated that Gracilaria sp. yielded the highest reducing sugar content. Optimal lactic acid fermentation parameters, such as cell inoculum, agitation, and temperature, were determined to be 6% ( v / v ), 0 rpm, and 30 °C, respectively. Gracilaria sp. hydrolysates fermented by lactic acid bacteria at optimal conditions yielded a final lactic acid concentration of 19.32 g/L.


Introduction
Lactic acid, a colorless and odorless monocarboxylic acid [1], has received considerable attention owing to its various applications in food and non-food industries [2]. For example, there has been an increasing demand of lactic acid in the manufacturing of biodegradable polylactic acid materials used as an alternative to petroleum-derived plastics [3]. It was estimated that the worldwide demand for lactic acid would reach 1,000,000 metric tons by the year 2020 [4].
Lactic acid can be produced by either chemical synthesis or microbial fermentation. Two optical isomers of lactic acid, L(+)-and D(−)-lactic acid, are produced during its chemical synthesis; of these, D(−)-lactic acid is considered to be the main entity involved in human acidosis [5]. As a result, a variety of microbial strains, such as the fungus Rhizopus [6][7][8], yeast Pichia stipitis [9], and lactic acid bacteria [10,11], have been used in microbial fermentation for lactic acid production. Fermentation parameters along with the medium compositions for the production of pure L(+)-lactic acid of microbial origin have been thoroughly optimized [7,[12][13][14]. Moreover, engineered microbial strains have been reported for the efficacious production of lactic acid. Liaud et al. [4] indicated that Aspergillus brasiliensis could produce more lactic acid as a result of the insertion of recombinant idhA genes obtained from R. oryzae. Metabolically engineered Saccharomyces cerevisiae and A. oryzae could produce lactic acid more efficiently if a bovine L-lactate dehydrogenase gene (LDH) was integrated into their genome [15,16]. Candida sonorensis expressing a heterologous lactate dehydrogenase from Lactobacillus helveticus could produce lactic acid by xylose fermentation [17].
Although extensive studies have been reported to efficiently improve lactic acid production by microbial fermentation, the high production cost of lactic acid still remains the major limitation for its application, indicating that the cost of carbon sources for the production of lactic acid has to be minimized for industrial application [18]. Many studies on lactic acid production by the fermentation of starchy materials, such as corn starch [19][20][21], rice starch [22], potato starch [23,24], and sweet sorghum juice [25], have been reported. However, scientists have been searching for a relatively cheap carbon source for the microbial fermentation of lactic acid. Renewable lignocellulosic biomass materials have been reported to be low-cost carbon sources and as feedstock for lactic acid fermentation, including waste sugarcane [18], corn stover [26], apple pomace [27], wheat straw [28,29], and wood waste [30]. However, producing lactic acid from lignocelluloses has limitations, given the inherent inefficiency of extracting lignin, a cell wall polymer of aromatic subunits and also a negative substance in the biochemical process during fermentation.
Marine algae are now attracting considerable attention as possible complementary/alternative lignocellulosic substrates because of several advantages. First, marine algae have a short generation cycle and can be easily cultivated in various aquatic environments. Second, they are not considered as a primary food crop and do not contain lignin, which makes saccharification more feasible. Third, they are rich in carbohydrates. Utilization of lignocellulosic biomass for biofuel or lactic acid production might incur environmental consequences and economic impacts associated with land use and water consumption [31]; clearing new land for lignocellulosic biomass production might have limitations, since greater needs for major food crops will certainly come with a growing world population. A lack of source availability of food crop and lignocellulosic biomass has encouraged researchers and industries to explore new feedstocks for microbial fermentation, such as seaweeds due to their favorable rapid growth rate and cultivation in the aquatic environment [32]. Jung et al. [33] indicated that the amount of some seaweeds is approximately four and six orders of magnitude greater than the lignocellulosic biomass, such as corn stover and switchgrass. In addition, seaweeds exhibited a higher photosynthetic efficiency than that of terrestrial biomass [34,35], leading to their high crop yields and carbon fixation. On the other side, lignocellulosic biomass is dominant in cellulose and hemicellulose, which break down into mostly glucose and xylose. However, seaweeds have versatile polysaccharides, such as fucoidan and laminaran in brown seaweeds, agar and carrageenan in red seaweeds, and ulvan and glucuronan in green seaweeds, and it is worth evaluating their potentials for lactic acid fermentation [36].
To date, marine algae have been used in the production of bio-based chemicals [37,38] and biofuels [39,40], which is an indication of their great potential as carbon sources for lactic acid fermentation. In this study, we used lactic acid bacteria for the fermentation of the marine algae Gracilaria sp., Sargassum siliquosum, and Ulva lactuca for lactic acid production.

Effect of Acid Concentration and Hydrolysis Time on the Dilute Acid Pretreatment of Gracilaria sp., S. siliquosum, and U. lactuca
Among the various pretreatments prior to enzymatic saccharification, those using dilute acids, such as sulfuric acid and hydrochloric acid, and those using alkali are considered to be effective [44,45]. As shown in Table 2, HCl concentrations of 0.2 and 0.4 N were used for the acid hydrolysis of 10% (w/v) of the marine algae Gracilaria sp., S. siliquosum, and U. lactuca at 121 °C for 20, 30, and 60 min, individually. For the HCl concentrations of 0.2 and 0.4 N, the higher the concentration of acid used, the higher the amount of reducing sugars obtained. In addition, 30 and 60 min proved to be optimal for acid hydrolysis with respect to obtaining the best reducing sugar content and yield. The former condition was adopted for the remainder of the study for dilute acid pretreatment after taking the energy cost into account. Thus, after optimum pretreatment (0.4 N HCl for 30 min), the reducing sugar contents of 23.32 ± 0.26, 12.18 ± 0.17, and 18.89 ± 0.39 g/L and yields of 36.05 ± 0.40, 19.06 ± 0.27, and 40.06 ± 0.89 g/L obtained by the dilute acid hydrolysis of the marine algae Gracilaria sp., S. siliquosum, and U. lactuca, respectively, were achieved. Dilute acid pretreatment could minimize the release of inhibitors, such as furans and organic acids, which are normally generated under a higher concentration of acid during hydrolysis as a result of sugar degradation caused by over acid-catalyzed hydrolysis. Wu et al. [39] previously reported that 10% (w/v) of the red alga P. capillacea was hydrolyzed using 2% (v/v) sulfuric acid at 121°C, and approximately 5 g/L of 5-hydroxymethylfurfural (5-HMF), 5 g/L of formic acid, and 7 g/L of levulinic acid were produced. Each value is mean ± standard deviation (n = 3); different superscript letters in the same column are significantly different (p < 0.05).

The Effect of Cellulase Hydrolysis on Marine Algae Saccharification
There have been a number of reports describing the strategy of using a combination of dilute acid/alkaline pretreatment and enzymatic hydrolysis for cellulosic biomass saccharification [43,44,46,47]. Alkaline pretreatment and enzymatic hydrolysis of wheat straw (50 g/L) can generate a reducing sugar of 13.82 g/L [43], and dilute acid hydrolysis followed by enzymatic hydrolysis for wood sawdust could generate glucose at 8.68 g/L [48]. However, few studies have reported the saccharification of marine algae. In this study, the dilute acid hydrolysate obtained from the marine algae Gracilaria sp., S. siliquosum, and U. lactuca was incubated with cellulase for further saccharification. As shown in Table 3, the reducing sugar contents of the hydrolysate of the marine algae Gracilaria sp., S. siliquosum, and U. lactuca were increased from 21.71 ± 0.84 to 31.13 ± 1.59, 12.75 ± 0.51 to 19.51 ± 1.68, and 12.89 ± 2.51 to 18.99 ± 1.59 g/L, respectively. This led to an increase in the reducing sugar yield of the marine algae Gracilaria sp., S. siliquosum, and U. lactuca from 33.56% ± 1.29% to 48.12% ± 2.45%, 19.95% ± 0.76% to 30.53% ± 2.60%, and 27.34%± 5.75% to 40.27% ± 3.63%, respectively. The hydrolysate of red seaweed Gracilaria sp. was chosen for the optimization of lactic acid fermentation in further studies. Each value is mean ± standard deviation (n = 3).

The Effects of Inoculum, Agitation, and Temperature on Lactic Acid Fermentation
It has been reported that not all of the lactic acid bacteria could ferment galactose; the dominant fermentable sugar from Gracillaria sp. hydrolysate; and some or all strains in Lactobacillus bulgaricus, Lactobacillus delbreckii, Lactobacillus lactis, and Lactobacillus brevis are unable to use galactose for lactic acid production [49]. Thus, the lactic acid bacteria species, such as L. acidophilus and L. plantarum, which can ferment galactose were selected, and the trials using single or combined strains of lactic acid bacteria for lactic acid fermentation of red seaweed hydrolysate were accomplished. As a result, our preliminary experiments showed that the combined use of L. acidophilus and L. plantarum had the best lactic acid production (data not shown). The effects of 1%, 3%, 6%, 9%, 12%, 15%, and 18% (v/v) inoculum of combined use of L. acidophilus and L. plantarum were tested based on cell growth, final pH, sugar usage, and final lactic acid concentration after a 24-h fermentation at 37 °C (Table 4). The 6% (v/v) inoculum of the combined lactic acid bacteria yielded the best lactic acid concentration of 15.02 ± 0.80 g/L; however, the difference among the combinations tested was not statistically significant. We also observed that a higher cell biomass was obtained using more inoculum of lactic acid bacteria, i.e., the highest cell biomass of 8.62 ± 0.16 log CFU/mL was obtained using an inoculum of 18% (v/v). Djukic-Vukovic et al. [50] indicated that 5% (v/v) inoculum of lactic acid bacteria exhibited the best lactic acid concentration when compared with the 2% and 10% (v/v) inoculums. Each value is mean ± standard deviation (n = 3); different superscript letters in the same column are significantly different (p < 0.05).
The effects of 0, 50, 100, and 150 rpm of lactic acid fermentation were tested based on cell growth, final pH, sugar usage, and the final lactic acid concentration after a 24-h fermentation at 37 °C (Table 5). The 0-rpm condition exhibited the best lactic acid concentration of 15.08 ± 0.25 g/L, indicating that the anaerobic environment was suitable for lactic acid fermentation. Lactobacilli are rod-shaped, gram-positive, and microaerophilic to strictly anaerobic microorganisms [51]. Each value is mean ± standard deviation (n = 3); different superscript letters in the same column are significantly different (p < 0.05).
The optimum temperature conditions for cell growth and metabolism are sometimes strain specific. The effects of temperature on lactic acid fermentation were tested based on cell growth, final pH, sugar usage, and the final lactic acid concentration after a 24-h fermentation at 26, 30, and 37 °C (Table 6). Our data indicated that 30 and 37 °C were optimum for lactic acid fermentation. Lactobacilli have been reported to grow well at 30, 34, and 37 °C but very slowly at 40 °C [52]. Each value is mean ± standard deviation (n = 3); different superscript letters in the same column are significantly different (p < 0.05).

Optimum Conditions for Lactic Acid Fermentation
Gracilaria sp. hydrolysate was added with 6% (v/v) of inoculum of lactic acid bacteria for lactic acid fermentation at 30 °C, 0 rpm for 72 h. Fermentation was monitored for the production of lactic acid, reducing sugar usage, cell count, and pH. As shown in Figure 1, lactic acid rapidly accumulated within 24 h and reached a plateau within 36 h, which could be due to the rapid decrease in pH. The lactic acid production had another increase by 15% when the pH was adjusted from 4.4 to 5.6 by adding CaCO3 at 36 h, and the highest lactic acid concentration of 19.32 g/L was attained at 72 h. The conversion yield from seaweeds to lactic acid is calculated to be 0.19 g/g. An initial concentration of 29.85 g/L of reducing sugars in the Gracilaria sp. hydrolysate was used in the lactic acid fermentation, and two-third of the reducing sugars were consumed in 72 h. The residual sugar in fermentation could be the non-fermentable sugar 3, 6-anhydrogalactose. Gracilaria sp. comprise polysaccharide chains made of repeating alternative units of fermentable simple sugar galactose and non-fermentable sugar 3, 6-anhydrogalactose [53]; however, the latter cannot be used by lactic acid bacteria. To date, lactic acid has been reported to be produced by starchy and lignocellulosic biomass sources, such as corn and sugar cane waste [18,19], but very few reports exist on the production of lactic acid by algae. Nguyen et al. [54] used L. paracasei LA104 for simultaneous saccharification and co-fermentation for lactic acid production using the enzymatic hydrolysates of the freshwater microalga Hydrodictyon recticulum, which contains a high amount of polysaccharides, mainly glucose and mannose. This is the first study that reports the lactic acid fermentation of seaweeds, indicating its potential as biomass for lactic acid production.

Source of Marine Algae and Lactic Acid Bacteria
Dried Gracilaria sp. was purchase from Yunlin country, Taiwan, and dried Sargassum siliquosum, and Ulva lactuca were purchased from a local market in Hoping Island, Keelung, Taiwan. The seaweed was ground, sieved (1 mm pore size), and stored at −20 °C before use. The proximate composition, including the moisture, crude protein, crude lipid, crude fiber, and ash of the marine algae, were analyzed according to Association of Official Analytical Chemists (AOAC) official methods of analysis [55]. The lactic acid bacteria strains Lactobacillus (Lb.) acidophilus BCRC 10695 and Lb. plantarum BCRC 12327 used in this study were purchased from Bioresources Collection and Research Center (BCRC) in Hsinchu.

Preparation of the Marine Algae Hydrolysate and Lactic Acid Fermentation
The experiments were accomplished according to a previous report [56] with some modifications. In this study, 10% (w/v) of marine algae were treated with 0.2 N and 0.4 N HCl for 20, 30, and 60 min for pretreatment. An optimum pretreatment condition was selected for dilute HCl hydrolysis. The pH of the acid hydrolysates was adjusted to pH 4.5 after pretreatment and incubated with 7.6 U/mL cellulase (Amano Enzyme, Elgin, IL, USA) at 37 °C for 48 h for enzymatic saccharification. 3% (v/v) inoculum of BCRC 10695 and 12327 was inoculated into the hydrolysate of Gracilaria sp. for lactic acid fermentation at 30 °C for 24 h [57].

Reducing Sugars Quantification Using DNS (3,5-Dinitrosalicylic Acid) Assay
The experiments were accomplished according to Miller [58], and the standard curve was accomplished by using simple sugar galactose (Formedium, Hungstanton, Norfolk, UK). In brief, the sample was centrifuged at 10,000× g at room temperature for 5 min. The supernatant was collected and mixed with an equal volume of DNS (3,5-dinitrosalicylic acid) reagent (Sigma-Aldrich, St. Louis , MO, USA). The mixture was heated at 100 °C for 10 min, and cooled prior to being mixed with an equal volume of deionized water. The mixture was measured at an absorbance at 546 nm, and the reducing sugar contents were calculated. The seaweed hydrolysate analyzed for reducing sugar contents, and the reducing sugar yields were calculated according to a previous report [59] as follows: Reducing sugar yield = reducing sugar content/weight of dried algae powder/ratio of carbohydrate × 100%.

Analysis of Lactic Acid Using HPLC
The lactic acid (g) was analyzed using an Asahipak GS-320 HQ (7.5 × 300 mm) (Showa Denko America, Inc., New York, NY, USA), along with a refractive index detector with a filtered distilled water eluent at a flow rate of 0.4 mL/min at 40 °C. All the tested samples were filtered through a 0.22-μm membrane filter prior to the HPLC analysis [60].

Effect of Inoculum, Agitation, and Temperature on Lactic Acid Fermentation
For optimizations of cell inoculum, 1%, 3%, 6%, 9%, 12%, 15%, and 18% (v/v) of inoculum at the density of 1 × 10 9 CFU/mL of lactic acid bacteria (BCRC 10695 and 12327, mixing ratio = 1: 1) was inoculated into Gracilaria sp. hydrolysate containing 0.5% (w/v) yeast extract (Formedium, Hungstanton, Norfolk, UK), and the fermentation mixture was incubated at 37 °C without shaking for 72 h. For optimizations of the agitation rate, 6% (v/v) of inoculum of lactic acid bacteria (BCRC 10695 and 12327) was inoculated into Gracilaria sp. hydrolysate containing 0.5% (w/v) yeast extract, and the fermentation mixture was incubated at 37 °C with a shaking rate of 0, 100, 150, or 200 rpm for 72 h. For optimizations of the fermentation temperature, 6% (v/v) of inoculum of lactic acid bacteria (BCRC 10695 and 12327) was inoculated into Gracilaria sp. hydrolysate containing 0.5% (w/v) yeast extract, and the fermentation mixture was incubated at 25, 30, and 37 °C for 72 h. The samples were collected at the end of fermentation for analyses of the cells counts, reducing sugar contents, pH, and lactic acid concentration [61].

Statistical Analysis
Data were analyzed statistically using SPSS Version 12.0 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to determine statistical differences between sample means, with the level of significance set at p < 0.05. Multiple comparisons of means were done by Duncan's test. All data are expressed as mean ± SD.

Conclusions
In this study, we evaluated red, brown, and green seaweeds for lactic acid fermentation. The optimum acid/enzyme hydrolysis condition for the saccharification of the seaweeds was determined, and the lactic acid fermentation parameters of the red seaweed hydrolysate were optimized. Our study identifies a practical approach to use seaweeds as a carbon source for lactic acid fermentation.