Journal of Ecological Engineering Volume 18, Issue 4, July 2017, pages 35–41 DOI: 10.12911/22998993/74308 Research Article BIOCONVERSION OF CITROFORTUNELLA MICROCARPA FRUIT WASTE INTO LACTIC ACID BY LACTOBACILLUS PLANTARUM Cesar V. Ortinero1,2, Alissandra Pauline B. Mariano1, Sofronio P. Kalaw3, Rosalie R. Rafael2 1 Department of Environmental Science, Central Luzon State University, Science City of Muñoz 3120, Nueva Ecija, Philippines, e-mail: [email protected] 2 Department of Chemistry, Central Luzon State University, Science City of Muñoz 3120, Nueva Ecija, Philippines 3 Department of Biological Sciences, Central Luzon State University, Science City of Muñoz 3120, Nueva Ecija, Philippines Received: 2017.04.25 ABSTRACT Accepted: 2017.06.01 The processing of Citrofortunella mircocarpa fruit juice generates large volume of solid Published: 2017.07.01 waste, causing disposal problem. Several studies have demonstrated that wastes from agricultural and food processing industries such as fruit and vegetable peels contain high amount of polysaccharides that can be transformed into useful chemicals, includ- ing lactic acid, through fermentation. Lactic acid is widely used in various industries, such as in the manufacture of biodegradable plastic, and the demand for this chemical justifies the search of renewable feedstock for its biotechnological production. This study aimed to produce lactic acid from C. microcarpa fruit waste biomass through fermentation with Lactobacillus plantarum. The hydrolysate from C. microcarpa fruit waste was prepared, inoculated with different amounts of L. plantarum cell suspen- sion, and incubated for three days. Lactic acid production was monitored daily. The lactic acid produced from the fermentation was recovered as calcium lactate and lactic acid crystals. The identity of the crystals was evaluated using Fourier transform infrared spectroscopy (FTIR) spectroscopy and paper chromatography. The highest lactic acid production was observed in fermentation mixtures containing the highest number of L. plantarum cells. Within three days of fermentation, the amount of lactic acid production increased with increasing period of incubation. Partial characterization of the crystals recovered from the fermentation mixtures by FTIR spectroscopy showed that the peaks in the spectrum were consistent with the chemical structure of lactate. Paper chromatog- raphy results likewise confirmed that the crystals are lactate. C. microcarpa fruit waste can afford lactic acid when fermented with L. plantarum. The results of the study may serve as basis for the development of technology for the utilization of C. microcarpa fruit waste biomass as renewable resource for industrial production of lactic acid. Keywords: Lactobacillus plantarum, Citrofortunella microcarpa, lactic acid; waste valorization; bioconversion INTRODUCTION packed juice drink products, and incorporated in various consumer products including flavored Citrofortunella microcarpa is among the most soy sauce, detergents and dishwashing liquid. widely cultivated fruit crop in the Philippines and Data from the Philippine Statistic Authority show disposal of wastes from its processing may pose that in the last quarter of 2016, the country’s pro- a serious environmental problem. The juice from duction of C. microcarpa fruits reached an esti- the C. microcarpa fruit is extracted and used as mated 28.13 tons (https://psa.gov.ph/fruits-crops- a common flavoring agent in Philippine cuisine. bulletin/calamansi). Thus, it can be assumed that It is also processed industrially into bottled or tons of wastes consisting of fruit peels and seeds 35 Journal of Ecological Engineering Vol. 18(4), 2017 are also produced from the industrial processing bulgaricus, and Bifidobacterium bifidum (Chick, of C. microcarpa fruit juice. Shin, & Ustunol, 2001) and Lactobacillus planta- Waste valorization, or the conversion of rum (Sreenath, Moldes, Koegel, & Straub, 2001). wastes into useful products (Arancon, Lin, Chan, It has previously been demonstrated that the Kwan, & Luque, 2013), is an alternative manage- fruit peel of C. microcarpa has volatile oil and the ment strategy to deal with the disposal challenges residue left after the extraction of oil can be used posed by wastes from food and agricultural in- in the production of pectin (Anzaldo & Briones, dustries. Because these wastes contain large 1993). The fruit peel of C. microcarpa contains amount of sugars that can be easily assimilated 16.12 g total sugar per 100 g (Samonte & Trinidad, by microorganisms, they can be transformed into 2013) and is therefore a potential substrate for the industrially important chemicals (Couto, 2008). microbial production of lactic acid. However, to For example, citric acid has been produced the best of our knowledge the production of lactic through the fermentation of banana (Karthikey- acid from the waste from the processing of C. mi- an & Sivakumar, 2010) and orange peel (Rivas, crocarpa fruit has never been attempted. There- Torrado, Torre, Converti, & Dominguez, 2008), fore, this study was conducted to investigate the both through the action of Aspergillus niger while potential of C. microcarpa fruit waste as substrate butanol has been obtained from the fermentation for the biotechnological production of lactic acid. of apple and pear peels by Clostridium aceto- butylcum (Raganati, Procentese, Oliveri, Russo, & Marzocchella, 2016). METHODOLOGY Among the industrial chemical that may be obtained from fruit and vegetable wastes is lac- The waste materials produced after the ex- tic acid. Lactic acid has numerous applications in pression of the juice from Citrofortunella micro- the food and cosmetics industries as well as in the carpa fruits were collected from food vendors manufacture of biodegradable plastics (Datta & inside the Central Luzon State University cam- Henry, 2006) and as a precursor of other chemi- pus in Nueva Ecija, Philippines. The seeds were cals and biocompatible materials with medical removed before the rest of the waste (rind, pulp applications (Martinez, et al., 2013). A common and juice vesicles) were cleaned of unwanted route for the production of lactic acid is through materials by washing with distilled water. The the hydrolysis of lactonitrile, which is obtained cleaned waste was used in the production of hy- from the reaction between acetaldehyde and hy- drolysate through steam explosion by modifying drogen cyanide (Huang, Jin, Lant, & Zhou, 2003; a previously described method (Pumiput, Chunt- Martinez, et al., 2013). Lactic acid may also be ranuluck, Kitpreechavanich, Punsuvon, & Vaith- produced from fermentation of various materi- anomsat, 2008). Forty grams of C. microcarpa als consisting of monosaccharides and disaccha- fruit waste was steam-exploded in an autoclave rides, starchy materials, and lignocellulosic hy- at 121oC for 20 min. Enough sterilized water was drolyzates by microorganisms such as lactic acid added to the steam-exploded waste to a volume bacteria (Martinez, et al., 2013). Previous studies of 1 L, and then the mixture was heated for 20 have shown that lactic acid can be produced from min at 80oC. The solid materials were separated mango peel waste by a consortium of indigenous from the supernatant with a cheese cloth. Acid microorganisms (Jawad, Alkarkhi, Jason, Easa, & hydrolysis was carried out by adding HCl (RCI Nirulaini, 2013) and from potato wastewater with Labscan, Thailand) to the supernatant to a final the aid of Rhizopus arrhizus (Huang, Jin, Lant, & concentration of 1% v/v and then autoclaving the Zhou, 2003). Various microorganisms have also mixture at 121oC for 30 min (Umesh & Preethi, been shown to be effective in converting different 2014). The pH of the hydrolysate was adjusted raw materials into lactic acid. These include or- to 6–6.8 using CaO (Sigma-Aldrich, USA) and ganisms of the following genera Carnobacterium, the CaSO4 preciptate was separated from the hy- Enterococcus, Lactobacillus, Lactococcus, Leu- drolysate through filtration (Mudaliyar, Sharma, conostoc, Oenococcus, Pediococcus, Streptococ- & Kulkarni, 2012). cus, Tetragenococcus, Vagococcus, Clostridium Lactobacillus plantarum (BIOTECH 1223) and Weissella (Huang, Jin, Lant, & Zhou, 2003). was obtained from the National Institute of Mo- Lactic acid has been produced by the following lecular Biology and Biotechnology, University species: Streptococcus thermophilus, Lactobacil- of the Philippines at Los Baños College, Laguna, lus acidophilus, Lactobacillus delbrukeii subsp Philippines. The bacterium was first grown on 36 Journal of Ecological Engineering Vol. 18(4), 2017 solid de Man Rogosa (MRS) medium (Scharlau, Statistical analysis of the data was performed Spain) before the preparation of innoculum ac- using Prism 7 (GraphPad Software, La Jolla, USA). cording to a previously described method (Umesh & Preethi, 2014) where L. plantarum was cultivat- ed in MRS broth prepared with the hydrolysate. RESULTS AND DISCUSSION After incubation for three days in a microaero- philic environment, the cell density was adjusted All fermentation mixtures, except the nega- to 1 x107 cfu/mL based on McFarland standard. tive control, produced lactic acid (Figure 1). The Fermentation mixtures were prepared by add- results suggest that C. microcarpa fruit waste can ing the L. plantarum cell suspension to the hy- serve as substrate for lactic acid production by L. drolysate to produce mixtures with final concen- plantarum. It was observed that the production trations of 3, 5