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3605

Citric Acid Fermentation From Spoiled Fruits And

Vegetables

Purackal Nima Mathew, Sourav Kumar Das, Gopal Tiwari, Aswini Kumar Das, Dr. Suneetha V.

Abstract: India is the largest producers of fruits and vegetables, and most are being thrown away because of spoilage, due to lack of adequate cold storage facilities or refrigerated transport. As fruit and vegetables contains lots of water, as a result they are prone to microbes which could easily grow and multiple hence resulting to spoilage. This work provides a review about the production of citric acid which is produced in tonnage and is extensively used in food & Pharmaceutical industry. In this study bacterial and fungal species are isolated from the spoiled part of the fruits and vegetables by various methods to have a pure culture of microbes and undergone submerged fermentation with a suitable fermentation media for production of critic acid by optimizing various factors like carbon source, temperature, pH, incubation time to recover maximum production. Then the final product is recovered and a comparative analysis was done between the amount of production of citric acid by bacterial species and fungal species.

Keywords: Spoiled fruits and vegetables, citric acid, bacteria and fungus, submerged fermentation, fruit extract, optimization, filtration, precipitation ——————————  ——————————

1 INTRODUCTION AND REVIEW:

Citrus extract (CA), a middle of the road of the tricarboxylic acid cycle, is found in an assortment of acidic organic product juices, especially in the citrus ones, in spite of the fact that its extraction from normal sources, basically lemon, was bit by bit supplanted by natural techniques, primarily dependent on the utilization of micro fungi, which are as of now the most generally utilized. It is generally utilized in the nourishment business on account of its high solvency, incredibly low poisonous quality, and acceptability; besides, models are given of some ongoing CA applications in the business of cleansers and beautifying agents, or as the dynamic fixing in some washroom and kitchen cleaning arrangements. Citrus extract is predominantly utilized in nourishment industry due to its charming corrosive taste and its high solvency in water. It is worldwide acknowledged as "GRAS" (for the most part perceived as protected), affirmed by the Joint FAO/WHO Expert Committee on Food Additives. The pharmaceutical and corrective ventures hold 10% of its use and the rest of utilized for different purposes [1]. The world’s demand for citric acid is more than 1.4 million tons annually. Presently, most of the citric acid employed in food industries comes from the fermentations processes especially especially with Aspergillus niger [2]. Although chemical synthesis of this organic acid is feasible, so far there is no alternative synthetic method developed which is superior to biological fermentation [3]. According to many journals and review papers,many microorganisms are evaluated for the citric acid production including Bacillus licheniformis, Bacillus subtilis and Cornynebacterium spp., Aspergillus niger, A. foetidus, A. awamori, Trichoderma viride, Penicillium restrictum, Mucor pyriformis and yeast like Candida lipolytica and also Saccharomyces cerevisiae.

Regardless of whether parasitic strains are great at aging essentially citrus extract which is being utilized since numerous years. But on the other hand it's to be viewed as that organism can undoubtedly taint people which can even prompts passing. Aspergillus molds exist pervasively as spores that are breathed in huge numbers day by day. While most are expelled by anatomical hindrances, infection may happen in specific conditions. Contingent upon the basic condition of the human insusceptible framework, clinical outcomes can result running from an inordinate safe reaction during unfavourably susceptible bronchopulmonary aspergillosis to the development of an aspergilloma in the immunocompetent state. The severest contaminations happen in the individuals who are immunocompromised where obtrusive pneumonic aspergillosis outcomes in high death rates [4]. Isolation of gram negative Bacillus from spoiled fruit and vegetables will be a suitable alternative for extraction of citric acid by fermenting these isolated bacterial strain on a suitable fermentation media made up extract from peels of fruits like pineapple, apple and orange along with some chemicals. The major mechanism of production of citric acid from aerobic bacterial species is the blocking of their TCA, cycle, during the process of fermentation by the above mentioned fermentation media. Bacteria are the major and important factor for fruit spoilage. They will survive in suitable temperature with the presence of food and water, which caused changes in the appearance, colour and smell of the fruits [5] .Fruits, provides the ideal environment for the survival and growth of many types of microorganisms especially bacteria. The internal fruits tissues consists high concentration of various types of sugars, minerals, vitamins and amino acids [6]. Commercial production of citric acid is generally by submerged fermentation of sucrose or molasses using the filamentous fungus A. niger or synthetically from acetone or glycerol [7]. Submerged fermentation was used in this study as it’s less expensive and can be easily accomplished[8].Many works had been done with apple, orange and pineapple which proved these best for carbon and nitrogen sources for bacterial strains: Orange (Citrus sinensis) peel was employed in this work as raw material for the production of citric acid (CA) (193 mg CA/g dry orange peel), corresponding to yields of product on total initial and consumed sugars (glucose, fructose and sucrose) of 376 and 383 mg CA/g, respectively [9]. Apple

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Purackal Nima Mathew, Sourav Kumar Das, Gopal Tiwari, Aswini Kumar Das, Dr. Suneetha V.*

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pomace proved to be the promising substrate for the hyper production of citric acid [10].Pineapple peel is a by-product resulting from the processing of pineapple into slices and represents about 10% w/w of the weight of the original fruit. The highest citric acid content achieved on pineapple waste was 16.1 g per 100 g dried pineapple waste , pineapple peels are 30% weight of the of the entire pineapple [11]. Peels of these fruits fruit should be used as fermentation media, suitable for citric acid production, since it contains carbon, nitrogen source which is essential for consumption of bacterial species and to carry on their metabolic cycle. Hence, our aim is to study and compare the production of citric acid by both fungal and bacterial species isolated from same spoiled fruits and give a brief idea about the, what are the parameters to be taken care to use bacterial species for citric acid fermentation rather than fungal species. Our study is also to produce large scale production of citric acid by using bacterial species from spoiled fruits and fruit wastes, without causing any dangerous health issues by fungus

2

MATERIALS AND METHODS

1. Sample collection

Spoiled fruits like apple, pomegranate, spoiled vegetables like tomato, potato, onion; were collected from the local market of Vellore, Tamil Nadu. The spoiled fruits were then brought to lab and processing of samples was done.

2. Isolation and screening of bacteria and fungus Serial dilution method was carried out to isolate bacteria from spoiled fruits. The spoiled fruits were crushed into pre -sterile mortar and pestle with distilled water to form suspension, which was serially diluted from 10-1 to10-5 dilutions. A 100 μL of each of diluted fruit suspension was spreader over specifically labelled nutrient agar medium for bacterial growth and for fungal growth the dilutions was spreader over specifically labelled potato dextrose agar medium. The nutrient agar medium plates where then kept for incubation at 37oC for 24 hour and fungal plates where kept for incubation at 27oC for 2 -3 days. Morphological analysis was observed conducted to determine based on different bacterial colonies and fungal growth and morphological variation of bacteria growth and fungal growth were noted. Consequently, bacterial isolates were sub-cultured, maintained and on nutrient agar medium for further use and fungal cultures on potato dextrose agar medium. All the media used for experiment are from HiMedia Mumbai.

3. Pure culture and biochemical study of bacteria The bacterial isolates were identified based on their colony characteristics, morphological and biochemical characteristics. Gram staining was conducted to study the cellular morphology of isolated bacteria. The bacterial species were further identified on the basis of biochemical characteristics via the determination of biochemical tests like catalase, triple sugar iron agar (TSIA), maltose and by using HiMedia KB002 HiAssorted TM Biochemical test kit (for Gram Negative Rods).

4. Fermentative production

The fermentation was carried out using submerged fermentation method. The fermentation media for production of citric acid was prepared with different constituents with mentioned amounts (Table1).An another fermentation media was prepared using the same constituents and but an additional constituent Fruit peel Extract was added to the media.( Fruit peels of orange and pineapple were dried for two days, finely powdered and filtered using distilled water). Two sets of each fermentation media was prepared and a standard for both the fermentation media was prepared. The pH of the medium was maintained at 7.0 using 0.1N HCl or 0.1N NaOH. The media was then incubated at room temperature in an orbital shaker for 5 days.

Table 1: Composition of the media

Serial

No. Components Amount

1 Sucrose 70gms

2 Ammonium Sulfate 1.15 gms

3 Magnesium Sulfate 0115 gms

4 Potassium Hydrogen

phosphate 0.5 gms

5 Fruit Extract 1 ml

6 Distilled water 500 ml

*gms= weight in grams and ml= volume in millitres

1) Optimisation:

Optimisation of different parameters were done like carbon source, pH, Temperature and incubation time period(1-5 days) to yield maximum production of citric acid, which is needed to be maintained during fermentation process.

Parameters for optimization.

Carbon and Nitrogen Source

Carbon source and nitrogen source plays a vital role in citric acid production and the production cost mainly depends upon the use of the carbon source. In this experiment we have used sucrose as the carbon source for efficient production of citric acid. In addition to sucrose a carbon source and ammonium sulphate a nitrogen source we also used orange and pineapple peel extract in the media. The orange peel consists of 1.15% nitrogen content and 35.89% of carbon content in it. The pineapple peel consists of 1.2 to 2.2% nitrogen and 35 to 40% of carbon.

Effect of pH

In the present study, the effect of varying pH (3.0 to7.0) on citric acid production was studied. Culture pH has a profound effect on citric acid production since certain enzymes within TCA cycle are pHsensitive. Therefore, maintenance of an appropriate pH for fermentation is vital for a good yield. A maximum citric acid yield was obtained when the initial pH of fermentation medium (optimized) was maintained at 5.0 and maximum citric acid yield was obtained when the pH of the fermentation medium was maintained at 3.0 for fungus and for bacteria the optimum initial pH was maintained at 7 and maximum yield was obtained at pH range of 4-5.

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3607 The optimum incubation time for the maximum citric acid

production varies both with the organism and fermentation conditions. In order to find the optimum incubation time bacterial and fungal fermentation media were kept at different incubation periods (for 1-7 days).

Effect of temperature

The incubation temperature of fermentation medium has a profound effect on citric acid production. It is one of the critical factors that have direct effect citric acid production. Temperature affects the nutrient requirements, the biomass composition and the nature of the metabolism in cultures. The effects of temperature on growth rate over most of its range can be predicted in terms of the activation energy for growth. This relation breaks down near to both the upper and lower temperature limits for growth. Above the optimum temperature, cell degradation probably becomes dominant over the growth process. Near the lower temperature limit the regulation of metabolism may fail. The maximum citric acid production was however obtained when temperature of medium was maintained between 25 to 30OC for fungal fermentation and 28OC to 37oC for bacterial fermentation. When the temperature of the fermentation medium was increased above the range, citric acid production was decreased gradually. It might be due to the fact that higher temperatures cause denaturation of enzymes. In a lower incubation temperature, citric acid production could be affected by a retarded germination of fungi and ceasing of metabolic activities of bacteria and low cell viability.

Accumulation and recovery of Citric acid.

The first step of citric acid recovery involves the precipitation of calcium acetate dihydride, possibly in the form of calcium citrate at low pH, and subsequent separation from the medium through rotating filters or centrifuges. Citric acid is then precipitated at pH 5.9 and 70-90 °C and recovered by filtration and drying. The citric acid then obtained and the comparative analysis between the recovery of citric acid from bacteria and fungus in the fermentation media was studied.

Biochemistry and accumulation of citric acid.

Citric acid is produced by the aerobic bacteria that can undergo TCA cycle as a part of carbohydrate metabolism in order to produce energy and this cycle need to be interrupted to accumulate and finally recover to get crude citric acid. It has been recommended that the amassing of citrus extract requires deactivation of the Krebs cycle catalysts liable for its debasement, aconitase as well as isocitrate dehydrogenase. However, there are confirmations that during the generation of citrus extract, the Krebs cycle is dynamic in the creation of intermediates required for biomass arrangement. Citrus extract aggregation may almost certain be the after-effect of improved (deregulated) biosynthesis instead of restrained debasement. [12,13].The first step of citric acid recovery involves the precipitation of oxalic acid, possibly in the form of calcium oxalate at low pH, and subsequent separation from the medium containing the mycelium through rotating filters or centrifuges. Citric acid is then precipitated at pH 7.2 and 70–90 °C and recovered by filtration and drying. If a purer product were desired, it is dissolved with sulphuric acid, treated with charcoal or ion exchange resins, and again crystallized as

anhydrous citric acid (above 40 °C) or as a monohydrate (below 36.5 °C) [14]

3 RESULTS AND DISCUSSIONS

1) Isolation and screening of bacteria.

Bacteria and fungi were isolated from spoiled fruits and vegetable samples for citric acid production. Characterization study was done and the colony characterization and morphological characteristics were observed under compound microscope stained with grams staining and lacto phenol cotton blue mounting for bacterial and fungal species respectively.

2) Pure culture study

Pure culture study was done by observing the colony characteristics, morphological characteristics and by performing biochemical tests. Gram negative bacterial species like Klebsiella species, Escherichia coliand gram positive bacteria like Bacillus species were isolated from nutrient agar plates. From fungal PDA plates yeast species was isolated.

3) Fermentation process

Fermentation process was carried out for 7 days under optimum parameters. The parameters maintained during the process gave maximum yield of the product. The comparison was then done between bacterial and fungal fermentation.

4) Citric acid recovery

The citric acid recovery was then done by precipitation and the production of citric acid obtained after filtering and drying was compared of both bacterial and fungal fermentation in the presence of fruit extract and without extract. It was observed that maximum citric acid production was obtained for bacterial fermentation in the presence of fruit extract medium than the media without extract. In fungus the citric acid production was obtained and when it was compared to bacterial extract medium only a small proportion of increase of citric acid was seen.

Table 2: Comparative results of citric acid

Fermentation Media Citric Acid Production(in grams)

Bacteria with fruit extract

(B/E) 0.245

Bacteria without fruit extract

(B/WE) 0.165

Fungal media with fruit extract (F/E) 0.195

Comparative results of citric acid production from different fermentation media using bacteria and fungi as inoculums.*(B/E) is bacteria with fruit extract , (B/WE) bacteria without fruit extract and (F/E) fungal media with fruit extract.

4 CONCLUSION

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from the fungus Aspergillus niger, but working with fungus is not so recommendable and it causes health issues, hence bacteria could be a better choice. So as an alternative to it and to produce best from the waste the citric acid production was carried out from spoiled fruits and vegetables using natural extract in the fermentation media. The various parameters process was optimized for the production. Further studies can be carried out to maximize the production of citric acid from this process and to convert it into an efficient alternative method for citric acid production. Our study could contribute by suggesting a cheapest source for citric acid production to meet the current needs.

Fig 9: Comparative study of production of citric acid from bacterial media without extraction, bacterial media with

extraction and fungal media with extraction.

5 ACKNOWLEDGEMENT

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3609 CONFLICT OF INTEREST:

The authors declare no conflict of interest. [NIL].

6 REFERENCES:

[1]. Belén Max, José Manuel Salgado, Noelia Rodríguez, Sandra Cortés, Attilio Converti, and José Manuel Domínguez, 2010, Biotechnological production of citric acid, Braz J Microbiol. 2010 Oct-Dec; 41(4): 862–875., Published online 2010 Dec doi: 10.1590/S1517-83822010000400005 [2]. Papagianni, M. (2007). Advances in citric acid

fermentation by Aspergillus niger: biochemical aspects, membrane transport and modeling. Biotechnology Advances, 25(3), 244–263.

[3]. Yuogo, Z., Zhao, W., & Xiaolong, C. (1999). Citric acid production from the mash of dried sweet potato with its dregs by Aspergillus niger in an external-loop airlift bioreactor. Process Biochemistry, 35(3), 237–242.

[4]. Sanjay H. Chotirmall, Mazen Al-Alawi, Bojana Mirkovic, Gillian Lavelle, P. Mark Logan, Catherine M. Greene, and Noel G. McElvaney, 2013, Aspergillus-Associated Airway Disease, Inflammation, and the Innate Immune Response, Biomed Res Int. 2013; 2013: 723129. Published online 2013 Jul 21. doi: 10.1155/2013/723129, PMCID: PMC3736487, PMID: 23971044, BioMed Research International.

[5]. Torres, N. V., Lopez, J. C., Rivero, M. G. and Rojas, M. G. (1998). Kinetics of growth of Aspergillus niger during submerged, agar surface and solid-state fermentations. Process Biochemistry 33, 103-107.

[6]. Stefan T. Jaronski, in Mass Production of Beneficial Organisms, 2014Malaysian Journal of Microbiology, Vol 6(2) 2010, pp. 161-165 161 Production of citric acid by Aspergillus niger using pineapple waste Kareem, S. O.*, Akpan, I. and Alebiowu, O. O. Department of Microbiology, University of Agriculture, P.M.B. 2240, Abeokuta, Nigeria. E-mail: sharafkareem @yahoo.co.uk Received 9 June 2009; received in revised form 22 December 2009; accepted 29 December 2009 [7]. G.S. Dhillon, S.K. Brar, M. Verma , R.D. Tyagi,

First published: 05 February 2011, https://doi.org/10.1111/j.1365-2672.2011.04962.x, Enhanced solid‐state citric acid bio-production using apple pomace waste through surface response methodology, journal of applied microbiology

[8]. Torres, N. V., Lopez, J. C., Rivero, M. G. and Rojas, M. G. (1998). Kinetics of growth of Aspergillus niger during submerged, agar surface and solid-state fermentations. Process Biochemistry 33, 103-107.

[9]. Frost, G.M. and Moss, D.A. (1987) Production of Enzymes by Fermentation. In: Rehm, H.J., Reed, G., Eds., Biotechnology, Vol. 7a, VCH, Weinheim, 65-102.

[10]. Jernejc K., Perdih A., Cimerman A. Biochemical composition of Aspergillus niger mycelium grown in

citric acid productive and non-productive conditions. J. Biotechnology. 1992;25:341–348. [11]. Dr. Suneetha V., High Performance Liquid

Chromatography analysis, production and brief comparative study o.f citric acid producing microorganisms from spoiled onions in and around vellore district, IJST (2014) 38A2: 193-197, Iranian Journal Of Science and Technology

[12]. Kubicek C.P. Organic acids. In: Ratledge C., Kristiansen B., editors. Basic Biotechnology. 2. Cambridge, UK: Cambridge University Press; 2001. pp. 305–324.

[13]. Marzona M. Chimica delle Fermentazioni & Microbiologia Industriale. 2. Italia: Piccin, Padua; 1996.

[14]. Ana Maria Tornado; Sandra Cortés; José Manuel Salado; Belen Maxi; Noelia Rodríguez; Belinda P. Bobbins; Attila Converted; José Manuel Dominguez, 2011, Citric acid production from orange peel wastes by solid-state fermentation, industrial microbiology(Brazilian journal of microbiology)

[15]. N.A. Has an and I.M.Zulkahar , Isolation and identification of bacteria from spoiled fruits, AIP Conference Proceedings 2020,020073 (2018); https://doi.org/10.1063/1.5062699, Published online 05th October 2018.

[16]. Pranav D. Pathak , Sachin A. Mandavgane* and Bhaskar D. Kulkarni, 2017. Fruit peel waste: characterization and its potential uses Department of Chemical Engineering, Visvesvaraya National Institute of Technology, Nagpur 440 010, India 2CSIR-National Chemical Laboratory, Pune 411 008, India, ResearchGate.

[17]. Utilization of Pineapple Waste: A Review ATUL UPADHYAY ,JEEWAN PRAVA LAMA and SHINKICHI TAWATA 2010* Department of Biochemistry and Applied Bioscience, The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065, Japan. Department of Food Technology and Quality Control, Ministry of Agriculture and Cooperatives, Kathmandu, Nepal. Faculty of Agriculture, University of the Ryukyus, Senbaru, Nishihara-cho, Okinawa 903-0213, Japan, ReearchGate.

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Patel, By: Patel, Rakesh., Material type: Book

Publisher: Delhi Aditya Book

Centre, 2009Edition: 5thed.Description: 206p.Subj ect(s): Microbiology, DDC classification: 579 [19]. Industrial Microbiology by A.H. Patel, Industrial

Figure

Table 1: Composition of the media
Table 2: Comparative results of citric acid
Fig 9: Comparative   study   of production of    citric acid from bacterial media without extraction, bacterial media with extraction and fungal media with extraction

References

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