FUNGAL CITRIC ACID PRODUCTION USING WASTE MATERIALS: A MINI-REVIEW

Omkar Sawant2, Sagar Mahale2, Vanitha Ramchandran3, Geetha Nagaraj4 and Ashok Bankar*1
Address(es): Dr Ashok Bankar* (Corresponding author),
1
MES Abasaheb Garware College, Department of Microbiology, Savitribai Phule Pune University, Karve Road, Pune, Maharashtra, Pune- 411004, Maharshtra, India.
Tel. No : +91-20-41038200.
2
KK Wagh College of Food Technology, Mahatma Phule Krishi Vidyapeeth, Nashik, Maharashtra, India.
3
Maharani’s Science College for Women, Department of Microbiology, University of Mysore, Mysuru, Karnataka, India.
4
Department of Studies in Biotechnology, University of Mysore, Mysuru, Karnataka- 570006, India.

*Corresponding author: [email protected] doi: 10.15414/jmbfs.2018.8.2.821-828

ARTICLE INFO ABSTRACT

Received 7. 3. 2018 Citric acid is one of the extensively used organic acids in many industries. There is a worldwide demand for citric acid consumption due
Revised 5. 9. 2018 to their many industrial applications. It is also considered as a Generally Recognized As Safe (GRAS) compound. In this review, current
Accepted 7. 9. 2018 developments in microbial fermentation processes for citric acid production have been discussed. In food, beverages, milk and sugar
Published 1. 10. 2018 processing several biodegradable organic waste materials are generated in large quantities. These include sugarcane bagasse, grape
pomace, apple pomace, pineapple pomace, vegetables, tapioca, coconut husk, banana peels, citrus peels, whey and decaying fruits are
found to be potential substrates for citric acid production. A significant effect of substrate concentration, inorganic salts, initial sugar
Review
concentration, moisture and additives etc on citric acid production have been highlighted for further improvement in fermentation
process. Recent developments in upstream and downstream processes for citric acid production are also deeply discussed. This review
gives insights for future possibilities of cost effective fermentation process for citric acid production from several cheap raw materials.
Formation of citric acid as an industrial byproduct will help to tackle waste disposal issue and also reduce the dependency of industry
over other citric acid producers. Thus, the industry would be benefitted ecologically and economically.

Keywords: Citric acid, GRAS, Fermentation, Substrates, Waste, Fungi

INTRODUCTION potential producer of citric acid (Selvankumar et al., 2014; Singh et al., 2016;
Alnassar et al., 2016). Commercial production of citric acid has been mainly
Citric acid (2-hydroxy propane 1, 2, 3-tricarboxylic acid) is one of the most carried out by microbial fermentation process, with increasing production by
important natural organic acids. Its name is derived from the Latin word ‘Citrus’ improving the environmental parameters and genetic manipulation of
and obtained naturally by metabolic pathways which are performed in living cells microorganisms which are used (Ali et al., 2016). Microorganisms have the
via tri-carboxylic acid cycle (Swain et al., 2012). Citric acid is one of the natural ability to utilize cheap raw waste materials and convert them into value added
organic acids known to be non-toxic and pleasant with sour taste. It is also products such as organic acid (Singh et al., 2016). Microbial fermentation may
considered as a Generally Recognized As Safe (GRAS) compound. It is a be carried out by using three main methods: 1) Submerged fermentation 2)
naturally occurring compound in citrus fruits like oranges, berries, limes, lemons, Surface fermentation and 3) Solid substrate fermentation (Gupta et al., 2015).
tangerines and grapes. Citric acid is commonly used as a preservative, acidulant, Solid surface fermentation is found to be a better alternative process to
antioxidant, emulsifier and buffer in food industry. For these reasons, citric acid conventional submerged fermentation. In this process, microorganisms utilize
is continuously used as a common ingredient in variety of food products. Thus, cheap waste materials as substrates. The waste materials generated in food
all over the world such beneficial compound is of a high demand for their daily industries are used as substrates by microorganisms for citric acid production. In
consumptions (Soccol et al., 2006; Radwan et al., 2010; Vasanthabharathi et food, beverages, milk and sugar processing several biodegradable organic waste
al., 2013). Citric acid has several applications in food, chemical and materials are generated in large quantities. These include sugarcane bagasse,
pharmaceutical industries. It is reported that 70% of citric acid produced by grape pomace, apple pomace, pineapple pomace, vegetables, tapioca, coconut
industries is used in food industries only and the remaining 30% is used in the husk, banana peels, citrus peels, whey and decaying fruits that can be used as
chemical, pharmaceutical, medical and other industries (Soccol et al., 2003). fodder and feedstock for biogas production (Hang and Woodams, 1984; Hang,
Food industry is the largest consumer of citric acid due to their several 1998; Murad et al., 2003; Kumar et al., 2003; Hamdy, 2013; Kareem and
advantageous properties (El-Hussein et al., 2009; Yalcin et al., 2010). Citric Rehman, 2011). Such nutrient rich and cheap wastes can be utilized as substrates
acid occupies a key position in the global market due to their heavy usage in by microorganisms for the production of citric acid as value-added product. In
many industries (Ali et al., 2016). this review, overview of bioconversion of different wastes into citric acid by
Production of citric acid was carried out by several physical and chemical microorganisms is highlighted.
methods. However, such conventional methods are found to be an expensive,
complex and not eco-friendly (Gupta et al., 2015; Yin et al., 2017; Yu et al., HISTORY OF CITRIC ACID PRODUCTION
2018). Moreover, annual production rate of citric acid is low while the rate of
demand is high. Hence there is a need to obtain continuous production of citric In 1784, citric acid was extracted from lemon fruits for the first time by Karls
acid to fulfil the market demand. Production of citric acid by using Scheels in England. The lemon juice was treated with calcium hydroxide to form
microorganisms is much more feasible when compared with plant and animal calcium citrate. It was treated with sulfuric acid to obtain citric acid. The
sources. There are several microorganisms with the ability to produce citric acid industrial production of citric acid was started in 1890 and the industrial process
such as Bacillus subtilis, B. licheniformis, Corynebacterium spp., Aspergillus was used until 1919. At that time, citric acid was sold at high cost due to Italian
niger, A. flavus, Mucor piriformis, Trichoderma viride, Penicillium janthinellum, monopoly. In 1917, James Currie was the first to observe that some strains of A.
Candida tropicalis, C. lipolytica and C. intermedia etc (Kapoor et al., 1983; niger have the ability to produce citric acid and can be used for commercial
Papagianni, 2007). Literature survey has revealed that A. niger is found to be a purpose. He investigated that these strains were able to grow well in acidic

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environment (pH 2.5-3.5) containing high sugar concentration. The columella at the tip (Singh et al., 2016). It has been reported that the mycelium
environmental conditions aforementioned were found to stimulate the citric acid form of fungus has a key role to obtain final yield of citric acid (Kristiansen and
production (Kristiansen and Sinclair, 1979). Around 1929, Pfizer started Sinclair, 1979). A. niger has been widely used in several industrial fermentation
commercial production of citric acid with surface fermentation techniques. In processes due to its ability to convert cheap substrates into value-added products
1948, technologies got developed where molasses was used as a cheap raw with higher yields. Being a dominant filamentous fungus, there are reduced
substrate instead of sucrose for citric acid production. Around late 60’s, n-alkanes chances of contamination and complications in handling it. Immobilization of A.
were effectively used as substrate by many bacteria and yeasts for citric acid niger is found to be useful method for continuous production of citric acid (Garg
production (Anastassiadis et al., 2008). All over the world, about 80% of citric and Sharma, 1992). A. niger is a commonly occurring natural flora of soil and
acid production was performed by submerged fermentation process (Yalcin et many edible products like bread, onions, banana and wheat bran etc. Serial
al., 2010). Citric acid production by submerged fermentation was strongly dilution of soil samples is carried out and allowed to grow on potato dextrose
influenced by several physico-chemical parameters such as aeration, pH, agar (PDA) (Selvankumar et al., 2014; Abbas et al., 2016a). Based on growth
temperature, trace elements, morphology of microorganisms, initial carbon and and distinct morphological characteristics, A. niger colonies are identified and
nitrogen concentrations (Dhulappa, 2016). allowed to grow on newly prepared PDA plates. Screening of citric acid
Currently, molasses is used as substrate for production of citric acid. In this producing strains of A. niger is identified by growing on Czapeck dox agar plates
process, there is a higher chance of high level of cations contamination and the containing bromocresol green. Yellow colored zone appears around fungal
needs continuous control on process parameters. These cations are usually colonies which have potential of citric acid production. All fungal isolates are
originated from insoluble residues formed by precipitation with potassium then preserved on PDA slants under refrigeration (4˚C). The isolates may also be
ferrocyanide. Therefore, due to the complexity of such pretreatment process stored in glycerol solution (30% V/V) at -76˚C (Selvankumar et al., 2014).
results into reduction of citric acid production. Thus, the search continues for new Pure culture of A. niger is inoculated on sterile slants of PDA and incubated at
potential alternatives. Solid substrate fermentation (SSF) is one of the methods 25-32˚C for 5-7 days. The inocula are obtained in 10 ml of sterile distilled water
that can be used for industrial fermentation. There are several advantages of SSF containing two drops of 0.1% tween 80 solutions. A small sterile inoculation
such as high productivities, low production costs, low water consuming, extended needle is used to scrap the spores of fungi with proper care to avoid
stability of products and eco-friendly etc (Hölker and Lenz, 2005). The citric contamination and without disturbing the agar surface. Usually, ≈ (1-2)×107
acid is a commercially valuable product. The worldwide demand for citric acid is spores are suspended into the sterile medium (Selvankumar et al., 2014;
around 3 million tons. The annual production of citric acid was reported as 700 Alnassar et al., 2016). The number of spores is counted by using
thousand tonnes in 1993, 1.4 million tonnes in 2004, 1.6 million tonnes in 2008 haemocytometer. The prepared inocula should be used within 7 days because
and 1.8 million tonnes in 2010 (Yalcin et al., 2010; Addo et al., 2016). The older spores have tendency of consuming citric acid which is initially produced
consumption of citric acid was 75, 10 and 15 % by food industries, before completion of fermentation. The starter culture can be prepared by using
pharmaceuticals and other industries, respectively (Ali et al., 2016). United States sucrose salt media. The culture is incubated at 30°C for 2-3 days and further used
was the largest producer of citric acid in the world. Chinese production volume of as per need (Alnassar et al., 2016; Abbas et al., 2016 a,b).
citric acid has surpassed the production in United States from 1995 due to the Strain improvement program is one of the approaches to increase the yield of
introduction of cheap raw materials as substrate for citric acid production that citric acid. It can be enhanced by modifying metabolism of the fungus, A. niger
reduced their cost. All over the world, the production rate of citric acid has been (Swain et al., 2012). Strain improvement can be achieved by mutagenesis
increasing at the rate of 3.5 - 4 % per annum (Amenaghawon et al., 2015) due to process. Various mutant strains of A. niger are obtained and used for commercial
their several applications (figure 1). production of citric acid (Jialong et al., 2000). Various parameters need to be
taken into accounts while selecting an improved strain which are as follows : 1)
Functions as Imparts dark Inversion of Efficient Citric acid with Strains should have stable physiological and biochemical characters 2) Citric acid
acidulants in color in hard sucrose in case of antimicrobial Biocarbonate should not be used by strains for any other purpose 3) Strains should not produce
food products boiled candies Jam and Jelly etc activity have enhanced other metabolic acids such as oxalic, gluconic and malic acid etc. (Yalcin et al.,
and cheese antimicrobial 2010). Mutagenesis can be achieved by either physical, chemical means and by
activity gene cloning. The most common physical mutagens used are gamma and UV
Gives flavor
radiations. Chemicals such as diethyl sulfonate (DES), N-Methyl-N-
Reduced cadmium
to beverages Nitrosoguanidine, ethidium bromide etc., are well known chemical mutagens
toxicity in plant
(Zia et al., 2010). Enzyme diffusion zone analysis is a specific method for
Applications Used for biodegradable screening and identification of mutated improved strain which is based on
Act as neutralizer enzymatic reaction on plate media (Prasad et al., 2014). Other two methods such
and buffer in of elastomers with enhanced
mechanical properties used in as Single Spore Technique (SST) and Passage Method (PM) are well known
pharmaceutical citric acid biomedical applications alternative methods for selection of improved strains (Soccol et al., 2006). Strains
and cosmetic
industry with robust citric acid production capacity would be possible by identifying
Used as photographic molecular mechanisms involved in citric acid production.
reagent in industrial
Activates sodium application
FERMENTATION PROCESSES FOR CITRIC ACID PRODUCTION
chlorite used as
disinfectant Used as a component in
anticancer composition
Liquid substrate fermentation

Remove metal oxides from Inactivates oxidative enzymes It is used as Preservative In liquid substrate fermentation process, a substrate used is soluble in liquid
the surfaces of objects of by lowering down pH of food in poultry products media. In this process, a lot of free flowing liquid is used without any physical
ferrous and other metals products support to the microorganisms. This process can be performed by two different
methods a) Submerged fermentation and b) Surface fermentation (Reddy, 2002)
Figure 1 Different applications of citric acid (Zimmermann and Wausau, as shown in the Figure 2.
1940; Leveskis, 1979; Soccol et al., 2006; Anastassiadis et al., 2008;Glevitzky
et al., 2009; EI-Sheikh et al., 2010; Javanmard and Endan, 2010; Swain et
al., 2012; Ioannou and Ghoul, 2013; Brima and Abbas, 2014; Singh et al., Fermentation
2016).

STRAIN IMPROVEMENT PROGRAM FOR CITRIC ACID

PRODUCTION
Liquid Solid
Different types of yeasts, molds and bacteria are being used in industry for substrate substrate
commercial production of citric acid including C. tropicalis, C. oleophila, C. fermentation fermentation
guilliermondii, C. citroformans, Hansenula anamola, Yarrowia lipolytica,
Saccharomicopsis lipolytica, . niger, A. carbonarius, A. aculeatus, A. awamori, ,
A. fonsecaeus, A. foetidus, A. phoenicis, Corynebacterium ssp., P. janthinelium,
P. restrictum, Talaromyces sp., T. viride, Ustulinavulgaris, B. licheniformis,
Arthrobacter paraffinens. However, several commercially developed strains of A. Submerged Surface
niger have been most ideally used for citric acid production (Sardinas, 1972; fermentation fermentation
Ikeno et al., 1975; Kapelli et al., 1978; Grewal and Kalra, 1995; Lu et al.,
1997; Abbas et al., 2016a; Alnassar et al., 2016).
Figure 2 Different fermentation processes for citric acid production
A. niger is a mold with asexual reproduction belonging to the class
Deuteromycetes. It gives rise to black colored erect conidiophores with globose

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Submerged fermentation process is commonly used for citric acid production. Solid substrate fermentation
They have advantages like higher yields and lower labour costs make it more
suitable for citric acid production (Rohr et al., 1983). In this process, anaerobic Solid substrate fermentation in which fermentation process is carried out on a
or partially anaerobic decomposition of carbohydrates is carried out by non-soluble material which acts as physical support as well as a source of
microorganisms in liquid medium containing a lot of freely available water nutrients instead of free flowing liquid (Pandey, 1992). This has been practiced
(Swain et al., 2012). The nutrient substratum is in liquid form which is also from ancient times, common examples including application of A. oryzae in koji
called broth and the organism grows inside the substratum. Spargers and impeller process for fermentation of rice and cheese production by P. roquefortii. In Koji
blades are used to maintain the culture conditions (Reddy, 2002). Aseptic and process, the mould (A. oryzae) is grown on a steamed rice medium. The rice is a
uniform conditions are maintained in the specialized vessels known as major source polysaccharide starch. The starch present in medium can not utilize
bioreactors which are required for fermentation. The performance of a bioreactor by yeast as an energy source. The enzymes such as glucoamylase and alpha
is dependent on different parameters such as biomass concentration, aseptic amylase used in Koji process are responsible for conversion of rice starch into
conditions, heat transfer and operation at optimum process conditions. Stirred glucose. Thus, glucose can be easily utilized as an energy source by yeast during
tank bioreactor and airlift bioreactor are commonly preferred for submerged the sake brewing process. Thus, this process is also known as saccharification
fermentation process. Stirred tank bioreactors have several advantages such as (Furukawa, 2012). Solid substrate fermentation can also be useful to obtain
high oxygen transfer rates, high biomass productivity, low investment and low variety of chemicals and value added products like enzymes, biologically active
operating costs for aerobic fermentation process. There are several complex secondary metabolites, amino acids, single cell proteins, mushroom and organic
medium ingredients such as corn steep liquor, molasses and soybean flour which acids etc. Recently, solid substrate fermentation is used for the production of
are used as inexpensive nutrition source in fermentation (Kristiansen and flavor compounds, enzymes, colorants, biopesticides and other industrially
Sinclair, 1979; Swain et al., 2012; Alnasaar et al., 2016). Airlift reactor can be important products. This process have some advantages such as low energy
advantageous if fermentations have low shear and energy requirements. In this requirements, high product yields, low risk of contamination, better product
reactor, high pressure sterilized air is provided from the bottom of the bioreactor characteristics, less efforts in downstream processing, less effluent generation
for uniform mixing and oxygenation of medium. It also has disadvantages like and simple operation as compared to submerged fermentation. It was found that
inadequate sterilization, higher capital investment, aeration requirements and the economic efficiency is 100 folds as compared to submerged fermentation.
high power requirements (Pometto et al., 2005). Solid substrate fermentation serves to give anchorage to filamentous fungi which
Surface fermentation is a stationary batch fermentation process in which results in good growth and high productivity. Thus, different solid nutrient rich
microorganisms grow on the substratum and derive nutrients from it. Nutrient agronomic wastes are effective and economic for solid substrate fermentation.
media like cane molasses, beet molasses, wheat bran, potato starch and glucose This process may have some drawbacks such as difficulties to scale up, low
syrup are commonly used (Kristiansen and Sinclair, 1979; Alnasaar et al., amenability of the process to standardization, difficult control of process
2016). Prepared media is placed in thin layers in shallow trays (1-2.5 cm depth) parameters and problems with heat buildup (Tengerdy, 1996; Holker and Lenz,
and inoculated with desired microorganism. Continuous aeration is necessary to 2005; Kapilan, 2015; Handa et al., 2016).
supplant the CO2 generated. Approximately 40-60% humidity is maintained to
prevent moisture loss from the surface of the medium. Once the fermentation WASTE MATERIALS UTILIZED FOR CITRIC ACID PRODUCTION
process is over, crude fermentation fluid and mycelial mats are separated.
Further, they need washing in order to remove any impregnated citric acid. The A variety of wastes are continuously produced from different types of industrial
disadvantages of this process are unequal distribution of air and nutrients, high processes. Such wastes can be cost effective substrates for microorganisms to
labour requirements and inefficient use of floor space (Reddy, 2002). This obtain value added products like citric acid listed in Table 1.
process is still used in small and medium scale industries due to their some
advantages such as low energy cost, low installation cost and less efforts for
operation (Swain et al., 2012).

Table 1 Waste materials utilized as substrates by fungi for citric acid production

Microorganisms Substrate/Media Citric acid production (g/l) References

A. niger (Van Tieghem MTCC 281) Waste apple pomace 4.6 g/100 gram of pomace Kumar et al., 2010
Y. lipolytica (NRRL Y-1095) Glycerol 59.00 Avila-Neto et al., 2014
A. niger (FUO2) Cassava peel malted sorghum 1.93 Adeoye et al., 2015
A. niger (FUO110) Cassava peel malted sorghum 9.40 Adeoye et al., 2015
A. niger Synthetic medium 14.68 Alsudani et al., 2015
A. niger (GCB 117) Cane molasses 14.17 Iqbal et al., 2015
Y. lipolytica (K-168) Carrot juice-based medium 80.53 Urak et al., 2015
A. niger (GMCC 5751) Liquefied corn medium 151.67 Wang et al., 2015
A. niger (ATCC 9142) Cocoyam starch 108.00 Amenaghawon et al., 2015
Y. lipolytica (SJW-1b) Corn steep liquor 27.5 Liu et al., 2015
Y. lipolytica CBS 2073) Crude glycerol 10.00 Ferreira et al., 2016
Y. lipolytica W29 (ATCC 20460) Crude glycerol 10.00 Ferreira et al., 2016
A. niger (KA88) Corn cobs solid substrate 138.24 Addo et al., 2016
A. niger Mango peels 7.52
Sweet orange peels 11.01 Abbas et al., 2016b

However, such substrates need pre-treatments prior their utilization as substrate. wastes such as dairy wastewater, cane molasses, rice straw and sugarcane
Dried wastes like coconut husk, coconut cake, sunflower cake and corn cobs etc bagasse need prior treatment before their use as substrate. Dairy wastewater has
could be cost effective substrates (Lingappa et al., 2009; Addo et al., 2016). 2.5 % reducing sugars in the form of lactose which are concentrated to get 5% of
Some of the wastes collected may have high moisture content which becomes lactose. The proteins and lipid content may get precipitated during sterilization
susceptible for contamination with undesirable microorganisms result into process. Therefore, the mixture needs to be heated at 90˚C and maintain their
uncontrolled microbial growth. These wastes include decaying fruits, pomaces of optimum pH 4.3. The separated proteins and lipids should be removed by
grape, apple, orange, sweet-orange, pineapple, vegetables, tapioca, spoiled centrifugation process (Kim et al., 2002). Cheese whey need fortification of
coconut and banana peels etc. Therefore, such wastes need to be dried until lactose, NH4NO3, KH2PO4, ZnSO4, K4 [Fe(CN)6] before their use as substrates
moisture content comes down to the desired level (Kumar et al., 2010; Abbas et (Alnassar et al., 2016). Rice straw are washed and dried at 70˚C overnight then
al., 2016a; Ali et al., 2016). Drying of wastes is commonly performed at 60˚C in pretreated with NaOH at room temperature for 1 h. Further, they are washed with
mechanical driers. Period of drying varies depending upon the nature of wastes. water, neutralized and kept for drying at 70˚C overnight. Straws are then
For example, decayed fruits need 2 days for proper drying while banana or citrus pulverized and added with pretreated molasses (Ali et al., 2011). Sugarcane
peels may require 8 h. These are then pulverized and sieved through suitable bagasse is dried, cut down into small pieces and grinded to obtain desired particle
sieve prior to use as substrate. Banana peels are supplemented with various size. These particles are moistened with 15-20% of sucrose solution or molasses
nitrogenous compounds such as ammonium phosphate and peptone (Kareem solution. Such substrates are fortified with NH4NO3, KH2PO4, MgSO4, and
and Rehman, 2011). Corn cobs also need (NH4)2HPO4 as a preferred nitrogen CuSO4 before their use as substrates (Kumar et al., 2002). Sugarcane bagasse
source (Addo et al., 2016). Such pulverized medium is added with sufficient extract may be processed for their use in submerged fermentation. Bagasse is
amount of water and autoclaved at 121˚C for 1 h. Such properly cooked media is sun-dried and grinded to obtain particle size of 1.2-1.6 mm. It is soaked in
useful for increasing the amenability of the media to microorganisms. Other distilled water and the extract is obtained after filtration process. Cane molasses

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need to be diluted to obtain 15% of sugar level and the pH of medium is adjusted and of same quality for every batch of production. Table 2 showed different types
using HCl (El-Hussein et al., 2009). Orange peels have also been reported for of microorganisms having ability to produce citric acid. However, literature
their use in submerged conditions. Orange peels are initially washed properly, cut survey revealed that fungi and yeasts are most commonly used for citric than
into small size, dried, powdered, sieved and fortified with glucose, sucrose, bacteria. Table 2 clearly indicates that fungi and yeasts are comparatively
NH4NO3, MgSO4 and (NH4)3PO4 (Singh et al., 2016). Moisture of orange peel is highest citric acid producers than the bacterial system.
maintained at desired level and autoclaved at 121˚C for 15-60 minutes.
Precaution is needed to be taken so that the substrate selected is easily available

Table 2 Citric acid production (g/L) by different microorganisms

Microorganisms Citric acid yield (g/L) Substrate References
Fungi
Penicillium oxalicum 530.00 Potato dextrose liquid medium Li et al., 2016
A. niger 173.20 Corn flour liquefied Wang et al., 2017
A. niger ATCC12846 162.70 Wheat bran extract Yu et al., 2018
A. niger H915-1 157.00 Corn steep liquor and corn starch Yin et al., 2017
A. niger MTCC 282 46.50 Colocassia antiquorum (10% w/v) Ganne et al., 2008

Yeast
155.20 Crude glycerol (86% wt/wt) Rywińska et al., 2010
Y. lipolytica Wratislavia 1.31
152.30 Canola oil Good et al., 1985
Saccharomycopsis lipolytica
Y. lipolytica 1.31
144.50 Crude glycerol(25% wt/wt) Rymowicz et al., 2005
Y. lipolytica NG40/UV5
140.00 Rrapeseed oil Morgunov et al., 2018
Candida lipolytica
47.00 n-paraffins Crolla and Kennedy, 2001
Bacteria
Bacillus licheniformis
42.00 Glucose Kapoor et al., 1983
Cornybacterium sp
40.00 Dodecanerice Kapoor et al., 1983
Acetobacter xylinum
2.20 Saccharificate medium Lu et al., 2016

Table 3 shows citric acid yield (g/L) in different fermentation processes like solid 3 showed that yield of citric acid was higher in soild substarte fermentation
substrate fermentation, surface fermentation and submerged fermentation. Table process when compared with surface and submerged fermentation.

Table 3 Citric acid yield (g/L) in different fermentation processes

Citric acid fermentation Yield of citric acid
Microorganisms Substrates/media References
processes (g/Kg) or (g/L)
A.niger NRRL2001 Apple pomace 766.00 Hang and Woodams, 1984
A. niger MTCC 281 Coconut cake 108.90 Lingappa et al., 2002
Solid Substrate Fermentation A. niger UABN 210 Banana peels 82.12 Kareem and Rahman, 2011
A. niger Oat bran 62.00 Rao and Reddy, 2013
A. niger Rice straw 50.20 Ali et al., 2012
A. niger RCNM 17 Sugar 24.37 Narayanamurthy et al., 2008
Surface Fermentation
A. niger ATCC 9142 Grain liquor 19.00 Roukas and Kotzekidou, 1986
A. niger ATCC 9142 Corn starch hydrolysate 2.70 Andrew and Corresponding, 2012
A. niger Corn flour liquefied 173.20 Wang et al., 2017
A. niger ATCC12846 Synthetic medium combined 162.7 Yu et al., 2018
Submerged Fermentation with wheat bran extract
A. niger MO-25 Molasses and chicken feather 68.8 Ozdal and Kurbanoglu, 2018
Native A. niger Banana peels 51.68 Abbas et al., 2016a

Moisture, process additives and pH

INFLUENCE OF DIFFERENT PARAMETERS ON CITRIC ACID
PRODUCTION PROCESSES In case of solid substrate fermentation process, the moisture content is maintained
in between 50-80%. Increase in the moisture content beyond 70% results into
Inoculum is added into media and kept for incubation at 30˚C for the decreased sugar consumption and citric acid production. Addition of methanol in
predetermined period. Various tests are carried out for process control. The yield media increases the required moisture content. In case of pineapple pomace, the
of final product is dependent on various parameters that are described below: highest yield (11.3g/100g) was achieved at 4% of methanol and 70% of moisture
content. In the absence of methanol, yield was found highest (9.7g/100g) at 60%
Initial Sugar Concentration moisture content (Kumar et al., 2003). Apple pomace shows similar result at 4%
methanol and 73% moisture content with the yield of 883 g/kg of sugar. In the
The major waste is generated by food industry during fruits and beverages absence of methanol, yield was reduced to 259 g/kg of sugar even at same
processing. Such waste is in the form of pomaces, peels and trimmed portions moisture content (Hang and Woodams, 1984). In case of grape pomace, the
etc. The pomace is the solid material obtained after extraction of fruits. It highest yield was 600g/kg of sugar consumed in presence of 3% methanol and
contains skin, pulp, seeds and stems of fruits. This pomace is considered as 65.4% moisture content. It was noticed that yield was reduced to 275 g/kg of
promising substrate for citric acid production. They are found rich in sugar consumed in the absence of methanol (Hang, 1998). When banana peel is
carbohydrates but low in proteins. It was observed that apple and grape pomace used as substrate and optimum moisture content is maintained which gives yield
containing about 30% of reducing sugars. For such substrates, supplementation of of 52.08 g/L (Abbas et al., 2016). Orange peel substrate gives yield of 640 g/kg
any particular nutrients is not needed since they are found to be in optimum at 65% w/v moisture content. However, the yield also depends on the strain of
concentration. Other substrates such as citrus fruit peels, rice straw and sugarcane microorganism used. In case of sugarcane bagasse, the highest yield 19.8 g/100g
bagasse have fewer amounts of fermentable sugars and therefore moistened with was obtained after treatment with clarified molasses and 4% methanol (Kumar et
sucrose solution to increase the level of sugar (Kumar et al., 2002; Ali et al., al., 2002). The yield obtained was 5.023 g/100g when rice straw was used as
2011). Sweet lime peel is supplemented with sucrose solution to achieve sugar substrate in optimized condition (Ali et al., 2011). There is no exact information
concentration of 31.8g/100g dry solid. Other substrates such as corn cob, orange of the effect induced by methanol. However, it is seen that various trace elements
peel and pomegranate peel is similarly treated (Hamdy, 2013; Addo et al., 2016; such as Fe2+, Mn2+ and Zn2+ are responsible for suppression. Methanol has
Prabha and Subbarangaiah, 2016). The reducing sugar content of dairy and dehydrating action on the substrates and also intensifies the outbound
cheese wastewater is increased to 5% and 15%, respectively (Kim et al., 2002; permeability of cellular walls for secretion of citric acid (Yadegary et al., 2013).
Murad et al., 2003). In addition, other lower alcohols such as ethanol and n-propanol may also induce
the same effect (Kareem and Rehman, 2011). There are several substrates such

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as dairy wastewater, cane molasses and cheese whey that are used in submerged most influencing parameter observed in citric acid production. It was seen that
fermentation processes. The yield obtained was about 4 g/L when dairy increasing the surface area to volume ratio from 0.25-2 cm2/mL showed
wastewater was used as substrate with 5% of sugar concentration without any increased citric acid production from 0-21 g/L (Mazinanian et al., 2015).
particular additions (Kim et al., 2002). On the other hand, various additions such
as methanol, ethanol, EDTA, and combination of methanol and EDTA were BIOREACTOR DESIGNS FOR CITRIC ACID PRODUCTION
tested when cane molasses was used as substrate. It was noticed that in the
presence of 3% of methanol, yield obtained was 61.62 g/L and was reduced to Bioreactor is a vessel in which a biological reaction or process is performed. It is
48.43 g/L when methanol was replaced by 4% of ethanol. When EDTA alone facilitated with accessories required to maintain suitable environment for growth
was added at the rate of 250 ppm, yield was about 59.74 g/L. Maximum yield of and activity of microorganism. Liquid substrates such as whey and molasses can
≈92.86 g/L obtained when whey was used as substrate supplemented with 15% of be typically fermented by using the conventional Fermenter with sophisticated
sugar. However, addition of riboflavin (10 mg/L) or 4% tri-calcium phosphate automations in case of submerged fermentation process (Lingappa et al., 2009).
can reduce the required sugar addition significantly to 10% but may also affect In case of solid surface fermentation, some limited options of bioreactors and
citric acid production (Murad et al., 2003). The experimental conditions for process controlling systems are available (Rodriguez and Sanroman, 2005). To
citric acid production by A. niger was optimized and the highest citric acid yield perform solid surface fermentation effectively at industrial level following
of 19.447 g/l was reported when the rice husks filtrate medium was used designs have been proposed as follow:
(Alsudani et al., 2015). Several mutants of A. niger were obtained and tested for
citric acid production. Amongst the wild strains, a strain (FUO 2) has been Tray
reported for the highest citric acid production (1.93 g/l) when grown on
hydrolyzed cassava peel medium. The mutant strain FUO1 10 was developed after This consists of flat trays to be incubated in a room provided with proper
exposure to UV-radiation and gave highest yield of 9.4 g/l. Thus, mutation ventilation and temperature control systems through circulation of humidified air.
showed improvement of 4.87 fold in citric acid production. The process The depth of shallow trays should not be more than 7 cm. Similarly, depth of the
optimization was done by using the mutant strain (FUO 110) on the input solid medium should not be more than 5 cm for proper air circulation into the
parameters such as substrate concentration, process time, inoculum size and solid medium (Okafor, 2007). However, this design is not found attractive due to
initial pH. The highest yield of citric acid was 88.73 g/l and thus, showed requirement of a large number of trays, labor and extra floor space.
improvement of 45.97 fold over the best wild strain (Adeoye et al., 2015). The
high level of ATP showed a strong inhibitory effect on citric acid production by Packed bed
A. niger. Thus, increasing NADH oxidation and reducing the level of intracellular
ATP can accelerate glycolysis and the TCA cycle to enhance citric acid It comprises of a column mostly made of glass or plastic. It has a perforated base
production (Wang et al., 2015). The solid substrate such as corn cobs was used where substrate is retained. Humidified air is continuously passed with pressure
for citric acid production by using A. niger KA88. One-factor-at-a-time (OFAT) through perforations. Temperature is maintained via water circulation by using
model was employed for optimization of the fermentation media to obtain the jacket. However, recovering of final product from this bioreactor is difficult due
maximum yield of citric acid. The final OFAT fermentation media gave 138.24 g to non-uniform microbial growth. Further, inadequate removal of heat from
citric acid/kg dry corn cob. Thus, corn cob proved to be a highly promising solid bioreactor might be harmful to fermentation process (Rodriguez et al., 2000).
substrate for production of citric acid (Addo et al., 2016). Similarly, sugarcane
molasses was also used as carbon source for maximum citric acid production Horizontal drum
(Iqbal et al., 2015). Additives like low molecular weight alcohols, trace metals,
phytate, lipids etc have been reported to stimulate citric acid production by A. Such rotating bioreactors are found to be useful for proper mixing and aeration
niger on cocoyam starch. A three variable, three-level Box-Behnken design with minimal damage to inoculum or formed products. Paddles and baffles are
(BBD) was used to develop a statistical model to test effect of Zinc (II), Iron (III) used for this purpose. Only drawback of this bioreactor is that substrate has to be
and methanol on citric acid production. Response surface methodology (RSM) filled only up to 30 % of the total capacity of vessel for proper mixing and
was also used for optimization of such stimulants. It was seen that citric acid aeration (Domniguez et al., 2001).
production was enhanced with increasing concentration of zinc and methanol.
The maximum citric acid production was 108 g/L (Amenaghawon et al., 2015). Fluidized bed
Mango peels and sweet orange as novel substrates were used for citric acid
production by A. niger and the fermentation parameters were optimized. In this bioreactor, there is constant flow of air provided with pressure to prevent
Maximum citric acid production was obtained by using mango peel 11%, sucrose coalescence of particles. Such bioreactors have several advantages such as higher
concentration 5%, inoculum 2%, potassium dihydrogen phosphate and mass, heat transfer, proper aeration, agitation and higher heat buildup due to
ammonium nitrate at pH 5 and 32°C after 8 days. In sweet orange peel shear forces. However, harm to inoculum may lower the rate of productivity
fermentation process maximum citric acid was obtained by using 11% sweet (Mitchell et al., 2003). The above described bioreactors are of basic designs. The
orange peel, disodium hydrogen phosphate, inoculum 2%, sucrose concentration lab scale design for bioreactors could not be entirely useful for scale up process.
25% at 32° C and pH 4 after 6 days (Abbas et al., 2016a). Citric acid production Initially, scale up process was characterized by thumb rules mainly including heat
was maximum by A. niger using banana peel 20%, inoculum 5%, potassium and mass transfer phenomena. Later on, researchers also began to develop
dihydrogen phosphate and ammonium nitrate at pH 5 and 32 oC after 8 days of quantitative approaches towards mass transfer, mathematical modeling and
fermentation (Abbas et al., 2016b). Generally, initial pH for citric acid analysis of process for development of scale up process (Durand, 2003).
production is adjusted below 4.5 because higher pH favors production of Microbes were studied for better understanding of behavioral pattern under
gluconic and oxalic acid. various environmental conditions.

Effect of trace elements, phosphate source and surface area to volume ratio TECHNIQUES FOR RECOVERY OF CITRIC ACID

The trace elements are one of the most important factors in citric acid production After the desired incubation period, the fermentation process is stopped by the
by A. niger (Hang and Woodams, 1998). In particular, metal ions like zinc, action of heat. The end product (citric acid) may contain mycelium and varying
manganese, iron, copper and magnesium are found to be crucial elements in citric amounts of impurities such as mineral salts, other organic acids and proteins etc.
facid production. Therefore, it is necessary to consider the interdependence of The method used for product recovery depends on the raw materials used in
medium components. The yield of citric acid can be increased when the level of fermentation (Grewal and Kalra, 1995). In solid substrate fermentation method,
the trace elements is kept optimum, mostly in case of submerged fermentation the contents of the bioreactor are dried in oven at 50˚C for 2 h. Distilled water is
(Soccol et al., 2006). It was clearly noticed that the presence of phosphate in then added in excess and thoroughly mixed by using mechanical shakers. After
production medium had a great impact on citric acid yield. The low concentration desired mixing, the contents are filtered. In the submerged fermentation, the
of phosphate has a positive impact on citric acid production. The phosphate mycelium biomass is heated up to 70˚C for 15 minutes which results into partial
enhances the enzyme activity and do not affect at the level of gene expression. coagulation of proteins. The oxalic acid formed in the fermentation process is
The presence of an excess amount of phosphate source in medium can decrease removed by increasing the pH with calcium hydroxide at 72-75˚C. Calcium
the carbon dioxide fixation which results into certain sugar acids formations and oxalate is further precipitated and eliminated by centrifugation or filtration
the stimulation of growth (Grewal and Kalra, 1995; Vandenberghe et al., process. Citric acid remains in solution in the form of calcium salt (mono-
1999; Soccol et al., 2006). It was observed that a strain of A. niger produced a calcium citrate). Further, extraction of citric acid is carried out by various
low amount of citric acids in submerged fermentation conditions. It was seen techniques such as precipitation, solvent extraction, adsorption/absorption on ion
that higher yield of citric acid was obtained under surface culture conditions exchange resin, electro-dialysis, ultra/nano-filtration or liquid membranes.
when compared with submerged conditions. This clearly revealed that citric acid
production is dependent on an appropriate supply of oxygen and surface area to Precipitation
volume ratio. It was seen that addition surface area provided to growth of
microorganisms, thus resulting in high yield of citric acid production It is one of the conventional methods used for the recovery of citric acid.
(Lakshminarayana et al., 1975). The surface area to volume ratio is one of the Equivalent amount of lime is added to the solution of mono-calcium citrate in

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order to precipitate it as tri-calcium citrate. The efficiency of the process depends processes are found highly efficient in separation but more energy consuming.
on several parameters such as citric acid concentration, pH, temperature and rate The membranes used in this technique are also expensive (Li et al., 2016).
of lime addition. Pure and large crystals are formed after milk of lime (180-250
kg/m3) is added gradually. Temperature needs to be maintained at or around 90˚C Methods of citric acid estimation
with pH 6. The process requires presence of more than 15% of citric acid
concentration within the solution. Due to the solubility of citric acid, the minimal The amount of citric acid in the filtrate was estimated by traditional method of
expected loss is ≈ 4-5% (Soccol et al., 2006). The precipitated calcium citrate is titration. In this method, citric acid was measured by titration with 0.1 N NaOH
washed with hot water (90oC) to remove impurities such as saccharides, chlorides against phenolphthalin as an indicator. The end point in this method was pink
and colored substances. Further, calcium citrate is treated with sulfuric acid and color. The yield percentage of citric acid was further calculated (Priede and
calcium sulfate precipitate formed (gypsum) is eliminated. After elimination of Latvian, 2005).The citric acid was also determined by spectrophotometrically by
precipitate, remaining solution contains ≈ 25-30% of citric acid. Further, the acetic anhydride-pyridine method and (Miller, 1958), gas chromatography
purification is carried out by using activated charcoal or ion exchange columns. (Jham et al., 2002), high-resolution nuclear magnetic resonance spectroscopy
Thus, purified solution is then concentrated in vacuum evaporator below 40˚C to (del Campo et al., 2006), high performance liquid chromatography (HPLC)
avoid caramelization and finally dried to obtain citric acid crystals. Two forms of method (Wang et al., 2017).
citric acid can be obtained during crystallization process. Citric acid anhydrate
form is obtained when temperature dependent crystallization process is CONCLUSION
performed above 36.5˚C. However, citric acid monohydrate form is obtained
when crystallization is carried out below 36.5˚C. The optimum temperature to Citric acid is one of the extensively used organic acids in many industries.
obtain 100 % yield of citric acid was 50°C for 20 minutes. The citric acid need Biodegradable wastes such as mango peel, orange peels, glycerol, cane molasses,
not undergo any phase transition and the product obtained is of high purity (Li et coconut cake, canola oil and banana peel etc are found to be potential substrates
al., 2016). Disadvantage of precipitation process is that a large quantity of waste for citric acid production. Formation of citric acid as a byproduct will reduce
is generated per ton of citric acid produced (Pazouki and Panda, 1998). waste disposal problems and also reduce the dependency of industry over other
citric acid producers. The biomass generated during citric acid fermentation can
Solvent extraction be effectively utilized for biogas production and also in fertilizers preparation.
Thus, the industry would be benefitted ecologically and economically. Some
Recovery of citric acid by solvent extraction is also commonly used. It is an strains of A. niger are found to be potential organisms for citric acid
extraction process in which solvent used with very low or no solubility in fermentation. Both solid state fermentation and submerged fermentation can be
aqueous phase. The solvent used should have maximum solubility with citric acid employed for citric acid production based on the substrate used. Only constraint
and fewer amounts of impurities. Each solvent used for extraction is which the industry needs to overcome is studying the actual mechanisms
characterized by its equilibrium distribution coefficient which is defined as the involved in A. niger for citric acid production. Therefore, designing of
ratio of acid concentration of the extract to the acid concentration of the aqueous appropriate bioreactors with precise control over process parameters is thus of
phase (Kristiansen and Sinclair, 1979). Solvent systems such as mixture of n- high importance. Significant efforts are needed towards improvements in scale up
octyl alcohol, tridodecylamine and isoalkane (Soccol et al., 2006), Alanine 336 in process. Elimination of such obstacles will be economically advantageous to the
heptane or xylene (Sirman et al., 1990), mixture of butylacetate and N, N- industry.
disubstituted alkylamide (Yi et al., 1987) have been used. Amine extraction has
been found to be a prospective method of separation of carboxylic or Conflict of interest: All authors have no conflict of interest.
hydroxycarboxylic acid from aqueous solution. The citric acid is recovered by
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