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Citric acid (CA) is a weak organic acid
found naturally in the citrus fruits with a wide range of applications,
including, preservation, flavor enhancement, bacterial inhibition, pH
regulation and as an antioxidant. The main goal of this work was to produce
citric acid from Ethiopian sugar cane molasses using an isolated fungal species
via submerged fermentation process. The organism was identified as Aspergillus niger by 16S rDNA
sequencing. In this study, the sugar cane molasses after dilution, is pretreated
with  35 mL of 1N H2SO4 per liter, boiled, cooled, neutralized
with CaO and clarified prior to fermentation. The effect of pH (5, 7 and 9) and
the fermentation time is recorded during the process and the yield of citric
acid by the fungal species is determined.

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Aspergillus niger,
16S rDNA, Cane Molasses, Citric Acid, Submerged Fermentation


cane molasses is a black, viscous liquid by-product of refining sugarcane into
sugar. It is  used as a substrate for the
production of ethanol and citric acid due to its high reducing sugar content
which is about 62%. This content may vary based on the amount of sugar, method
of extraction and age of plant (cane). Citric acid (CA) has more health and
economic benefits 1. They exist in two forms,
monohydrous and anhydrous, which differs in their degree of hydration 2.Conventional citric acid production is carried out
in three ways, from the citrus fruit extracts, chemical synthesis and by using
carbohydrate source material such as sugar cane molasses, and other fruit peels. 

all the methods, fermentation is the most economical method for producing
citric acid and is a predominant way of producing of citric acid as it accounts
for about 90% of world production. Though all the three types of fermentation
processes have been widely employed, including the submerged, surface
fermentation and solid state fermentation 3,7,
in the present study submerged fermentation was used to produce citric acid due
to the liquid nature of the substrate, the sugar cane molasses. Submerged fermentation
technology has been reported by several workers as an
attractive process to produce citric acid due to its advantage of low cost,
short period and high yield. There are many sugar manufacturing industries in
the country, which provide cheap and ample cane molasses making the production
of citric acid reliable and consistent. The most important aspect  during the citric acid production is the
clarification of the raw molasses that comes from the sugar milling factories
and its pretreatment with sulfuric acid to form complex compounds of trace
elements  in the molasses and thereby removing
niger is one of the most common species used to utilize starchy and sugar
substrates like molasses and converted into different products . It is the most
promising microorganism used in production of citric acid compared to other
microorganisms due to its ability of utilizing starchy and sugar substrates 5. The production of citric acid using submerged
fermentation depends strongly on an appropriate strain and on operational
conditions such as aeration, type and concentration of carbon source, nitrogen
and phosphate limitation, pH, concentration of trace elements and morphology of
the fungal species.

2. Experimental section

Microorganism-Inoculation and culture conditions

fungus used in this study was obtained from the Ethiopian Biodiversity
Institute, Addis Ababa, Ethiopia.  The isolates were initially grown on
Potato Dextrose Agar at 328 K for 24 h. The fungus was further sub-cultured on
the Potato Dextrose Agar at regular intervals and incubated at 313 K.  For further propagation, the minimal medium composition employed in mg/L was,  0.223 NH4NO3,
0.1 K2HPO4, and 0.023 MgSO4.7H2O. Cane
molasses was employed as the chief carbon source.
All  the submerged fermentations were
carried out using the isolated fungus.  The strain was maintained on PDA slants at 30°C for 6 d.
The strain was prepared as conidial suspensions by washing slant cultures with
5 mL sterilized water. Spore suspension was counted at 25×106
spores/mL by Haemacytometer.  All the trials were carried out in 250 mL
Erlenmeyer flasks containing 100mL of molasses, at pH between 5 and 96. The flasks were
sterilized prior to the inoculation with three milliliters of prepared spores
and incubated at a temperature of 28°C on a shaker incubator at100 rpm for 11
days in succession.

2.2. Feedstock-Molasses

Cane molasses contains the following contents: water,
20%, sugar, 62%, and non-sugar, 10%, and inorganic salts (ash contents), 8%. It
is a blackish homogenous liquid with high viscosity20. Ash contents include
ions such as Mg, Mn, Al, Fe and Zn in variable ratios. Sugar content was
diluted to 24% with  deionized water. This
was followed by the addition of 35 mL of 1N
H2SO4 per liter, boiling for half an hour, cooling
and neutralizing using the lime-water (CaO). The contents were then left to
stand overnight for clarification 7. The
clear supernatant liquid was further diluted to 12-24 % sugar using deionized

2.3. Process
variables for citric acid production

            Citric acid
production by fermentation can be divided into three phases, which includes
preparation and inoculation of the raw material, fermentation, and recovery of
the final product. After the pretreatment of the molasses,
nutritive salts (like ammonium nitrate) are added and it is diluted with
distilled water to make a solution 8. This solution
is sterilized and after cooling down to 30°C, it is transferred to a sterilized
submerged fermenter and inoculated with the spore suspension of Aspergillus niger.  pH of the substrate (solution) was adjusted
to 5.5-5.9, and the temperature 28°C, the most suitable for the germina­tion of
the conidial aggregation. This was followed by the addition of nutrients and
microorganism to the bioreactor. Then the parameters such as pH and temperature
were adjusted to an initial value of 2.5-3.0 and 28°C respectively. The fermenter
is then aerated, when a thin film of mycelium is created in 24 h (germinating
period). This was followed by the production period, when the mycelium gets
stronger, and the temperature of the fermented solution rises9. During this phase, the exothermic citric acid
production is initiated by the environment.

3. Results and discussions

3.1. Microscopic observation and
molecular testing

The isolate was identified based on the colony
morphology, microscopic observation and molecular identification 12. The fungus was identified as Aspergillus niger based on the
production of clear carbon black /brown spores from the biseriate phialides. The
colonies were fast-growing, whitish to blackish or brownish, and usually thick
13. The morphology of the isolate was
examined on potato dextrose agar (PDA in the dark. Colonies on PDA were fast
growing with sporangiophores measuring 2.3 to 3.6 mm long and 1.2 to 2.9 mm
wide after 3 days. DNA was extracted from the hyphae of a 36 h culture on PDA slants
and suspended in UltraPURE distilled water in 2 ml Eppendorf tubes, each
containing one sterile 4.5-mm steel shot pellet.

3.2. Fungal strain identification

For the nucleotide sequence analysis, fungal genomic
DNA was purified using the Fungi Genomic DNA Isolation Kit (MTK 08) (Modern
Science Co., Nasik). The fungal primer pairs annealing at the 50 and 30 end of
respective-ly, were used for amplification. The PCR was run for 35 cycles in a
DNA thermal cycler (Thermal Cycler Applied Biosystems 2720, USA). Amplified PCR
products were then analyzed in a 1% (w/v) agarose gel and purified. Purified
products were cloned and subsequently sequenced using an automated DNA
sequencer (ABI 3130 Genetic Analyzer, USA). The 16S rDNA sequence obtained was
compared with the sequence obtained from the nucleotide database of the National
Center for Biotechnology Information (NCBI)12.
The phylogenetic analysis of the strain, using its nucleotide sequence data
showed that this strain had the highest homology of 98% and 99% with the Aspergillus niger strains,  Aspergillus niger SH-2 and Aspergillus
niger ATCC 10864, respectively. Based on the evolution distance and
partial sequencing, this strain isolated was identified as Aspergillus niger.

3.3. Citric
acid production

            After the citric acid production by the submerged
fermentation, the fungal mycelium is separated from the solution, washed to
prevent loss of considerable quantity of acid contained by the mycelium. After
the hydrolysis, the liquid fraction of the hydrolysate samples were analyzed
for their reducing sugar content was determined using the phenol-sulfuric acid
method. The absorption values of the samples were recorded at 490 nm on a
spectrophotometer 3. The acid solution is
then separated from the mycelium and is reconditioned through filtering to
remove the by-products such as oxalic acid. The calcium citrate is precipitated
using lime and then the resulting solution is filtered. The resulting solution
is decolorized batch-wise by the addition of activated carbon. The percentage
of citric acid produced by Aspergillus
niger is then determined by the titrimetric method4.
After the decolorization, the
solution is concentrated
by evaporation and crystallized at a temperature of 22-26°C to form a white
monohydrate citric acid powder as the final product.

3.4. Analysis of total reducing
sugar content

In this study, the amount of
reducing sugar in the molasses was investigated. The total sugar content of molasses
samples was determined using phenol sulfuric acid method. During the phenol
sulfuric acid method, the reducing sugar content in the samples is dehydrated
due to the reaction with sulfuric acid and produced furfural derivatives3. Further reaction between furfural derivatives and
phenol develops a detectible Yellow-Orange color. The concentrations of unknown
sugar content of samples were determined from the standard curve of glucose.
The resulting molasses substrate was subjected to fermatation by the Aspergillus niger.

3.5. Analysis of citric acid

The fermented samples were
subjected to the assay for citric acid by the titrimetric method using
phenolphthalein as an indicator. The
filtrate obtained is titrated against an alkali of known strength using
phenolphthalein as indicator. The end point is the formation of pale pink
color. The volume of alkali used for neutralization is used to find the
normality and the percentage of acid in the sample. In this study, a solution
of 2.1g citric acid per 100ml distilled water is prepared. And from the
prepared solution, 10 mL of solution was pipetted in to a conical flask, with
an addition of 2-3 drops of indicator, titration was carried out against 0.1N
NaOH in the burette till a pale pink color was formed4.
The titration was repeated till expected value was obtained. During the
sampling, a solution was prepared using 5 mL of the sample from each test tubes
of citric acid per 20 mL distilled water. 
From the prepared solution, 10 mL of solution was pipetted into a
conical flask and 2-3 drops of indicator were added and titrated following the
procedure previously mentioned. The percentage of citric acid produced is then



3.6. Effect of pH on the citric
acid production

In this study, a considerable amount of
citric acid was produced using molasses
as a carbon substrate by the submerged fermentation from the molasses
containing about 62% of sugar.  Stoichiometrically,
by the conversion of 100 g sucrose with oxygen, 123g monohydrate or 112g
anhydrous citric acid can be obtained. Biochemical conversion of sucrose into
citric acid by the microorganism is shown below,


During the fermentation process, the pH
of the medium was found to decrease initially at non regular intervals, from
the initial pH of 7.0 to 4.32 on the eleventh day, indicating the acidity of
the media and more evidently the citric acid stable pH (Table
1). Figure. 1 shows the effect of
fermentation pH on the citric acid production.

Table 1. Data collected throughout the fermentation process


weight (g)







































production of citric acid increased exponentially until the day 9, at which
maximum yield was observed. But as the fermentation time exceeded the day 9,
the yield decreased due to the possible conversion of Citric acid into its
byproducts. The production of citric acid was maximum at pH 7, implying the
stability of the product at the favorable pH value of around 710.

Fig.1.  Effect of pH on the citric acid production

the present batch-wise fermentation of citric acid, the production started
after  the lag phase of 1 day and
reached maximum at the onset of stationary phase or late-exponential phase.
Further, it was observed that there was no enhancement in the citric acid
production during the increase in the incubation period. It may be due to the
age of fungus used and depletion of sugar contents in the culture broth. The
acid production was found to start at the initial stage of the idiophase
(between 80-120 h) of fungal growth. At this stage, the pH was five. The
yield gradually increased and reached to the maximum at the late idiophase (180-220 h).
In this stage, the pH must be below 3 in order to suppress oxalic acid and
gluconic acid formation. The acceleration of fermentation can be explained by
the higher starting biomass concentration in fermenter and by the adaptation of
biomass to very high osmotic pressures11.

3.7. Effect of Temperature on
Citric acid production

plays an important role in the citric acid production. Temperature between 25°C
and 30°C is usually employed for culturing the Aspergillus niger. The
optimum temperature for citric acid production is 28°C, but during the citric
acid production, the temperature of the medium increases above 30°C and the
biosynthesis of citric acid decreases. This may be due to high temperature,
which causes the denaturation of enzyme-citrate synthase and accumulation of
other by-products such as oxalic acid. In addition to this, enzyme catabolite
repression, could be a possible inhibitor8.

Effect of fermentation time on the production of citric acid

is another limiting factor in the citric acid production. Nitrogen is usually
supplied in the form of ammonium nitrate, which is completely metabolized
during fermentation periods. Citric acid starts to appear when the nitrogen
concentration falls below a low limiting value6,11.
Figure. 2 shows the effect of fermentation time
on the citric acid production at a pH of 7. Earlier studies have reported that
the factors affecting citric acid production by fermentation includes, the
nutrient composition of the media, environmental conditions, deficiency of
manganese, types and concentration of sugars, chelating effect of metal ions,
ammonium nitrate and aeration10. The optimum
time of incubation for maximum citric acid production varies with the organism
and fermentation conditions used.4. Conclusions

study shows that the concentration and type of molasses, influences the yield
of citric acid produced by Aspergillus
niger. In the controlled production medium, the initial pH of 7 was
found to decrease to 4.32 during fermentation confirming the production of
citric acid. Sucrose in the molasses is the substrate responsible for the citric
acid production in this medium. Aspergillus
niger utilizes sucrose and produces 67.2% citric acid. Based on our
study, the maximum amount of this citric acid was produced in 9 days at the pH


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