Production of Oxalic Acid by Aspergillus niger
using Chlorella Vulgaris Grown with an
Industrial Effluent as a Potential Feedstock
Chioma DM* and Agwa OK
Department of Microbiology, University of Port Harcourt, Nigeria
Submission: May 03, 2018; Published: July 02, 2018
*Corresponding author: Chioma DM, Department of Microbiology, Faculty of Science, University of Port Harcourt, PMB 5323 Port Harcourt, Rivers state, Nigeria, Tel: +234-8035757992; Email: email@example.comfirstname.lastname@example.org
How to cite this article: Chioma DM, Agwa OK. Production of Oxalic Acid by Aspergillus niger using Chlorella Vulgaris Grown with an Industrial Effluent
as a Potential Feedstock. Curr Trends Biomedical Eng & Biosci. 2018; 16(1): 555928. DOI: 10.19080/CTBEB.2018.16.555928.
The potential of Chlorella vulgaris as a feedstock for production of oxalic acid through fermentation process was investigated. Chlorella vulgaris was obtained by blooming in 60:40 industrial effluents and freshwater under natural illumination by submerged fermentation at a retention period of 14days. The Aspergillus niger spores were standardized and inoculated into the algal formulated medium for oxalic acid production and the oxalic acid produced was assayed using gas chromatography-mass spectrophotometry. Result showed that the total carbon, nitrogen and phosphates content of the algal slurry were 9.85ppm, 18.73ppm and 1.75ppm respectively. The optimum temperature for the production of the acid was 30 °C and pH 6 respectively. The GC-MS result revealed that the algal formulations produced 8.48mg/g of oxalic acid. This work indicates that algal formulations would serve as a cheaper substrate for oxalic acid production and recommended for industrial application.
Oxalic acid is a soluble, dicarboxylic acid with the formula, H2C2O4 which can also be called ethanedioic acid. Oxalic acid has wide applications in food as a preservative in post-harvest ripening of banana and can serve as anti-browning agent , agricultural and textile industries. Its dissociation in bioleaching of iron and different metals has made it important in hydrometallurgy ; a major cleansing agent  and has been used to remove rust from pipes . Conventionally, most oxalic acids are synthesized by chemical processes which include oxidation of olefins and glycols, oxidation of carbohydrates with trioxonitrate (v) acid, decomposition of formates followed by tetraoxosulphate (vi) acid treatment . Unfortunately, these chemical processes have been reported to be eco-harmful thus, the need for a more sustainable and environment-friendly approach.
Biosynthetically, oxalic acid can be produced by certain microorganisms, plants and animals . Some microorganisms that have produce oxalic acid include: Aspergillus ficuum ; Glyophyllum trabeum ; Paxillus involutus ; Penicillium oxalicum  and Aspergillus niger . Among these microorganisms, A. niger has been reported to give the highest
amount of oxalic acid, hence its preference over other isolates. The organism is generally accepted because of ease of handling, rapid growth and ability to ferment versatile cheap raw materials [9,10].
In a bid to produce oxalic acid from cheap substrates, a variety of substrates such as: milk whey ; cashew apple juice [5,9]; molasses ; sweet potato hydrolysate  and corncobs have all been investigated. However, no research on oxalic acid production has been conducted with algal biomass slurry as source of carbon. Chlorella sp. is a microscopic cell (<10μm diameter) with physiological characteristics similar to plants , an eukaryotic green microalga which replicates, grows optimally in aerated and well humified environment . Chlorella vulgaris can be cultivated by photoautotrophic, heterotrophic and mixotrophic methods and can be used as animal feeds ; in the pharmaceutical, agricultural and nutraceutical industries as food for man. Hence, it can serve as a good nutrient source for fermentation processes. This study was conducted to assess the potential of Chlorella vulgaris biomass for oxalic acid production using A. niger in submerged fermentation technique.
The effluent and freshwater samples for the algal cultivation
were obtained from a food industry within Choba, Port Harcourt,
Rivers State Nigeria. The samples were filtered using Whatman
filter paper, sterilized and stored in a refrigerator at 4 °C until
The microalgae used in this study were obtained from
the department of microbiology, University of Port Harcourt,
Nigeria. A pure culture of the organism was obtained by
repeated sub-culturing of the isolate on nutrient agar using
spread plate technique, a mixture of chloramphenicol (62.5μg.
ml) and nystatin (100μg/ml) was added to the culture medium
to ensure free fungal and bacterial cultures. The algal strain
was selected after preliminary screening using biochemical and
morphological characteristics and was finally maintained on
agar slants until when required .
Five milliliters of the bloomed culture was aseptically
inoculated into flasks containing effluent-and freshwater
medium. About 1ml of the bloomed culture was inoculated
into a defined synthetic medium (0.132g/L Potassium nitrate,
0.066g/L sodium silicate, 0.066g/L monosodium phosphate and
0.066g/L EDTA. The pH of the medium was adjusted to 6.5 with
4M NaOH prior to autoclaving at 121 °C for 15 minutes . The
setups were maintained at 28±2 °C under natural illumination
and aerated intermittently by shaking at interval for 14days.
Samples were periodically removed every 48h to monitor
changes in algal concentration, optical density and biomass as
dry weight was determined.
The American and Public Health method were used
to determine the chemical composition of the effluent. All
experiments were designed in triplicates. The Statistical
Software of Statistical Package for Social Sciences (SPSS) was
used for the statistical analysis. The Posthoc test was used to test
for the significant difference at p-value<0.05 within the group
measured at 95% confidence level.
The oxalic acid producing strain of Aspergillus niger
used in the study was locally sourced from the department
of Microbiology, University of Port Harcourt, Choba, Nigeria.
Spores of A. Niger were grown on Potato dextrose agar (PDA)
for 5-7days at 30 °C. Afterwards, the spores were aseptically
transferred into 100ml sterile distilled water flask .
A slight modification of Emeko et al.  was adopted for
the medium formulation and fermentation study. The medium
consisted of 50g of algal slurry, 1.6g/L of yeast extract,
0.025g/L of yeast extract, 0.025g/L of MgSO4.7H2O and 0.5g/L
KH2PO4. The medium was adjusted to pH 6.0 using 4M NaOH
solutions prior to sterilization. In this work, the oxalic acid
produced was measured using the gas chromatography-mass
spectrophotometry and the catalytic kinetic spectrophotometry
documented by Jiang et al. .
The nutrient index observed in this investigation have
been shown to be necessary for the growth of micro algae. The
dissolved oxygen for industrial effluent was 2.87 0.03ppm while
that of the freshwater was shown to be 5.17 0.04ppm and there
exists a significant difference between samples. The pH of the
industrial effluent and freshwater samples were observed as
follows 7.75±0.01 and 6.89±0.03 respectively and the result
indicates existence of a significant difference between both
samples and regulatory standard (FEPA) of pH 6.0 as shown in
Table 1. The salinity for effluent and freshwater samples were
reported to be 35.65±0.78ppm and 252.5±3.4ppm and this
varied from FEPA standard of 90ppm. The phosphate contents
of the effluent and freshwater samples were 18.6c and 2.15, the
values of the samples varied from the FEPA standard of 7.0. The
optical density of Chlorella sp. revealed an increase from 0.145
abs at 620nm to 0.789 abs at 620nm after 14days of culture
while the cell dry weight also increased from 0.112mg/10ml
to 0.267mg/10ml as shown in Figure 1 & Figure 2 respectively.
Table 2 below shows the physico-chemical characteristics of the
algal biomass slurry used in this work. The result shows a high
concentration of calcium (19.1mg/g) and total nitrogen content
Furthermore, the study revealed that the best conditions for
oxalic acid fermentation were pH 6 and 30 °C as both conditions
gave the highest oxalate concentrations of 10.55ppm and
8.98ppm respectively after 10 days of fermentation as seen in
Figure 3 and Figure 4. The final oxalic acid produced at 30 °C
and pH 6.0 was reported to be 8.48mg/g with retention time of
12.621. Other organic acids like pyruvic, citric, malonic acids
were also synthesized along the oxalic acid.
Effluent treatment and disposal especially from industrial
activities have become a huge challenge to manufacturing
processes all around the world. Stiffer limits have been proposed
to both safeguard the discharge of harmful materials into
ecosystems and possibly reuse them for industrial benefits. The
growth of Chlorella sp. using industrial effluent medium revealed
inherent micronutrients found within the effluent proved its
suitability as growth medium. The report concurs with Grima et
al.  who showed that microalgae can be grown in sea water
supplemented with phosphates and nitrates. Amanullah (2007)
& Iyoyo et al. (2010) opines that most microalgae can be grown
with wastes rich in phosphorus and nitrogen. These nutrients
innate within the waste were necessary for the growth of the
organism . Poultry waste extracts and industrial dairy waste
have also been shown to support the cultivation of microalgae
[17,20]. Enitan et al.  observed that effluents could be
applied to the production of a wide variety of bioactive materials
as a cheap source of nutrients. These findings all agree with
the need and viability of microalgae cultivation with cheap and
inexpensive materials (Figure 5).
The reportedly low pH levels in the effluent in this study
could suggest a lower carbon concentration, in the form of
carbonates and bicarbonates. Conversely, Roselin (2015)
reported that pH of sewage water could range from 6.3-7.3. The
increased nitrate level in the effluent suggests the presence of
high nitrogen-containing compounds in the wastewater. The
nitrate levels reported in this work also disagrees with Ahmad
et al.  who reported a higher nitrate content of 1.19ppm.
However, the nitrate content reported in this work is similar to
that of Nwosu et al.  that reported a nitrate concentration
of 0.68 3ppm from a food factory during the rainy season and
higher than the nitrate content of 0.106 (Figure 6).
It has been well reported that fermentation medium for
oxalic acid production must contain carbon and nitrogen sources
. Algal starch has been reported to be readily used by yeast
via fermentation for the production of ethanol and organic acids
[24,25]. The biochemical composition of algae is a function of
the species, temperature, light and growth stage. A variation in
the biochemical composition due to growth stage is frequently
affected by the stage of the culture and nutrient exhaustion
. Basically, algal cultures become depleted in nutrients as
they enter stationary stages of growth, while protein content
declines, total carbon content increases [27,28].
The study reported a high concentration of total nitrogen
content (18.73ppm) in the algal slurry which is in contrast with
the report of Dineshkumar et al.  on algal biomass slurry.
The difference could be attributed to the difference in nature of
biomass used in the investigation. Dineshkumar et al.  also
reported a higher carbon concentration of 20.42±0.33mg/g on
dry biomass. This disparity could be as a result of the difference
in the algal growth medium. Furthermore, Giselle et al. 
reports a carbohydrate content of 7.09±0.84 and protein content
of 6.07±1.14 in Chlorella sp that has attained stationary growth
phase. This study also differs with Lum et al. 2013 who reported
12-17% carbohydrates dry weights, 14-22% lipids and 51-58%
proteins in Chlorella sp. The disparity could be attributed to the
fact that the microalgae were possibly in the exponential phase of
growth. Also, factors such as environmental factors, nutritional
factors and protein production can affect the carbohydrate
content and ultimately total carbon content in microalgal species
Abdelkhalek et al. .
The optimum pH and temperature reported in this work
agree with Rujiter et al. ; Mandal & Banerjee [4,33]. The
yields reported in this work do not corroborate with Betiku et al.
 who reported an oxalic acid yield of 38mg/g from molasses
and sweet potato starch hydolyzate with oxalic acid yield of
1,038mg/g respectively. This disparity could be attributed to
the choice and availability of the feedstock for metabolism by
the fungus. Furthermore, the relative reduced yield in this
work could also be linked to the non addition of methanol
to the fermentation medium. Betiku et al.  reported an
increased yield of oxalic acid after addition of 1% methanol to
the fermentation medium. In addition , Cameselle et al. 
reports that one of the challenges with oxalic acid formation via
fermentation is the simultaneous production of gluconic at pH 7,
thus, recommends a strict control for the medium environment
for optimum production (Figure 7).
The prospect for submerged fermentation using Chlorella
vulgaris biomass as feedstock for oxalic acid production was
explored in this study [35-40]. The results showed that algal
biomass had the necessary nutrients for oxalic acid production
at pH 6 and temperature of 30 °C for 10days [41-45]. The oxalic
acid concentration reported at the end of the study period was
8.48mg/g. Algal biomass remain an untapped resource for the
production of industry-important substances .