Short CommunicationEnhanced malic acid production using Aspergillus niger coupled with in situ product recovery
Introduction
Currently, the quest for renewable fuel production has received massive attention due to the major concern on depletion of nonrenewable fossil fuels. The progress of industrialization including transportation depends on the fossil fuels as the source of energy. However, excessive usage of fossil based fuels creates environment pollution, increased demand for fuel and biotic health problem (Yin et al., 2020). It is also projected that the exhaustion of fossil fuels could appear within 100 years (Liguori and Faraco, 2016). In order to meet such energy crisis, production of alternative renewable fuel is unavoidable. Biofuel serves as best promising fuel and about 82% of total biofuel is represented by biodiesel. Importantly, the growth of biodiesel production industry is increasing extremely in order to combat with traditional fossil fuel industry (Kumar et al., 2019). Biodiesel production principally relies on the process through transesterification of triglycerides which are derived from various sources like animal fats, vegetable oils and algal oils. Process consisting of 100 kg resulted in the production 90 kg of biodiesel and 10 kg of crude glycerol (Vivek et al., 2017).
Recently, crude glycerol has been reported as a very good source of carbon for bioconversion. Crude glycerol was used as substrate for the production of biobutanol (Xin et al., 2017), 1,3-dihydroxyacetone (Dikshit et al., 2017), citric acid (Rzechonek et al., 2018) and succinic acid (Sadhukhan et al., 2016). Glycerol serves as carbon source that are utilized by the microorganisms. When compared with glucose, glycerol has increased degree of reduction (Garlapati et al., 2016). The availability of various impurities present in the crude glycerol is the major task. Hence, various pretreatment methods were proposed to remove the contaminants present in the crude glycerol (Vivek et al., 2017). But the processes should be considered with devoid of pretreatment methods in order to attain cost effective methods.
Malic acid is a highly versatile product with various industrial applications such as acidulant and preservative in food industry, feedstock for polymer synthesis, bioavailability of calcium in the form of calcium malate and as antimicrobial agent (Chi et al., 2016, Iyyappan et al., 2019a). Currently, the commercial production of malic acid is performed using petro chemically derived maleic acid or fumaric acid as the feedstock and accordingly hydration method is carried out by means of chemical synthesis (Wang et al., 2016). Furthermore, environmental issues occur due to usage of petro chemically derived fumaric acid and maleic acid. Hence, there is a need for malic acid production using fermentation methods from cost effective substrates like crude glycerol.
The major limitation of organic acid like malic acid production through fermentation is product inhibition which affects the product establishment. In-Situ Product Recovery (ISPR) enables recovery from fermentation broth with simultaneous reduction in product inhibition. Consequently, the growth of the microorganism is not affected due to the recovery of product from fermentation broth and productivity can be increased using ISPR (Van Hecke et al., 2014). Adsorption, extraction and precipitation are the some of the organic acid recovery methods. Extraction of malic acid can be performed by reactive extraction method, ion-exchange chromatography and aqueous two phase systems (Gao et al., 2012, Lopez-Garzon and Straathof, 2014). Previous studies were performed to separate malic acid using model (aqueous) solution to validate the efficiency of malic acid separation methods. With these backgrounds, the present study was focused on the liquid-liquid extraction of malic acid from the fermentation broth containing crude glycerol using Aspergillus niger at the same time as when it is produced.
Section snippets
Microorganism and culture conditions
Aspergillus niger PJR1, a previously generated mutant strain was used in this study for malic acid production (Iyyappan et al., 2018a). The strain was cultivated in potato dextrose agar (PDA) medium (HiMedia, India) and incubated at 25 °C for 168 h. The seed culture development was performed by the same method as mentioned by Iyyappan et al., (2018b).
Biocompatibility test
Screening of solvents was performed with Aspergillus niger PJR1 using shake flask studies. Shake flasks containing 90 mL of statistically
Biocompatibility test of various solvent mixtures on A. niger
Octylamine, dioctylamine and trioctylamine in two solvents namely 1-octanol and n heptane were selected for toxicity test on A. niger PJR1. The selection of solvent mixture was based on the literature reports (Kaur and Elst, 2014), in which these solvent mixtures were used as the reactive extraction system for malic acid. Totally six organic mixtures namely octylamine in 1-octanol, octylamine in n-heptane, dioctylamine in 1-octanol, dioctylamine in n-heptane, trioctylamine in 1-octanol and
Conclusions
In-situ product recovery was applied to overcome the problem of product inhibition for malic acid production. Trioctylamine in 1-octanol was employed for in-situ recovery of malic acid. During batch extractive fermentation, malic acid titer and productivity were reached 115.67 ± 3.5 g/L and 0.5 g/L.h, respectively. Fed-batch extractive fermentation resulted in malic acid titer and productivity of 131.48 ± 3.4 g/L and 0.45 g/L.h, respectively. Further, continues malic acid production should be
CRediT authorship contribution statement
J. Iyyappan: Investigation. G. Baskar: Writing - review & editing, Supervision. B. Bharathiraja: Conceptualization, Methodology, Writing - review & editing, Supervision. M. Gopinath: Validation.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
This work was financially supported by grant from Department of Science and Technology, Science and Engineering Research Board (SERB), India (No. EEQ/2017/000200). The authors thank DST-SERB, India for granting financial support for this work.
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