Wignyanto Dept. Agro-Industrial Technology Brawijaya University

Industrial Fermentation Batch-fed and continuous fermentation processes are common In the batch-fed process, sterilized nutrients are added to the fermenter during the growth of the biomass. In the continuous process, sterilized liquid nutrients are fed into the fermenter at the same flow rate as the fermentation broth leaving the system. This will achieve a steady-state production. Parameters like temperature, pH, oxygen consumption and carbon dioxide formation are measured and controlled to optimize the fermentation process.

Major Industrial Fermentation Products Non-Food Applications Antibiotics Vitamins Amino Acids Food Applications Organic Acids Enzymes Polysaccharides Oils and Fatty Acids Colors Flavors

Organic acids Used as food acidulants Most versatile ingredients in industry because they are: – Soluble and hydroscopic – Buffers and chelators Organic acids produced by fermentation and commonly used as food acidulants include: – Citric – Lactic – Gluconic – Propionic

What is Citric acid? Produced by several molds and bacteria froma variety of substrates 2-hydroxy-1, 2, 3-propane tricarboxylic acid first isolated from lemon juice Natural intermediate in Krebs cycle Naturally non-toxic due to its widespread presence Pleasant taste and property of enhancing existing flavours have ensured its dominance in industry produced in anhydrous or monohydrate 270,00 tonnes worldwide/year=$ 1.4 billion produced in anhydrous or monohydrate 270,00 tonnes worldwide/year=$ 1.4 billion Andhydrous form is obtained by crystallization from hot aqueous solutions. Monohydrate is obtained by crystallization at temperature below 36.6°C Andhydrous form is obtained by crystallization from hot aqueous solutions. Monohydrate is obtained by crystallization at temperature below 36.6°C food, confectionery and beverages (75%) pharmaceutical (10%) industrial (15%) food, confectionery and beverages (75%) pharmaceutical (10%) industrial (15%)

Stabilizer of oils and fats, ascorbic acid.Emulsifire in processed foodstuffs, e.g. cheese.Mixtures of citric acid and its salts have good buffering capacityMonostearyl citrate as an antioxidant in oils and fats Citric acid esters as non-toxic plasticizers in plastic films used to protect foodstuffs Citric Acid Function

Methods for Citric Acid Production Submerged or surface fermentation process, batch fermentation Production media: Beet molasses or glucose syrup as raw materials Nitrogen added as ammonium nitrate or sulphate Metals are often removed from raw material as high levelsof iron appear to inhibit citrate production Fermentation: Inhibition of formation of long hyphae (which would resultin dramatic increase of viscosity of fermentation medium) Low pH (pH is controlled at 2.2 –2.6 by addition of NH3), dissolved oxygen concentrations, and temperature controlare important

PRODUCTION PROCESSES- FERMENTATION A. niger – (i)Surface fermentation using beet molasses – (ii) submerged fermentation using beet or cane molasses or glucose syrup. Submerged processes using sucrose as carbohydrate source are also belived to be running in areas where sugar is cheap Yeast – Submerged fermentation using beet molasses or glucose syrup Over production of citric acid in A. niger requires several pathways and pathway modifications: High flux of metabolites through glycolysis, glucose transport Block of TCA cycle reactions that degrade citrate Over production of citric acid in A. niger requires several pathways and pathway modifications: High flux of metabolites through glycolysis, glucose transport Block of TCA cycle reactions that degrade citrate

Schematic: Citric Acid Fermentation

Continuous Production of Citric Acid from Dairy Wastewater Using Immobilized Aspergillus niger ATCC 9142 Se-Kwon Kim Pyo-Jam Park Hee-Guk Byun

In addition, whole metabolic pathway of microbe or part of it required for the production of a particular compound, can be efficiently used over a longer period under immobilized state. Immobilized Biocatalyst Technology a means to utilize  as efficient and heterogeneous catalyst for a multitude of industrial and medical applications a means to utilize  as efficient and heterogeneous catalyst for a multitude of industrial and medical applications Enzymes Whole cells & found to be more advantageous than enzymes as in situ regeneration of activity is merely by supplementing suitable nutrients

produce Modern Dairies Very high quantities waste (thousand cubic meters / day) Very high quantities waste (thousand cubic meters / day) which is High concentration in organic matter Problem on sewage treatment systems Main organic load contributor of lactose, fats, and proteins originated from milk PURPOSE: Examine the potential of dairy wastewater as “a source for citric acid production by Aspergillus niger via a continuous reactor as well as to study the effect of various fermentation parameters, such as the dilution rate, pH, and temperature” PURPOSE: Examine the potential of dairy wastewater as “a source for citric acid production by Aspergillus niger via a continuous reactor as well as to study the effect of various fermentation parameters, such as the dilution rate, pH, and temperature”

MATERIALS AND METHODS Microorganism and Inoculum Preparation – Aspergillus niger ATCC 9142 (American Type Culture Collection, Rockville, MA, USA) Medium – The dairy wastewater contained about 2.5 % reducing sugar mostly as lactose that had moisture rate of 97%. – The total reducing sugar was concentrated up to 5 % for the experiment, and sterilized at 121 o C for 20 min. – To remove protein and lipid, the initial pH was identified as 4.3 by 4 N of HCl, and carried out the activating charcoal treatment. Cell Immobilization – The cell immobilization for the continuous flask culture was achieved by putting 4 % sodium alginate (cell : alginate = 1 : 10, w/w) into ATCC 9142 strain growing in a 500 mL flask at 30oC for 72 h, then the cell-alginate mixture was extruded into a 2% CaCl2 solution through a needle. The Ca-alginate beads with entrapped cells of Aspergillus niger exhibited a typical shrinking behavior, and the sizes of the beads were from 2.5 mm to 3.5 mm particle diameter.

Bioreactor and Fermentation The Ca-alginate beads entrapped with immobilized cells were packed up to 30% in the column. The shake-flask experiments were performed in 500 mL Erlenmeyer flasks containing 150 mL of the medium mixture (bead volume : medium volume = 1 : 2). The flasks were incubated at 30 o C on a rotary shaking incubator 200 rpm total working volume was 250 mL (300 mm length and 25 mm diameter) operation temperature by pumping temperature-controlled water through a jacket a positive displacement peristaltic pump was used to vary the liquid feed flow rate

Analytical Techniques The citric acid  colorimetric method of Marrier and Boulet The reduced sugar  colorimetric method with 3,5-dinitrosalicylic acid (DNS) as the color reagent All analyses were performed in duplicate.

Effect of pH on Citric Acid Production The pH of the substrate is an important factor that affects the performance of dairy wastewater fermentation Citric acid concentration and yield were highest at pH 3.0 consumption of sugar was highest at pH 2.0 pH did not directly influence the citric acid production mechanism but rather affected the enzymes which were active in degrading the substrate and/or the permeability of the cell membrane of the substrate and product pH did not directly influence the citric acid production mechanism but rather affected the enzymes which were active in degrading the substrate and/or the permeability of the cell membrane of the substrate and product In general, a low pH is essential for achieving the maximum production of citric acid Low initial pH has the advantage of checking contamination and inhibiting oxalic acid formation. In general, a low pH is essential for achieving the maximum production of citric acid Low initial pH has the advantage of checking contamination and inhibiting oxalic acid formation.

Effect of Temperature on Citric Acid Production Temperature is also an important factor for citric acid production. The maximal citric acid production was obtained at 30 o C Higher temperatures  very rapid process & abundant mycelial growth = consuming large amounts of sugar thus lowering the yield of citric acid Lower temperatures  higher yields of citric acid are possible, by prolonging the fermentation process

Effect of Dilution Rate on Citric Acid Production The dilution rate is the medium flow rate per effective reactor volume The optimum citric acid productivity and yield were 160mg L -1 h -1 and 70.3% at a dilution rate of h -1

The current study attempted to improve the citric acid productivity by a continuous fermentation process using calcium-alginate immobilized cells of Aspergillus niger in a bioreactor Major Objective Of Cell Immobilization Technology Major Objective Of Cell Immobilization Technology achieve a reactor system with a high productivity that can be maintained for extended time periods Material should be inexpensive Actual immobilization procedure and subsequent reactor operation should be simple

Comparison of Citric Acid Production between Shake-Flask and Continuous Fermentation In a batch shake-flask fermentation, the productivity (63.3 mg L -1 h -1 ) and yield (51.4%) of citric acid reached a maximum after 3 days, and the citric acid concentration and residual sugar concentration after 20 days were 1.38g/L and 8.85 g/L, respectively. Meanwhile, the yield (70.3 %) of continuous citric acid production when using immobilized Aspergillus niger reached a maximum level after 9 days (Fig. 5). In addition, the productivity value (160.0 mg L-1 h-1) was twice that of the batch shake-flask culture (Table 3)

Conclusion important aspects of citric acid production from dairy waste-water by immobilized Aspergillus niger : – The optimum conditions were pH, 3.0 – temperature, 30oC – dilution rate, h-1. In addition, the productivity of citric acid by the immobilized Aspergillus niger in a continuous reactor was more than two-fold higher than that in a the shake-flask culture. The results suggest that the bioreactor used in the current study could be potentially used for continuous citric acid production from dairy wastewater by calcium-alginate immobilized Aspergillus niger.

Citric Acid: Downstream Processing Three separation methods – Direct Crystallization Most successful with highly refined raw materials – Precipitation as calcium citrate tetrahydrate Dominant process Calcium hydroxide is added to filtrate – Liquid extraction Suspended particles are removed under vacuum Precipitate is washed to remove impurities Solution is concentrated using evaporators Fed to a crystallizer, Crystals arecentrifuged, dehydrated, and ground

Citric Acid Plants

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