5.1 Introduction
The first intensive work on citric acid production by the fungus using the solid state process was that of Lu ( 1 995). A medium prepared from sweet potato (kumara, Ipomoea batatus) was used as the solid substrate for the process which employed A .niger Yang No.2 as the producing organism. Initially the fermentation was conducted in flasks, after which the process was scaled up in various types of reactor. Although representing a systematic investigation, this report provided little detail on the biological aspects of the process. As emphasized earlier, the interaction between the fungus and the solid substrate particles is very complicated. This leads to difficulty in obtaining pure active fungal biomass from the fermented sample and may have contributed to the lack of a biochemical study of the process. Although Lu ( 1 995) was able to determine growth of the organism by measuring the mycelial
dry
weight, the data were achieved indirectly and the procedure was inapplicable for obtaining viable biomass. In the present study, petri-dishes, rather than flasks, were employed as the reactor for performing the fermentation in which the solid substrate was arranged as a layer resembling an agar medium. In this manner, the fungus grew covering the surface of the substrate layer and formed a single piece of mycelial sheet which could subsequently be easily removed for the biochemical assays.This chapter will describe the solid state fermentation for citric acid production using the two high-citric acid-accumulating strains of A .niger, Yang No.2 and MH 1 5- 1 5, and the two low-accumulating mutants, SL- l and SL-2, which had been selected through the mutation programme reported in Chapter 4. All the fermentations were performed in petri-dishes with sweet potato being used as the solid substrate. The
Production of Citric Acid Using A.niger in Solid State Fermentation 73 progress of the fermentation, the kinetic parameters, and the growth morphology of each fungal strain will be described. Furthermore, a comparison between the petri dish and the flask systems for the fermentation using A .niger Yang No.2 wiII also be made. Thus, this investigation provided necessary background knowledge of all the tested strains for further biochemical work.
5.2 Physical Properties of The Solid Substrate
The solid substrate used for the fermentation was prepared from sweet potato, as described i n Section 3.2.4. It was packed in a petri-dish as a layer of about 0.4 em depth. In this manner, the upper surface was the only side exposed directly to the atmosphere. The surface area of the substrate layer was approximately equal to the circular area of the dish (diameter of 9.0 em), which was 63.6 cm2• The den sity of spores inoculated on this surface was about 790 spores/cm2, given that 50,000 inoculated spores were uniformly distributed over the surface.
Since this substrate was prepared from fresh tubers of sweet potato, it would normally vary in water content and nutrient composition. Thus, to diminish this problem and to ensure the reproducibility of the fermentation the substrate was prepared in a large quantity, sufficient for the entire experiment. Before conducting any fermentation, the major physical properties of the substrate were u sually recorded. It was found that the substrate usually contained an initial moisture content of about 70 to 72 % (w/w). The initial starch content varied in the range of 200 to 230 glkg substrate on wet weight basis, as determined by the enzymatic method (Section 3.2. 1 0), with a small amount of free glucose, at 6.0 to 6.4 glkg wet weight of the substrate. The pH before starting the fermentation, measured after diluting 40 g of the substrate with 1 50 ml of water, was between 6.0 to 6.4. These physical parameters of the substrate were in the optimum ranges for initiation of citric acid production, as recommended by Lu ( 1 995).
Production of Citric Acid Using
A.nigerin Solid State Fermentation
745.3 Growth Morphology on Sol id Substrate i n Petri-dishes
The morphological development of each fungal strain during the progress of the
fermentation were observed over a period of 1 0 days. The parameters examined were
intensity of growth, pigmentation and sporulation.
Figures 5 . l a to 5 . l d display the morphology of
A .niger
Yang No.2 during the course of the fermentation. Spore germination was observed after about 1 8 h incubation, followed by a rapid surface extension of the fungal hyphae creating patches of thin mycelium after about 24 h. By 48 h incubation, the entire surface of the substrate was covered with yellow mycelium and conidiophore development could be observed (Figure 5 . l a). Upon further incubation, the mycelial sheet became thicker with patches of black spores expanding with the incubation time (Figures 5. l b,c). By day8, the entire surface of the thick mycelium sheet was covered with black spores (Figure 5 . l d).
Production of Citric Acid Using
A.nigerin Solid State Fermentation
75a. b.
c. d.
Figure 5.1 Growth morphology of
A.niger
Yang No.2 during solid state fermentationfor citric acid production
a. day 2 c. day 6
b. day 4 d. day 8
For the morphological appearance of strain SL- l , there were some variations from that of its parent, strain Yang No.2. In the early phase of growth, the mutant developed a yellow mycelial sheet over the substrate layer. Upon further incubation, the mycelium became thicker, however, no sporulation was observed. B y the end of the incubation, this mutant strain had developed a thick, cream-coloured mycelial sheet, with very short aerial hyphae, indicating incomplete development i nto spores. These morphological descriptions are illustrated in Figures S.2a to S.2d.
a. b.
c. d.
Figure 5.2 Growth morphology of A . niger S L- l during the solid state fermentation
for citric acid production
a. day 2 c. day 6
b. day 4
Production of Citric Acid Using
A.nigerin Solid State Fermentation
77For the morphological development of
A.niger MH
1 5 - 1 5 during the solid state fermentation, a wrinkly, yellow-brown mycelial sheet was formed after the germination of spores. As the cultivation proceeded, there was an increase in the formation of white aerial hyphae creating a fluffy mycelium. Sporulation, which commenced on day 2, was dark-brown in colour and less profuse than that of strain Yang No.2 upon further incubation. These growth characteristics are shown in Figures 5.3a to 5.3d.a. b.
c. d.
Figure 5.3 Growth morphology of
A .niger MH
1 5 - 1 5 during solid state fermentationfor citric acid production
a. day 2 c. day 6
b. day 4 d. day 8
In the case of the growth development of strain SL-2, there was no major difference to that of its parent, strain
MH 1 5- 1 5 ,
except a small variation in colour of the mycelium. In addition, fewer spores were produced by the mutant even at later stage of growth. These growth characteristics are displayed in Figures 5.4a to 5.4d.a. b.
c. d.
Figure 5.4 Growth morphology of
A .niger
SL-2 during solid state fermentation forcitric acid production
a. day 2 c. day 6
b. day 4