Effect of medium composition on Succinic bacteria performance
During the pre-evaluation, the bacteria life cycle was about two days. When 36 h was reached, the three bacteria entered the stationary phase. Eventually, the population growth declined between 40 h to 48 h and reached the death phase. The death phase occurred even though the media component was still not fully consumed. All the results demonstrate the effect of mediums on the bacteria performance by four main indications 1, SA production, 2, cell dry weight, 3, biomass and 4, Total sugar. All the mediums in the study exerted a positive effect on SA production. The results indicate similar behavior, which supports reliable outcomes across the parameters.
Fig 1 shows the results of medium for all bacteria. Actinobacillus succinogenes
(S02) has higher than the Mannheimia succiniciproducens
(S03) then Basfia Succiniciproducens
(S01). In (Fig 1-a), the SA produced by S02 is 6 g/L, which is around 2 g/L more than S01 and S03. Looking at (Fig 1-b), up to 25% additional dry cell weight was achieved by the S02 compared to S01 and S03. The biomass of S02 is more than the other two bacteria. This is because medium one contains 60 g of sugar as the primary source of carbon.
Fig 1: Succinic acid productions by 3 Strains selected using medium 1.
These findings confirm that the Actinobacillus Succinogenes
have high sugar consumption during the fermentation. The findings came similar to the observation of the A. succinogenes
in rumen. The microorganism was adapted to a symbiotic role in the rumen and produced 74 g of SA (Guettler et al., 1999
). In the current study, the mechanism is expected to adhere to the glucose uptake by the A. succinogenes. The A. succinogenes’s
ability to produce large amounts of succinate is attributed to its genome, which encodes a sugar transport protein (McKinlay et al., 2010
). However, it is still plausible that the total sugar consumed by Actinobacillus succinogenes
can be increased under different condition (Almqvist et al., 2016
The results reported in (Fig 2) shows that Actinobacillus succinogenes
(S02) produced 3.50 g/L. So in order to judge the result, it is crucial to bear in mind that medium two does not contain sugar or glucose, but uses Polypeptone as the primary carbon source. The mediocre succinic production for all the strains is due to the nature of Polypeptone. It is known that the biological constituents of Polypeptone have a mixture of casein peptone and meat peptone. The component of Polypeptone can increase cell density for several bacteria. Specific recent studies conducted on Actinobacillus succinogenes
with Polypeptone proved that cell growth had accelerated and production increased (Pinkian et al., 2018
; Kanchanasuta et al., 2020
Fig 2: Succinic acid productions by 3 Strains selected using medium 2.
Regarding the cell dry weight, the recorded quantities of different strains seem far less than what has been obtained by medium one. For each strain, the results were lowered to 2.50 g/L, 1.75 g/L and 1.00 g/L. The total sugar consumed by S02 is 6.50 g/L compared to 4.25 g/L and 6.00 g/L for the other bacteria.
In Fig 3 the effect of glucose as the main component of the medium is demonstrated. The SA production exceeded what has been obtained by mediums one and two for all the strains. By analyzing S02, SA production is around 12.5 g/L, which is 60% more than S01 and S03. Furthermore, the cell dry weight recorded the quantities of the three types of strains as follow: 8.50 g/L, 5.00 g/L and 3.50 g/L for S02, S01 and S03, respectively. Regarding sugars utilization, the total sugar consumed by S02 is 7.50 g/L compared to 6.25 g/L for S03 and 5.00 g/L for S01.
Fig 3: Succinic acid productions by 3 Strains selected using medium 3.
S03 revealed weakness in attaining satisfactory SA production in all the mediums. S01 performance was moderate with potentiality at medium three. S02 had the utmost SA production quantity. The S02 has great performance in medium three especially, which attributes to the superiority of glucose over sugar and Polypeptone. Because glucose better absorbed in the reaction and accelerated bacteria growth (Liu et al., 2008
; Ferone et al., 2019
; Lin et al., 2020
Our findings appear to be well substantiated by the pathway of the Actinobacillus succinogenes in
Glucose medium (McKinlay et al., 2007
), (Bradfield et al., 2014
). It has been proved that sufficient Glucose utilization as a substrate, can enhance the succinate yield and improves the quality (Liu et al., 2013
). In fact that associated with the low glucose concentration, so the SA would not be affected by cell fluctuation or inhibitors (Li et al., 2010
S01 and S03 could be beneficial only if further investigations of carbon source utilization were conducted. Lastly, the second part of the study will only focus on S02 optimum at medium three.
Effect of raw materials on Succinic acid production
At this point, different types of sugars were involved in the process and were used as a carbon sources. By scrutinizing the typical raw material components, the corn fiber consists of 35% hemicellulose, 18% cellulose and 20% remaining starch (Gáspár et al., 2007
). In contrast, Cane Molasses has glucose and fructose with an average concentration of 8.1 and 5.3%, respectively (Palmonari et al., 2020
). the orange peel contains 16.9% soluble sugars, 9.21% cellulose and 10.5% hemicelluloses (Badaró et al., 2020
). This variation in the composition revealed an impact on the magnitude of SA production. As seen in (Fig 4), the SA production vs material concentration manifests different SA levels. There is no question that the corn fiber produced the highest Succinic acid. The total SA production increased by 6 g/L, approximately more than medium three. For the Cane Molasses, an addition of 1 g/L was observed. Surprisingly, the orange peel did not show any addition, but it seems to produce less than medium three.
Fig 4 elucidates a semi linear relation between the Orange Peel concentration and SA production. The second observation is that cane molasses stabilized production when the concentration reached 8 g/L. This means that any further addition of cane molasses will not be fermented. These results imply that S02 could efficiently ferment the combination of sugars in corn fiber.
Effect of raw materials on cell dry weight
Fig 4: Succinic acid production after cultivation for corn fiber, cane molasses and orange peel at 37°C for 48 h.
In Fig 5, cane molasses and corn fiber are showing inconsistency between the increasing concentration and the cell dry weight. The results show that the intermediate content of sugar in cane molasses resulted in a slight drop in the middle concentrations. The lowest dry cell weight obtained is for the orange peel. When the corn fiber was used as a carbon source, the operation changed and the cell dry weight is 33% more than the cane molasses and 100% more than the orange peel. By comparing the previous cell dry weight obtained by medium three and the current carbon sources, 60% and 18% increase by corn fiber and cane molasses, respectively. The results in Fig 6 show high biomass retained from all the examined cultures.
Fig 5: Dry cell weight after cultivation for corn fiber, cane molasses and orange peel at 37°C for 48 h.
Effect of raw materials on total sugar
Fig 6: Total sugar after cultivation for corn fiber, cane molasses and orange peel at 37°C for 48 h.
The specific sugar consumption is a significant indication of cell growth. As much sugar is consumed SA production increases. The initial total sugar content varies as the carbon sources vary in the examination. However, at the end of the 48 h a significant trend appears, as seen in Fig 6.
Fig 6 illustrates the relationship between the sample concentration and the sugar consumed by S02. A clear consumption pattern, especially for the corn fiber, supports the possibility of high SA. The corn fiber reached the total sugar content of zero; this was interpreted by the S02 entirely consuming the culture content. However, this is not the case for the orange peel and the cane molasses. The sugar depletion follows a linear trend for increasing the concentration for all samples.
The findings support the study hypothesis. The corn fiber has successfully attained all the criteria required to obtain SA in the flask experiments. No consideration could limit the ultimate SA production by the S02 at the corn fiber medium. Thus, it will be continued to the 4-L bioreactor examination to predict the industrialization benefits.
Succinic acid production and kinetics of bioprocess parameter in 4-litter bioreactor
Corn fiber was selected to continue the scale-up examination for the large-scale fermentation process. 10 g/L of Corn fiber was utilized to isolate Actinobacillus succinogenes
. in the 4-L Bioreactor. The medium used in the process is composed of Corn fiber 10.0, 10.0 g yeast extract, 8.0 g NaHCO3
, 8.5 g NaH2
O, 15.5 g K2
, 1.0 g (NH4
, 0.2 g MgCl2
O, 0.2 g CaCl2
. The bioreactor ran under controlled pH conditions, where the initial pH sat at 7.0. The cultivation process was taken after 48 h; as had been practiced in the flask experiments.
It was noticed that the pH reduced to 6.5 after 3 h of cultivation. From the observation, the cells reached the stationary phase only after 3h. The cells entered the exponential phase with a growth rate of 0.175 [g/L.h]. The maximum growth rate and specific SA production are 1.024 [g/L] and1.175 [g/g], respectively.
During the process, the SA increased over time, as illustrated in Fig 7. SA production is 5.75, 11.30, 15.50, 22.00 g/L at 6, 12 and 24h. This indicates that the Bioreactor was able to give a similar result to the shake flask only after 12 hours. It is a quarter of the time needed previously. The maximal SA production value is 25.00 g/L. Besides, the cell dry weight and biomass, as seen in (Fig 7), were escalated during the fermentation process and reached a maximum value of 19.00 g/L and 20.50 g/L, respectively.
Fig 7: SA production, cell dry weight, biomass, total sugar and dissolved oxygen changes in Bioreactor 4-L cultivation.
Apparently, both values are 50% higher than reported for the shake flask culture. The total sugar in batch bioreactor 4-L cultivation is reducing gradually from 18.00 g/L to null. Presumably, the sugar diminishes has occurred approximately between 36 h to 48 h. The quantity of dissolved oxygen (DO) decreased gradually from 100% to 25.00% at the end of fermentation. All the findings at the bioreactor stage could be explained by the effects of corn fiber (sugar complexity) and the production of many acids during the Actinobacillus succinogenes
fermentation pathway (Alsaheb et al., 2020
; Raje Nimbalkar et al., 2022