Dear All,
I am performing saccharification of biomass with the help of commercial cellulase enzyme. But when I am treating biomass with enzyme and taking reading at 0 hr it shows a pick of sugar also and with increasing incubation time sugar concentration is lowering.
So please suggest me any troubleshooting method if any?
Firstly, If your biomass had been previously pre-treated (by aqeous chemical or autohydrolytic methods) , then some level of sugars are expected in your biomass. That may account for the sugar at zero hr.
Secondly, most cellulase enzyme preparations are also not devoid of sugar components.
It is always advisable to run blanks of both enzyme without substrate and substrate without enzyme. These values will be substracted from the total resulting sugar at any time T.
As for the reduction in total sugar after time 0. There could be two possible explanations:
1- Activities of the enzymes invoved in lignocellulosic hydrolysis (usually cellulases) are non-linear. Cellulose has been defined to posses two distinct regions, the amorphous region, which is often subject to and is readily hydrolysed by endoglucanases, and the crystalline region , which needs a combinational effect of complementary enzymes and time before being hydrolysed. Hydrolysis of the amorphous region usually gives false alarm as the enzyme activity drops sharply as soon as the region is completely hydrolysed. You may check Ghose et al 1986, for more explanations on this.
2. The efect of microbial contamination may also contribute to the low amount of sugar obtained as fermentation proceed. Some percentage of antibiotics (0.2%) is recommended by the NREL proceedure for enzymatic saccharification of biomass.
May I know whether your biomass is pretreated before enzymatic saccharification?
Dear Mr. Ajijolakewu Kamoldeen,
I can understand your point. but in enzymatic saccharification sugar concentration should be increase with time which is not happening.
Is this problem happened with anyone else.
Dear Dr. Selvaraju Sivamani,
Yes, biomass is pretreated by acid hydrolysis.
Dear Mr. Mustafa Vohra,
I am using HPLC to measure sugar concentration.
Have you monitored the pH of the broth in which the saccharification was performed? It may happen that there is a shift in the pH as you are using acid pretreated bagasse and over a period of time the acid would have leached at higher temperature.
Dear Nilesh,
Did you implement a neutralization step to wash away the acid from the pretreatment? A reduction in sugars can be because they are being degraded to furfural/hmf by the acid. The other possibility is microbial contamination as mentioned above.
Hope you will get your experiment to work!
I can confirm that most of the commercial preparation of enzymes contain a lot of simple sugars, so you need to carry out two blanks one of pure enzyme and one pure substarte. If you do not want to add antibiotic in your mixture, be sure of pasteurise the substrate and the bioreactor/piping: lactic acid production i very competitive!
There are two occurrences might have happened.
1. I hope you have pretreated your biomass prior to EH. So there are chances to form inhibitory compounds like furfural, HMF, etc. Thus, your sugars may considerably converted to phenolic compounds. So care must be taken while doing pretreatment. And, as mentioned by Tim, neutralization or detoxification of pretreated biomass may significantly decrease inhibitor formation.
2. Overload of enzymes may fail to do its job. So go through some articles to find out the desired enzyme load.
All the best !!!
if your enzyme hydrolysis environment are gentle, it may can be happended that some bacterials, which have the ability consumping the reducing suger at the enzyme hydrolysis time, multiply rapidly in the liver on your sugar. maybe you should put a little ethyl acetate in this system at the first? trying again.
all the best!!!
Dear Mr Sharma,
Some commercial enzymes cocktails there are sugars in your content. This is due during the cultivation of the fungi used to this porpose (Aspergilus sp., Trichoderma sp., etc). Along the cultivation the microorganism secreted enzymes- degrating cellulose and hemicellulose releasing sugars (glucose and xylose). These sugars will be used for the growth. In the moment of extracting the enzymes, some residual sugars can come together with the enzymatic extract and maybe this occured with your HPLC experiments.
I hope that response help you.
Best wishes,
Had the same issue and found out most commercially produced cellulases have a maltodextrin extender
Ohamobi has rightly quoted. Most of the enzymes which are sold in powdered form use maltodextrin as a binding agent during spray drying and therefore for such enzyme assays, enzyme blank becomes a very important issue. I think the commercial enzyme at you are using also has a good amount of maltodextin in it. In that case you can go for glucose estimation specifically rather than total reducing sugar or even may go for HPLC determination of sugars.
In case you are not getting increasing sugar concentration over increased time of hydrolysis, there may be some error in the optimization and maintenance of temperature and pH conditions for the said enzyme
Like Deepti quoted, the optimisation is wrong. The already present glucose (maltodextrin) is acting as a catabolite repressor to the cellulase enzyme thereby reducing the rate of hydrolysis. Try increasing the hydrolysis time and see if that helps.
If your enzyme hydrolysis conditions are not sterile, it may be happening that some microbial contaminations are there, which is consuming the produced sugar with the increasing enzyme hydrolysis time. Try autoclaving the pretreated biomass and treating with detoxifying agents.