Induction model

We developed the induction model to predict the amount of the lactose needed to open the lac promoter, and thus triggering the turning point from cellulose degradation to production. The prediction was necessary since the lactose we use in real condition were not as stable as IPTG as a inducer, and there were no theoretical relationship between the natural and artificial inducer. We performed parallel experiment to investigate the activation efficiency of two inducer, and by comparing the data collected from the references, we modified several relevant parameters.

By doing so, we expected to repress the expression of endoglucanase and exoglucanase and activate the expression of beta-glycosidase and cellulose synthases with least lactose to cut expense and offered a guidance to our wet experiemnt.

Enzymatic activity adjustment model

We developed an enzymatic activity adjustment model to predict the most desired pH and temperature condition to balance the inactivation of cellulase with maintaining stable proceeding of cellulose synthesis. Since the cellulase and cellulose synthase were functionally antagonistic with each other, denoting the latter produced bacterial cellulose might be easily degraded if the former secreted cellulase were not effectively inhibited. Nevertheless, adding cellulose inhibitor and regulating the reaction condition were both feasible to decrease cellulase activity, the latter one seemed to be a more economic method.

We performed numerical simulations on the model and sensitivity analysis on some key parameters, and design several reaction conditions to test the accuracy of the equations. By conducting wet experiment on CMCNase activity test, we got the specific activity data of cellulase and compared them with the simulated one to adjust some key factors to better conform to our engineered bacteria.

Finally, we estimated the best condition to meet this double demands was at pH6.5 and temperature 28℃, in this case, the specific activity of cellulase could dropp to 15% ~ 10% of the normal activity at pH7 and temperature 37℃.

Conversion model

We developed the conversion model to optimize the substrate cellulose input as the material to manufacture bacterial cellulose. Since an excessive amount of cellobiose could drastically decrease the activity of endoglucansse(CenA) and exoglucansse(Cex), we must chose a appropriate amount of wastepaper pulp containing suitable cellulose content.

T, we had to clear how much sugar was used to produce bacteria cellulose.When cellobiose in fermentation liquor entered E.coli, with expression of Cellobiose Phosphorylase and Bacterial Cellulose Synthase, the strain began to produce bacterial cellulose utilizing cellobiose. So, we analyzed the metabolic pathways from glucose to UDPG and from UDPG to bacteria cellulose, predicting the varieties of the concentration of cellobiose and bacteria cellulose.

Fermentation model

To ascertain the suitable strain concenstration and enzyme activity,we.built follow models.Genes of endocellulase and exocellulase expressed conducted by λ promoter at first.Without inhibition for λ promoter,genes of endocellulase and exocellulase transcribed and translated as E.coli strain grew.So we aimed to incubate E.coli at liquid medium and predicted the concentration of bacteria and activity of enzymes with Logistic equation and Luedeking-Piret equation.As a result,we assumed the relationship between concentration of E.coli and enzymes was semi-relative and predicted how much we could get enzymes.

Then how to ascertain the best.concenstration of cellulose to predict the concenstration of cellobiose.With results of activity of enzymes and concentration of cellulose,we had used Langmuir adsorption equation and modeled Michaelis-Menten equation to predict concentration of cellobiose.Langmuir adsorption equation was commonly used for enzyme adsorption to solid substrate.Especially,for cellulose,just when enzymes adsorbed long chains of cellulose they could work.Michaelis-Menten equation was a classical equation for enzyme reaction,but because of inactivity in process of enzyme adsorption,it was modified to match the actual enzyme reaction rate.To calculate the concentration of cellobiose,we applied the empirical second exponential decay equation which could predict the maximum concentration of cellobiose under different concentrations of cellulose.We found the most suitable concentration of cellulose was 20g/L and time of fermentation was 24hours.

Bio-manufacturing by fermentation

how we make paper transformer work and maximize our profit by recycling and reuse waste paper,so we build a factory to produce bacteria cellulose according to recent statue of waste paper utilization.

By investigation, we find pretreated waste-paper pulp contains 65% cellulose and its pH is 2~3 and after adjustment,it can act as substrate for fermentation.So we design a set of 500L fermentor as a factory model to produce bacteria cellulose.We use waste-paper pulp and industrial molasses as substrate for E.coli to produce and in the early fermentation we use airlift fermentor to keep strain grow and maintain suitable level of cellulase concentration for catalyzing cellulose to cellobiose.When the concentration of cellobiose does not change,we change fermentation liquor condition and control the temperature and pH to open later-stage fermentation switch by adding lactose as a inducer to synthesize BC.

But how to ensure the concentration of cellulose and get maxium quality of cellobiose,we build logistic model for strain growth,cellulase enzymatic reaction equation model and the mathematical models for transformation from cellulose to cellobiose.Finally,we consider the concentration of cellulose is 20g/L and can get 6g/L cellobiose after 24-hr fermentation under 37℃ in the first fermentation.Then,we analyze the metabolic pathway of UDPG which transfers about 5% cellobiose and other sugar to BC and will get 1.3g/L cellobiose in the end.To avoid to destroy the length and diameter of BC,we decline the ventilation in the airlift fermentor for latter process.

We analyze the expense of all materials and items to evaluate economic benefits,if we build a factory and have such a produce process in 200 m3 fermentor ,we can make a profit 8062780 dollars per year,decrease waste water 412200 ton ,reduce waste paper pulp 2061 ton,so environmentally friendly and economical!