Effect of co-substrate on xylitol production by

Debaryomyces nepalensis NCYC 3413: A

cybernetic modelling approach
Presented by
Hrithik Baradia
BT22M003
Introduction
• Agriculture and allied industries generate a large amount of waste amongst which the lignocellulosic waste
holds the major proportion.
• Increased research interests are present for production of bio-energy and high value fine chemicals at large
scale and economically using lignocellulosic waste due to their abundant availability and renewable nature
(Asgher et al., 2013).
• Various works are reported for utilization of these waste for production of xylitol, an alternative to
conventional sweetener.
• Xylitol is used in odontological preparations due to its anti-carcinogenic properties
• It is also used as a sugar substitute for diabetic patients and people suffering from deficiency of glucose-6-
phosphate dehydrogenase (Mussato et al., 2008).
• Traditionally, xylitol production was done by a well established process involving the reduction of xylose
present in wood hydrolysates in presence of catalysts such as nickel, palladium at high temperatures and
pressure, thus the making the process energy and cost intensive (Parajó et al., 1995).
• An attractive alternative to the chemical production of xylitol is its production using biotechnological methods.
But, the poor fermentability of lignocellulosic waste at high substrate concentration is a major bottle neck for
this alternative method.
Introduction

• The pre-treatment of lignocellulosic waste generates large amount of inorganic salts inhibiting the growth of
microbes (Kumar and Gummadi, 2011).
• Debaryomyces nepalensis NCYC 3413, isolated from rotten apple which is a halotolerant yeast could be an
ideal strain for xylitol production.
• The properties and composition of hydrolysate varies depending on the raw material and method of
hydrolysis.
• Initial substrate concentration and media composition are important factors to be considered for large scale
production of xylitol using biotechnological methods along with other common factors such as pH,
temperature (Sampaio et al., 2008).
• The complete knowledge of xylose metabolism in presence of other substrates could be used to enhance the
production of xylitol by directing the metabolic flow towards xylitol production (Lee et al., 1998).
• There is little information about the effect of co-substrate on xylose bioconversions using different yeast
strains and also little is known about the influence of co-substrate on activity of key enzymes involved in xylitol
production.
Introduction

• Modelling is vital in analyzing and understanding of a system.

• In bioprocess, it helps in getting knowledge about the dynamics of cell growth, product formation, regulatory mechanisms
in cell and interactions of cell with the environment in bioreactor.
• Models can be used for developing better optimization strategies ensuring economic feasibility (Ghaly et al., 2005).
• The major problem in developing models is taking into considerations the complex metabolic regulations logically (Young,
2015).
• Hypothesizing that microbes utilize the available substrate in an optimal manner so as to maximize their growth,
cybernetic modelling can be considered appropriate for describing complex metabolic behavior (alexander et al., 1991)
• A cybernetic model including rate of biomass formation, substrate consumption and metabolite production and
concentration of key enzymes representing lumped enzyme pools are termed as lumped cybernetic models (LCM) (Song et
al., 2013)
• lumped hybrid cybernetic model are those where metabolic flux analysis is coupled with cybernetic models (Song et al.,
2011)
• In this study the effect of co-substrate (glucose and or arabinose) on xylitol production by Debaryomyces nepalensis NCYC
3413 in shake flasks and the effect of co-substrate on levels of key enzymes of xylose metabolism were evaluated.
• Also, a cybernetic model was developed to study the effect of glucose/ xylose ratio on xylitol production by D. nepalensis in
batch bioreactor
• The model describes the diauxic growth of D. nepalensis as well as sequential utilization of glucose followed by xylose and
also elucidates the xylitol production
Overview of Metabolic Pathway
Materials and Methods

• Debaryomyces nepalensis NCYC 3413, isolated from rotten apple, was streaked on a plate with YEPP media (pH
7.0) and incubated at 30 °C for 24 h and stored at 4 °C.
• A single colony was transferred from a fully-grown culture plate into the YEPD medium and incubated for 12 h
at 30 °C at 180 rpm.
• . Shake flasks studies were conducted in 1 L conical flasks with 200 ml working volume at 30 °C, pH – 4.9 and
180 rpm
• The batch fermentation was carried out under varying substrate concentrations in 2 L bioreactor equipped
with a dissolved oxygen probe, pH probe and a temperature sensor at 30 °C, pH – 4.9, 500 rpm and 0.5 vvm.
• Samples were collected at regular time intervals and centrifuged at 10,000 rpm for 10 min
• The supernatant was used for analysis of xylitol production and the cell pellet was used to quantify growth.
• The pellet was washed twice with 50 mM phosphate buffer (pH 7.2) and resuspended in the same buffer
• The cell suspensions were subjected to mechanical disruption using glass beads (0.1–0.3 mm in diameter) in
centrifuge tubes under vortex agitation in a proportion of 1:1(v/v) of cell suspension to glass beads.
• Cell disruption period was 5 min separated by 30 s interval in an ice bath.
• The samples were then centrifuged (8000 × g; 15 min; 4 °C) to remove cell debris and the supernatant was
collected for protein estimation and assayed for enzyme activities
• Protein was estimated by bicinchoninic acid (BCA) method using bovine serum albumin as the standard
Material and Methods

• Enzyme activities of cell-free extracts were measured with Lambda 25 UV/VIS spectrophotometer (rate of change in absorbance at
340 nm through the oxidation or reduction of the coenzymes NADPH or NADP is recorded. The rates were measured for 1 min )
• Glucose 6 phosphate dehydrogenase (G6PDH) activity was measured at 30 °C by monitoring the reduction of NADP+ in the assay
mixture which contains 50 mM Tris – HCl buffer (pH 7.4), 7 mM glucose 6 phosphate, 1 mM NADP+, 5 mM MgCl2 and 0.25 mg of
enzyme in a final volume of 0.5 ml
• The activity was calculated using the molar extinction coefficient of 6220 M−1 cm−1 for NADP+. One unit of G6PDH (U) was defined
as the amount of enzyme required for the reduction of1 μmol of NADP+ per minute at the assay conditions
• Xylose reductase (XR) was measured by monitoring the consumption of xylose-dependent NADPH in the reaction mixture containing
100 mM phosphate buffer (pH 7.2), 0.17 mM NADPH as a reductant, 170 mM xylose and 0.25 mg of enzyme in a final volume of 0.5
ml at 30 °C.
• XR activity was calculated with a molar extinction coefficient of NADPH (6220 M−1 cm−1 ). One enzyme unit (U) was defined as the
amount of enzyme catalyzing the oxidation of 1 μmol of NADPH per minute at the assay conditions
• Arabinose reductase (AR) activity was assayed based on the xylose reductase assay, with xylose replaced by 170 mM arabinose. The
activity was expressed as units (U)/g cell dry weight (CDW), one unit is equivalent to the amount of enzyme required to oxidize 1
μmol of NADPH per minute at the assay conditions
• Xylitol dehydrogenase (XDH) activity was measured at 30 °C by monitoring the reduction of xylitol-dependent NADP+. The reaction
mixture (0.5 ml) contained 100 mM Tris buffer (pH 8.8), 150 mM xylitol, 1.5 mM NADP+ and 0.25 mg of enzyme.
• The activity was calculated using the molar extinction coefficient of NADP+ (6220 M−1 cm−1 ). A unit (U) of xylitol dehydrogenase is
defined as the amount of enzyme catalyzing the reduction of 1 μmol of NADP+ per minute at the assay conditions.
• Arabitol dehydrogenase (ArDH) activity was assayed based on the xylitol dehydrogenase assay, with xylitol replaced by 150 mM
arabitol
Materials and Methods

• Optical density was measured at A600 and cell dry weight was
calculated from pre-calibrated graph for D. nepalensis (A600 of 1.0
corresponds to 0.34 g cell dry weight per liter culture).
• The concentration of xylose and metabolites (xylitol and glycerol) are
estimated by HPLC equipped with refractive index detector and Aminex
HPX-87H column at 45° C with 0.01 N H2SO4 as mobile phase at a flow
rate of 0.6 ml min−1 .
 Cybernetic Model

• Assumptions:
1. The growth of Debaryomyces nepalensis on mixed substrates follows a diauxic pattern, where utilization of glucose (S1)
followed by xylose (S2).

2. Growth rates are given by modified Monod rate equation which is proportional to the intracellular concentration of key
enzyme responsible for the assimilation of a particular substrate.

3. Glucose 6 – phosphate dehydrogenase (G6PDH) is considered as the constitutive enzyme and also responsible for the
metabolism of glucose

4. NADPH dependent xylose reductase (XR) and NADP+ dependent xylitol dehydrogenase (XDH) is responsible for xylose
metabolism and these enzymes are repressed in the presence of hexose sugars

5. XR is considered as the key enzyme for the production of xylitol and the ratio of XR to XDH is crucial for xylitol production

6. As the consumption rate of by-products (ethanol and glycerol) as secondary carbon source is very less when compared to
consumption of xylitol as secondary energy source, the production of by-products (ethanol and glycerol) were modelled
but their degradation was not considered in this model
Model equations

• Rate equation for growth is given as:

• Rate equation for enzyme synthesis is given by

Model Equations

• Dynamic growth of Debaryomyces nepalensis is given by:

• Xylose consumption is described by the maintenance energy model:

Model Equations

• All products formation are assumed to be growth associated and hence related to biomass formation as:

• Actual rate of enzyme synthesis is given by:

• ui and νi are the cybernetic variables which represent the optimal strategies for enzyme synthesis and enzyme
activity respectively. The regulatory action of activity and expression of key enzymes are introduced into the
model equations with cybernetic variables ui and νi.;
Estimation of model Parameters

• The parameters in the model equations for each run were estimated from the
experimental data using integral method
• Estimation procedure involves varying the parameters values in order to
minimize the difference between the experimentally observed profiles and
model predicted profile
• This was carried out with a Nelder-mead simplex algorithm.
• Model equations were solved simultaneously with the initial conditions using
fourth-order Runge-Kutta method with variable space step size defined by the
function ode45 in MATLAB
• The error between the predicted and experimental values were evaluated and
according to simplex method the parameters were actualized. The procedure
is continued iteratively until the final value of the parameter is reached
Statistical Analysis
• The experimental values and the model predicted values were analyzed
using t-test and corresponding p-values were calculated to check the
adequacy of the model. For best fit of the model, p-value should be
greater than 0.05.
Results

• Effect of single and multiple carbon source on growth of D. nepalensis, metabolite production in shake flasks
Results
Results
Results
Results
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