Kinetic Analysis of Alcohol Production from Agro-Waste Hydrolysates

This report presents a study on the kinetic parameters of alcohol production using Saccharomyces cerevisiae and mixed agro-waste hydrolysates. The research explores the impact of different pre-treatment methods (N. mirabilis/CP and HWP/DAP/CP) on microbial growth, substrate utilization, and product yield. The study compares the effectiveness of these methods, focusing on the generation of inhibitory by-products and the overall efficiency of alcohol production. The report details the experimental methods, including the identification of the yeast strain, the determination of kinetic rates, and the modeling of substrate consumption. The findings reveal that the N. mirabilis/CP pre-treatment method yields less inhibitory by-products and exhibits a better substrate utilization rate, leading to comparable or slightly improved alcohol production compared to HWP/DAP/CP. Furthermore, the study highlights the use of the Luedeking–Piret model for assessing total reducible sugars (TRS) consumption and discusses the relative differences in kinetic parameters between the two pre-treatment systems. The report concludes that pre-treatment of mixed agro-waste with N. mirabilis/CP is a promising method for producing hydrolysates suitable for alcohol production in biorefineries.

Kinetic Parameters of Saccharomyces
cerevisiae Alcohols Production Using
Nepenthes mirabilis Pod Digestive Fluids-
Mixed Agro-Waste Hydrolysates
Objective
The goal of this study was to see how microbial growth, substrate use, and other factors
influenced microbial growth. The kinetic characteristics of product production during
fermentation operations using hydrolysates of. In comparison to hot water/dilute
acid/cellulase, N. mirabilis/cellulase (N. mirabilis/CP).
Abstract
Using different hydrolysates in a single pot system, microbial growth kinetics and modelling of
alcohol generation using Saccharomyces cerevisiae were studied in this study. In the
fermentations utilised to produce the alcohols of interest, mixed agro-waste hydrolysates from
diverse pre-treatment procedures, such as N. mirabilis/CP and HWP/DAP/CP, were used as the
only nutrient supply. When comparing the HWP/DAP/CP hydrolysates to the N. mirabilis/CP
cultures, the maximum Saccharomyces cerevisiae concentration was 1.47 CFU/mL (1010), a
relative difference of 21.1 percent; the product yield based on biomass generation was
relatively (20.2 percent) higher for the N. mirabilis/CP cultures. There was a 24.6 percent
difference in total residual phenolic compounds (TRPCs) generation between the N.
mirabilis/CP and HWP/DAP/CP pre-treatment systems, implying that N. mirabilis/CP generates
less inhibitory by-products. This was further demonstrated by the N. mirabilis/CP cultures
having the lowest substrate utilisation rate (3.3 104 g/(Lh)) while reaching roughly similar
product creation rates to the HWP/DAP/CP cultures. When predicting substrate usage for N.
mirabilis/CP cultures, a better correlation (R2 = 0.94) was obtained. In general, pre-treatment of
mixed agro-waste with N. mirabilis/CP appeared to be a successful way to produce hydrolysates
that Saccharomyces cerevisiae can employ to produce alcohol in the biorefinery business.
Introduction

Fermentation is a well-known method for making bioproducts such as bioethanol, biobutanol,
isobutanol, lactic acid, citric acid, and so on from glucose and/or lignocellulosic biomass
hydrolysates. However, the use of lignocellulosic biomass (agro-waste) hydrolysates as a sole
carbon source is largely dependent on extractable and fermentable constituents in the biomass,
i.e., holocelluloses, which can be extracted using pre-treatment technologies involving physical,
chemical, and enzymatic hydrolysis, followed by fermentation to produce products such as
alcohols using commercial strains of Saccharomyces cerevisiae. S. cerevisiae, on the other hand,
is the most often employed yeast for the industrial manufacture of bioethanol, which uses
easily fermentable components in a broth.
The hydrolysates' inhibition during fermentation is substantially to blame for the issues
associated with fermenter performance for alcohol generation. Inhibitors have also been linked
to hydrolysis procedures used to extract fermentable total reducible sugars (TRS) from
lignocellulosic biomass, resulting in stunted fermenter cell growth and low bioproduct
concentration and fermenter productivity. Inhibitory chemicals are divided into three
categories: 1) phenolic compounds (as determined in this study), 2) furan derivatives, and 3)
weak organic acids, which are primarily produced during lignocellulosic biomass hydrolysis
among fermentable holocellulose constituents such as galactose, mannose, and xylose [10],
with cellulose producing glucose primarily. Overall, the applicability of pre-treatment/hydrolysis
methods such as biological, physical, and chemical ways to lower the toxicity of constituents in
the ensuing pre-treatment hydrolysate has not been developed. (Meintjes, M.M.2011)
Chemical hydrolysis, as compared to biological hydrolysis, has the capacity to eliminate
inhibitory by-products, which has a good impact on productivity and biomass generation during
alcohol synthesis. Hydrolysis is the only method used in most research. Cellulases are
commonly utilised, however there are other enzyme combinations that can be used as well.
Delignification and holocellulolysis of renewable resources like lignocellulosic biomass without
the use of synthetic chemicals or high-energy procedures, including agro-waste These enzymes
are responsible for cocktails found in the pods of Nepenthes mirabilis have been discovered to
be suited for because they include -glucosidase, xylanases, and carboxylesterase, they are
suitable for holocellulolysis. However, the fermenter performance in the hydrolysate collected
from the digestive fluid of N. mirabilis pods. The hydrolysates of combined traditional hydrolysis
procedures for example hydrolysis, must be compared with cellulases, hot water, and dilute
acid. This is a kinetic evaluation that can be understood assessments of model parameters.
Appropriate mathematical kinetic models and performance parameter determination are
required for effective performance parameter determination. Hydrolysates, for example, are
utilised in experimental designs to test the impact of fermentation conditions are necessary.
The output of the kinetic models can help with determining the best circumstances and the
efficacy of system control, including medium (hydrolysate) selection. Previously, Monod,
Moser, Tessier, Logistic, and Leudeking-Piret models were used to describe the microbial
growth, substrate consumption, and product formation rates. Therefore, they can be used to

comparatively analyze hydrolysate suitability. However, the selection of these models depends
on the required purpose of the individual studies.
Questions
1) What could be the possible methods for this study?
a) Confirmatory Identification of the Commercial Yeast Used for Fermentation
The extraction of genomic DNA (gDNA) followed a methodology similar to that
described in Zymo Research Catalogue No. D6005. The ZR DNA Kit was used to
extract DNA from the 24 hour YPD pure yeast culture (Zymo Research, Catalogue No.
D6005, Irvine, CA, USA). The ITS target region was amplified with One Taq Quick-
Load 2 Master Mix (NEB, Catalogue No. M0486, Ipswich, UK) and primers ITS1-50-
TCCGTAGGTGAACCTGCGG-30 and ITS2-50-TCCTCCGCTTATTGATATGC-30, followed
by repeated sequencing with forward 27F-50-AGAGTTTGATCMTGGCTCAG-30 and
reverse 1492R-50-GGTTACCTTGT (Zymo Research, Catalogue No. D4001, Irvine, CA,
USA). After running the PCR results (i.e., extracted fragments) on a gel, the
ZymocleanTM Gel DNA Recovery Kit was used to extract the DNA. PCR was carried
out in 100 L reactions with 100 ng of gDNA. The PCR conditions were set to 36 cycles
of 98°C denaturation for 30 seconds, 60°C primer annealing for 20 seconds, and 72°C
elongation for 60 seconds. The resulting extracts were then gel extracted (Zymo
Research, Zymo CleanTM Gel DNA Recover kit) and purified (Zymo Research, ZR DNA
sequencing clean-up kit Catalogue No. D4050, Irvine, CA, USA), before being
sequenced (forward/reverse direction). Following that, the ABI PRISM 3500xl
Genetic analyzer was used to do the analysis. The PCR products were purified
further with a Zymo Research ZR-96 DNA Sequencing Clean-up kit (Catalogue No
D6006, Irvine, CA, USA) before being processed on the CLC main workbench.
Following that, the produced sequences were compared to existing nucleotide
sequences in the NCBI Genbank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi)
for confirmation of the S. cerevisiae strain utilised, and an accession number of
KT32652.1 was assigned. A commercial yeast grower in South Africa provided the S.
cerevisiae. (Jiménez-Islas, D.; Páez-Lerma, J.; Soto-Cruz,2017)
2) Based on the case study above predict the result of microbial growth Parameters
Using Mixed Agro-Waste Pre-Treatment Hydrolysate.
The kinetics of cellular growth, substrate utilisation, and alcohol production in
hydrolysates obtained from the pre-treatment of mixed agro-waste constituted of peels
of C. sinensis and M. domestica, including cobs of Z. mays and yard waste from Q. robur,
were determined in this study using a commercial S. cerevisiae strain in hydrolysates
obtained from the pre-treatment of mixed agro-waste constituted of peels of The newly
proposed N. mirabilis/cellulases pre-treatment approach, which was compared to
standard HWP/DAP/CP methods for the pre-treatment of lignocellulosic biomass, is one

source of the hydrolysates. Using cellular counts, the maximum S. cerevisiae (VIN13)
growth was found, a fermenter chosen for its quick fermentations at ambient
temperature with a high TRS and alcohol tolerance (see Figure 1 and Table 1). This
resulted in a maximum cellular concentration of 1.47 1010 CFU/mL for the HWP/DAP/CP
hydrolysates, compared to 1.16 1010 CFU/mL for N. mirabilis/CP, indicating a 21.1
percent difference in cellular concentration. This was related to the greatest TRS
concentration achieved during the HWP/DAP/CP pre-treatment. This method is not
specialised to holocellulose extraction, although it does degrade lignin more than the N.
mirabilis-based pre-treatment method, which is more holocellulose-targeted. This
indicates that the S. cerevisiae strain had a sufficient supply of substrate during the
fermentation process. In addition, TRS use has been linked to a faster advancement of
the fermentation cycle. This demonstrated that the S. cerevisiae strain's metabolism
remained intact, allowing for ongoing product synthesis during the fermentation
process. Unless there are inhibitory chemicals in the hydrolysates, in which case the
fermenter will consume most of the available TRS to offset the inhibitors' effects, a high
concentration of TRS results in a high volume of alcohols being generated. At the end of
the N. mirabilis/CP and HWP/DAP/CP fermentations, the residual TRS concentrations (S)
were 0.075 and 0.439 g/L, respectively, with initial TRS concentrations (So) of 0.311 and
3.22 g/L. The suitability of applying HWP/DAP/CP techniques for delignification-
cellulolysis operations was further validated. Previously, the consumption of TRS during
fermentation was investigated utilising lignocellulosic biomass hydrolysates as the
primary substrates and S. cerevisiae as the fermenter. The efficiency of the pre-
treatment methods used was credited with the successes. By offering a new way of pre-
treatment, the resulting hydrolysates must either perform similarly to existing methods
with added benefits, or outperform existing methods, as in the case of the N.
mirabilis/CP pre-treatment proposed here. In addition, a comparison of the alcohols
produced utilising N. mirabilis/CP and HWP/DAP/CP hydrolysates revealed that
hydrolysates from the N. mirabilis/CP pre-treatment systems produced 5.2 percent
more alcohol than hydrolysates from the HWP/DAP/CP pre-treatment system (Table 1)
(Table 1). The HWP/DAP/CP pre-treatment system's greater TRS content, 3.22 g/L, did
not result in significantly higher alcohol generation. (Jung, Y.H.; 2011)

3) Determine the kinetic rate based on the study above.
During the formation of alcohol, the kinetic rates of S. cerevisiae aided fermentation
were examined. The Malthus model was utilized to determine and describe the
fermenter's microbial growth. The specific and maximal specific growth rates (Table 1)
for N. mirabilis/CP and HWP/DAP/CP, respectively, were 1.76 and 1.58 h1, with the N.
mirabilis/CP hydrolysates having the greatest max and a significant relative difference of
12 percent and 9.1 percent for the specific growth rate. The maximum value attained in
this investigation is comparable to that reported in recent studies using S. cerevisiae as a
fermenter with routinely used (refined) medium. However, the maximum growth rate of
S. cerevisiae in the batch system under acidic circumstances was only 0.5717 h1 in
certain investigations. Table 2 also shows other kinetic characteristics that were
important in this investigation, such as a biomass formation rate of 1.61 and 2.04 108
CFU/mL/h for N. mirabilis/CP and HWP/DAP/CP cultures, respectively, with an absolute
difference of 21.1 percent. Both N. mirabilis/CP and HWP/DAP/CP had very low product
formation rates (0.025 and 0.027 area percent /h, respectively), with a little 5.26
percent relative difference regarded inconsequential at laboratory size, but a high
product margin estimated to be large at an industrial scale. Similarly, substrate
consumption rates of 0.0033 and 0.0387 g/(L•h) for N. mirabilis/CP and HWP/DAP/CP,
respectively, showed that a large portion of the energy source was utilized for metabolic
biomass maintenance tasks rather than product formation. The elevated TRPCs in the
hydrolysates of the HWP/DAP/CP pre-treatment regime were previously attributed to
this abnormality.

4) In Kinetic Parameter determination, Modelling TRS Consumption for Simultaneous
Biomass and Product Formation and Determination and Data Handling, Relative
Differences, and other Kinetic Parameter has specific formula for the parameter
determination. Describe the model and state the formula.
a) Modelling TRS Consumption for Simultaneous Biomass and Product Formation
With TRS consumption in fermentations is principally and
directly related to biomass generation and product formation. As a result, the
Luedeking–Piret model (Equation (3)) was used to assess TRS consumption in this
investigation, assuming that product production is directly related to biomass
generation and product formation. (Khamaiseh, E.I, M.S. 2014)
whereby p = 1/Yx/s (g/CFU), while q is the product formation coefficient (1/h).
Therefore, Equation (3) can be rearranged as shown in Equation (4):
Equation (5) was constructed by substituting Equation (2) into Equation (4) and then
integrating using the initial conditions of t = 0 and S = S0 to estimate overall TRS
consumption and residual TRS concentration.
b) Data Handling, Relative Differences, and other Kinetic Parameters
Microsoft Excel 2013 was used to compute and analyse the experimental data and
kinetic models, while Microsoft Excel Solver® was used to calculate and analyse other
model parameters. The relative differences between the hydrolysates of the N.
mirabilis/CP and the HWP/DAP/CP agro-waste pre-treatment systems were also
determined (Equations (7) and (8)) to highlight the significance of the differences

discovered. The log10 (CFU/mL) = 2 log10 (CFU/mL) = 2 log10 (CFU/mL) = 2 log10
(CFU/mL) = 2 log10 (CFU/mL) = 2 log Other analysed kinetic parameters are presented in
Equations (9)–(12), with S. cerevisiae (VIN13) fermentations employing hydrolysates
from the HWP/DAP/CP-agro waste pre-treatment system serving as the reference
quantity for both absolute and relative differences. (Khamaiseh, E.I, M.S. 2014)
References
Meintjes, M.M.2011. Dissertation: Fermentation Coupled with Pervaporation: A Kinetic
Study/Meintjes MM; North-West University: Potchefstroom, South Africa.
Jiménez-Islas, D.; Páez-Lerma, J.; Soto-Cruz,2017. Modelling of ethanol production from
red beet juice by Saccharomyces cerevisiae under thermal and acid stress conditions.
Food Technol. Biotechnol, 52,93–100.
Jung, Y.H.; 2011 Ethanol production from oil palm trunks treated with aqueous
ammonia and cellulase. Bioresour. Technol. 102, 7307–7312.
Khamaiseh, E.I, M.S. 2014. Enhanced butanol production by Clostridium acetobutylicum
NCIMB 13357 grown on date fruit as carbon source in P2 medium.