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18 Advancements in Bio-hydrogen Production from Waste Biomass

Surface Methodology (RSM) has been reported in several optimization studies for

H2 production as it is a standard method to assess the individual and interactive

effects of the variables with minimum error. It can be inferred from the literature

that for H2 production, the major processes for optimization have been done using

Plackett–Burman design followed by Central Composite Design/Box–Behnken

design. Plackett–Burman applies a first-order polynomial model for studying the

effects of different parameters based on experimental results. It is a two-level

fractional factorial design used to select the main parameters for further analysis.

CCD and Box–Behnken (BB) designs are the second-order polynomial model used

for estimating the relationship between the major factors and the response and

obtaining optimum values. Contour or surface plots can be used to display this

second-order polynomial. The significant factors can be determined by using an

analysis of variance (ANOVA) of the model.

18.5.3

Metabolic Flux Analysis

MFA plays a vital role in the genetic engineering of microbes as it provides prior

information on the effects of targeted genetic modification on microbial growth or

target production. It provides an in-silico-based evaluation of intracellular fluxes

within a metabolic pathway, either to boost the product yield or to analyze the effects

of genetic engineering [45]. It thus helps to elucidate the central metabolic pathway

by considering the rates of consumption and production of metabolites within a bio-

logical network.

Metabolic fluxes are quantified by two model-based approaches ¹³C MFA and

Flux Balance Analysis (FBA). Both methods use thermodynamic, stoichiometric,

and experimental constraints to obtain a range of feasible intracellular fluxes within

a metabolic system followed by determining the flux distributions across the pro-

vided space to optimize the objective function. However, these methods differ in the

type of objective function optimized.

Similarly, MFA approach is also necessary to overcome the limitations of lower H2

yield by the biological route. An extensive investigation and understanding of the

biohydrogen pathway in microorganisms are essential, which may provide insights

to reconstruct the existing metabolic network toward the maximization of H2 yield.

The intracellular consumption and production rates of metabolites (fluxes) could

be analyzed by solving the mass balances of metabolites. MFA approach has been

widely used in most research to maximize the production of various products such

as acetate [46], lysine [47], and ethanol [48]. Literature reports many articles on MFA

of H2 production by C. butyricum W5, Clostridium thermosuccinogenes, and C. ace-

tobutylicum [49]. It was well presented by Oh et al. (2008) that H2 production can be

maximized to 8.7 mol-H2/mol-glucose if glucose flux is redirected toward the pen-

tose phosphate pathway in Citrobacter amalonaticus Y19 [50]. Cheng et al. (2013)

reported MFA application of C. tyrobutyricum, which revealed that hydraulic reten-

tion time (HRT) significantly affects the flux toward H2 [51]. A similar report exists

on MFA application in C. butyricum W5, which suggested that pH has a significant

impact and initial glucose has less effect on H2 production.