Monitoring of bacterial activity and diversity during treatment may be helpful
during adjustment of the process condition such as type and amount of applied
organic matter, C:N:P ratio, aeration, humidity adjustment, or the effect of
bioaugmentation. Microbiological monitoring assays investigate the effect of the
contaminant on microbial communities or single bacterium species. To estimate the
changes in microbial communities during bioremediation, community-level multivariate profiling techniques are used (Bundy et al. 2004). The investigation of soil
microbial community is a challenging task since currently available methodologies
do not provide the tools to examine the entire soil microflora. The two main
approaches in microecology, medium culture-based or DNA and no-DNA molecular
methods, have some limitations (Bundy et al. 2004). Culture-based methods are
based on the cultivation of soil microorganisms in laboratory conditions. Common
counting methods based on bacteria cultivation are colony forming unit (CFU) and
most probable number (MPN). Through the selection of appropriate growth medium
it is possible to estimate the CFU of total heterotrophic bacteria as well as the amount
of bacteria utilizing particular hydrocarbons as a carbon source (Wang et al. 2016).
However, it is estimated that only less than 1% of soil bacteria are cultivable, thus the
interactions in soil ecosystem will not be reproduced and observed. Culture-based
studies may easily provide useful information about the bacterial populations’
dynamics and intensity of metabolism, but the obtained result should not be considered as a representative for entire soil ecosystem and accurate measurement of the
biodegradation (Margesin et al. 2000).
Implementation of metagenomics surveys with the application of 16S rRNA gene
sequences used as genetic markers resulted in acceleration and significant improvement of the ecological overview in the analysis of the qualitative changes in the
microbial communities (Fuentes et al. 2014). Popular genetic markers applied in the
analysis of hydrocarbon-degrading microbes are sequences encoding the oxidizing
enzymes such as monooxygenases, e.g., alkB genes and dioxygenases, e.g., phnAc,
nah genes, because of the essential role in hydrocarbon degradation and high
divergence (Fuentes et al. 2014; Shahi et al. 2016). The abundance of genes involved
in hydrocarbon degradation becomes common in the monitoring of TPH bioremediation (Fuentes et al. 2014). Abundance of catabolic genes such as alkB, xylE, and
nahAc, involved in hydrocarbon metabolism was positively correlated with the
degradation of hydrocarbons (Salminen et al. 2008). In recent years, the common
methods for detecting microorganisms able to degrade alkanes in environmental
samples are based on the detection of the particulate alkane hydroxylases (pAHs)
encoding genes. The pAH together with rubredoxin and a rubredoxin reductase,
constitutes a complex of integral-membrane non-heme di-iron monooxygenase
(AlkB), which is a first well-described monooxygenases system (van Beilen and
Funhoff 2007; Fuentes et al. 2014). Other known hydroxylation systems include
methane monooxygenases (MMOs) and cytochrome P450 protein superfamily
(Fuentes et al. 2014). Among DNA-based molecular methods polymerase chain
reaction (PCR) followed by denaturing gradient gel electrophoresis (DGGE), temporal temperature gradient gel electrophoresis (TTGE), and terminal restriction
fragment length polymorphism (T-RFLP) are commonly used (Liu et al. 2014;
5 Potential Use of Waste-to-Bioenergy By-Products in Bioremediation of Total. . .
269
during adjustment of the process condition such as type and amount of applied
organic matter, C:N:P ratio, aeration, humidity adjustment, or the effect of
bioaugmentation. Microbiological monitoring assays investigate the effect of the
contaminant on microbial communities or single bacterium species. To estimate the
changes in microbial communities during bioremediation, community-level multivariate profiling techniques are used (Bundy et al. 2004). The investigation of soil
microbial community is a challenging task since currently available methodologies
do not provide the tools to examine the entire soil microflora. The two main
approaches in microecology, medium culture-based or DNA and no-DNA molecular
methods, have some limitations (Bundy et al. 2004). Culture-based methods are
based on the cultivation of soil microorganisms in laboratory conditions. Common
counting methods based on bacteria cultivation are colony forming unit (CFU) and
most probable number (MPN). Through the selection of appropriate growth medium
it is possible to estimate the CFU of total heterotrophic bacteria as well as the amount
of bacteria utilizing particular hydrocarbons as a carbon source (Wang et al. 2016).
However, it is estimated that only less than 1% of soil bacteria are cultivable, thus the
interactions in soil ecosystem will not be reproduced and observed. Culture-based
studies may easily provide useful information about the bacterial populations’
dynamics and intensity of metabolism, but the obtained result should not be considered as a representative for entire soil ecosystem and accurate measurement of the
biodegradation (Margesin et al. 2000).
Implementation of metagenomics surveys with the application of 16S rRNA gene
sequences used as genetic markers resulted in acceleration and significant improvement of the ecological overview in the analysis of the qualitative changes in the
microbial communities (Fuentes et al. 2014). Popular genetic markers applied in the
analysis of hydrocarbon-degrading microbes are sequences encoding the oxidizing
enzymes such as monooxygenases, e.g., alkB genes and dioxygenases, e.g., phnAc,
nah genes, because of the essential role in hydrocarbon degradation and high
divergence (Fuentes et al. 2014; Shahi et al. 2016). The abundance of genes involved
in hydrocarbon degradation becomes common in the monitoring of TPH bioremediation (Fuentes et al. 2014). Abundance of catabolic genes such as alkB, xylE, and
nahAc, involved in hydrocarbon metabolism was positively correlated with the
degradation of hydrocarbons (Salminen et al. 2008). In recent years, the common
methods for detecting microorganisms able to degrade alkanes in environmental
samples are based on the detection of the particulate alkane hydroxylases (pAHs)
encoding genes. The pAH together with rubredoxin and a rubredoxin reductase,
constitutes a complex of integral-membrane non-heme di-iron monooxygenase
(AlkB), which is a first well-described monooxygenases system (van Beilen and
Funhoff 2007; Fuentes et al. 2014). Other known hydroxylation systems include
methane monooxygenases (MMOs) and cytochrome P450 protein superfamily
(Fuentes et al. 2014). Among DNA-based molecular methods polymerase chain
reaction (PCR) followed by denaturing gradient gel electrophoresis (DGGE), temporal temperature gradient gel electrophoresis (TTGE), and terminal restriction
fragment length polymorphism (T-RFLP) are commonly used (Liu et al. 2014;
5 Potential Use of Waste-to-Bioenergy By-Products in Bioremediation of Total. . .
269