142
be combined with polarization magic-angle spinning to avoid the cell lysis step
(Jacob et al. 1986).
Colony and/or cell staining is a popular method where Nile red, Nile blue A, or
Sudan black dye is added into the growth medium. The staining method followed by
fluorescent microscopy is used for visualization of polyhydroxyalkanoates when
Nile red or Nile blue A is used, or visible light microscopy with Sudan black. The
dyes are more soluble in polyhydroxyalkanoates and, therefore, make the cells more
visible (Koller et al. 2011). While this method is rapid and suitable for a large number of samples, it is prone to inaccuracies as other lipophilic nonpolyhydroxyalkanoate- containing groups could be stained in the process (Koller
et al. 2017). Transmission electron microscopy (TEM) allows direct visualization of
polyhydroxyalkanoates, but requires the use of radioactive chemicals for sample
preparation and is time-consuming (Tan et al. 2014). These mentioned methods
allow the detection of polyhydroxyalkanoates-producing microorganisms, but cannot quantify the amount of polyhydroxyalkanoates produced nor its monomeric
composition on their own. Many experiments have coupled these methods with
image analysis in order to extract the amount of polyhydroxyalkanoates accumulation in cells (Elain et al. 2015; Takahashi et al. 2017).
Gravimetric methods are among the first employed methods to evaluate the
quantity of polyhydroxyalkanoates produced. First, chloroform is used to extract
polyhydroxyalkanoates from lyophilized bacterial cells, and the acetone or diethyl
ether is used for the precipitation of polyhydroxyalkanoates. Turbidity measurements can also predict polyhydroxyalkanoates amounts after lysis of cell material
(Koller et al. 2011). Flow cytometry, spectrofluorometry, and Fourier transform
infrared (FTIR) spectroscopy are all methods that allow to measure polyhydroxyalkanoates quantity without breaking cell walls (Koller et al. 2011). Moreover, FTIR
has the advantage of detecting different types of polyhydroxyalkanoates monomers
without use of hazardous chemicals in a short time and no cell lysis is required
either (Tan et al. 2014). Flow cytometry and spectrofluorometry provide results
after 5 to 30 min (Godbole 2016; Alves et al. 2017). Of these methods, staining
techniques using fluorescence-based quantification such as flow cytometry or spectrofluorometry are most suitable for online analysis at a treatment plant.
5.8 Polyhydroxyalkanoates Processing and Recovery
Polyhydroxyalkanoates are an intracellular product stored inside the cells. Therefore,
its separation and harvesting methods are expensive and complex. There are five
steps involved in recovery of polyhydroxyalkanoates, which start by harvesting,
followed by an optional pretreatment, then retrieval, accumulation, and finally purifying and drying (Kourmentza et al. 2017; Tan et al. 2014). Figure 5.6 summarizes
the steps followed to recover polyhydroxyalkanoates granules.
The first step is harvesting, and it consists of collecting the biomass from the
fermenter via centrifugation or filtration (Kourmentza et al. 2017). This step aims at
S. Sali and H. R. Mackey
be combined with polarization magic-angle spinning to avoid the cell lysis step
(Jacob et al. 1986).
Colony and/or cell staining is a popular method where Nile red, Nile blue A, or
Sudan black dye is added into the growth medium. The staining method followed by
fluorescent microscopy is used for visualization of polyhydroxyalkanoates when
Nile red or Nile blue A is used, or visible light microscopy with Sudan black. The
dyes are more soluble in polyhydroxyalkanoates and, therefore, make the cells more
visible (Koller et al. 2011). While this method is rapid and suitable for a large number of samples, it is prone to inaccuracies as other lipophilic nonpolyhydroxyalkanoate- containing groups could be stained in the process (Koller
et al. 2017). Transmission electron microscopy (TEM) allows direct visualization of
polyhydroxyalkanoates, but requires the use of radioactive chemicals for sample
preparation and is time-consuming (Tan et al. 2014). These mentioned methods
allow the detection of polyhydroxyalkanoates-producing microorganisms, but cannot quantify the amount of polyhydroxyalkanoates produced nor its monomeric
composition on their own. Many experiments have coupled these methods with
image analysis in order to extract the amount of polyhydroxyalkanoates accumulation in cells (Elain et al. 2015; Takahashi et al. 2017).
Gravimetric methods are among the first employed methods to evaluate the
quantity of polyhydroxyalkanoates produced. First, chloroform is used to extract
polyhydroxyalkanoates from lyophilized bacterial cells, and the acetone or diethyl
ether is used for the precipitation of polyhydroxyalkanoates. Turbidity measurements can also predict polyhydroxyalkanoates amounts after lysis of cell material
(Koller et al. 2011). Flow cytometry, spectrofluorometry, and Fourier transform
infrared (FTIR) spectroscopy are all methods that allow to measure polyhydroxyalkanoates quantity without breaking cell walls (Koller et al. 2011). Moreover, FTIR
has the advantage of detecting different types of polyhydroxyalkanoates monomers
without use of hazardous chemicals in a short time and no cell lysis is required
either (Tan et al. 2014). Flow cytometry and spectrofluorometry provide results
after 5 to 30 min (Godbole 2016; Alves et al. 2017). Of these methods, staining
techniques using fluorescence-based quantification such as flow cytometry or spectrofluorometry are most suitable for online analysis at a treatment plant.
5.8 Polyhydroxyalkanoates Processing and Recovery
Polyhydroxyalkanoates are an intracellular product stored inside the cells. Therefore,
its separation and harvesting methods are expensive and complex. There are five
steps involved in recovery of polyhydroxyalkanoates, which start by harvesting,
followed by an optional pretreatment, then retrieval, accumulation, and finally purifying and drying (Kourmentza et al. 2017; Tan et al. 2014). Figure 5.6 summarizes
the steps followed to recover polyhydroxyalkanoates granules.
The first step is harvesting, and it consists of collecting the biomass from the
fermenter via centrifugation or filtration (Kourmentza et al. 2017). This step aims at
S. Sali and H. R. Mackey