**4. Applications of crude glycerol in biological processes**

Glycerol can be consumed by microorganisms in biological processes to generate byproducts with added value, such as ethanol, 1,3-propanediol (PD), H2 and organic acids, among others. PD may be used in industrial applications such as polymers, cosmetics, foods, adhesives, lubricants, laminates, solvents, antifreeze and pharmaceuticals [9]. Ethanol has been used in the pharmaceutical industry, solvent, cleaning products and personal hygiene. In Brazil, its use is remarkable mainly due to the sugarcane producing. In 2011 over 27 billion liters of ethanol was produced in Brazil from sugarcane. Most of it was destined for use as a fuel[18], mainly hydrid vehicles that can be driven by mixtures of gasoline and ethanol [19]. H2 has been utilized as a reactant in the chemical and petroleum industries during the production of ammonia, petroleum processing and methanol [2].

The energy content of the pure glycerol is 19.0 MJ/kg, however for crude glycerol it is 25.30 MJ/kg, possibly due to presence of methanol and biodiesel [10]. Such high energy content of crude glycerol indicates its potential to be an effective carbon source for hydrogen, PD and ethanol bioproduction.

The microorganisms involved in hydrogen production may be classified in four groups: strictly anaerobic, facultative anaerobic, aerobic and phototrophic [22], for example, green algae, cyanobacteria, phototrophic bacteria and fermentative bacteria. However, higher yields of H2 generation are obtained by fermentation processes. The main fermentative bacteria known to produce hydrogen include *Enterobacter* sp.*, Bacillus* sp., *Clostridium* sp., *Klebsiella* sp. and *Citrobacter* sp.[9,21]. The process of dark fermentation from crude glycerol may be followed by fotofermentation [9] because phototrophic bacteria grow with organic acids (the possible metabolites from fermentation) and they may produce more hydrogen [22].

crude glycerol and can be removed by precipitation from the liquid medium through pH adjustment. Sodium ions can be removed from crude glycerol by neutralization with addition of phosphoric acid and lime in excess, in order to crystallize/precipitate hidroxyapatite [17]. However, these treatments are costly and not economically justifiable. Alternatively, there are many studies covering the use of crude glycerol for bio-hydrogen production without pretreatments [11]. These bioconversions of the crude glycerol may be suitable and economically

**Source of biodiesel Glycerol content w/wProduct Yield Impurities Ref.**

supernatant)

(4.91 g l-1)

H2. m3

Docosahexaenoic acid

H2 dm3

Glycerol can be consumed by microorganisms in biological processes to generate byproducts with added value, such as ethanol, 1,3-propanediol (PD), H2 and organic acids, among others. PD may be used in industrial applications such as polymers, cosmetics, foods, adhesives, lubricants, laminates, solvents, antifreeze and pharmaceuticals [9]. Ethanol has been used in the pharmaceutical industry, solvent, cleaning products and personal hygiene. In Brazil, its use is remarkable mainly due to the sugarcane producing. In 2011 over 27 billion liters of ethanol was produced in Brazil from sugarcane. Most of it was destined for use as a fuel[18], mainly hydrid vehicles that can be driven by mixtures of gasoline and ethanol [19]. H2 has been utilized as a reactant in the chemical and petroleum industries during the production of

The energy content of the pure glycerol is 19.0 MJ/kg, however for crude glycerol it is 25.30 MJ/kg, possibly due to presence of methanol and biodiesel [10]. Such high energy content of crude glycerol indicates its potential to be an effective carbon source for hydrogen, PD and

The microorganisms involved in hydrogen production may be classified in four groups: strictly anaerobic, facultative anaerobic, aerobic and phototrophic [22], for example, green algae,

d-1 MeOH, NaOH and sodium

methylate [13]

NaSO4, MeOH, Water, [14]

Soap, FFA, MeOH, Mono, Di or Triglycerides [15]

medium OMNG, ash and methanol [16]

attractive alternatives to the industrial processes.

84%

Biodiesel factory, Portugal 86% 710.0 cm3

ammonia, petroleum processing and methanol [2].

69.5% 0.41 m3

OMNG - Organic Matter Not Glycerol; FFA – Free Fat Acids; MeOH – Methanol

**Table 1.** Impurities present in the crude glycerol during the biodiesel production

**4. Applications of crude glycerol in biological processes**

67.5±3.2% Phytase (1125 U ml-1

Nittany Biodiesel, State

480 Biofuels - Status and Perspective

Integrity Biofuels, Indiana,

Virginia Biodiesel Refinery (West Point, VA, USA)

ethanol bioproduction.

College, PA

USA

Glycerin may have different yields of hydrogen per mole of organic substrate, depending on the route of the fermentation used and the exact composition of the end products. According reference [9], the end products of the fermentation process may be acetic acid (equation 1); butyric acid (equation 2), butanol (equation 3) and ethanol (equation 4). Furthermore, as reported by several authors, it may also generate PD (equation 5),[23,24]. Generation of acetic acid (equation 1) and butyric acid (equation 2) are accompanied by higher yields of H2, as observed in the fermentation of sugars [25].

$$\rm{C}\_{3}\rm{H}\_{8}\rm{O}\_{3} + \rm{H}\_{2}\rm{O} \rightarrow \rm{CH}\_{3}\rm{COOH} + \rm{CO}\_{2} + 3\rm{H}\_{2} \tag{1}$$

$$2\,\mathrm{C}\_{3}\mathrm{H}\_{8}\mathrm{O}\_{3} \rightarrow \mathrm{C}\_{4}\mathrm{H}\_{8}\mathrm{O}\_{2} + 2\,\mathrm{CO}\_{2} + 4\,\mathrm{H}\_{2} \tag{2}$$

$$2\,\mathrm{C}\_{3}\mathrm{H}\_{8}\mathrm{O}\_{3} \rightarrow \mathrm{C}\_{4}\mathrm{H}\_{10}\mathrm{O} + 2\,\mathrm{CO}\_{2} + \mathrm{H}\_{2}\mathrm{O} + 2\,\mathrm{H}\_{2} \tag{3}$$

$$\rm C\_3H\_8O\_3 \rightarrow C\_2H\_6O + CO\_2 + H\_2 \tag{4}$$

$$2\,\mathrm{C}\_{3}\mathrm{H}\_{8}\mathrm{O}\_{3} \rightarrow \mathrm{CH}\_{3}\mathrm{COOH} + \mathrm{C}\_{3}\mathrm{H}\_{8}\mathrm{O}\_{2} + \mathrm{CO}\_{2} + 2\,\mathrm{H}\_{2} \tag{5}$$

Glycerol can also produce lactate (equation 6) and succinate (equation 7) [26].

$$\rm{C}\_{3}\rm{H}\_{8}\rm{O}\_{3} \rightarrow \rm{C}\_{3}\rm{H}\_{6}\rm{O}\_{3} + \rm{H}\_{2} \tag{6}$$

$$2\,\mathrm{C}\_{3}\mathrm{H}\_{8}\mathrm{O}\_{3} \rightarrow \mathrm{C}\_{4}\mathrm{H}\_{6}\mathrm{O}\_{4} + 2\,\mathrm{CO}\_{2} + 5\,\mathrm{H}\_{2} \tag{7}$$

Fermentation processes of hydrogen production using anaerobic acidogenic bacteria have been extensively described by several authors [27-31]. Additionally there are several key intermediate products created during the fermentation of glycerol: mainly organic acids, such as acetic acid and butyric acid and alcohols, such as ethanol and PD [9].

The following metabolites are obtained from the fermentation of glycerol: dihydroxyacetone, succinic acid, citric acid, docosahexaenoic acid, propionic acid, hydrogen, ethanol, and PD. Figure 2 demonstrate that during the oxidative metabolism of glycerol, pyruvate is formed as an intermediate.

**Figure 2.** Glycerol metabolism during anaerobic fermentation (adapted from [10])

The production of PD is achieved through a reductive pathway in the anaerobic fermentation of glycerol. However, production of H2 and other metabolites (ethanol, butanol, acetone, acetate, butyrate and lactate) compete with the production of PD by oxidative pathways [24].

However pyruvate formed during the conversion of glycerol (using the oxidative route) may be employed in various ways by microorganisms. The pyruvate is responsible for the forma‐ tion of numerous metabolites such as lactate, ethanol, acetone, butanol and butyrate, as demonstrated during glucose metabolism (Figure 3). A similar metabolism using glycerol first produces pyruvate, before conversion to different metabolites and H2 [10].

In many bacteria there are two biochemical pathways for glycerol metabolism: an oxidative pathway, where H2 is generated and a reductive pathway leading to PD generation. When both pathways exist in the same microorganism PD production is preferential against H2 [10].

**Figure 3.** The glucose metabolism to pyruvate and hydrogen (Adapted from [10])

Figure 2 demonstrate that during the oxidative metabolism of glycerol, pyruvate is formed as

**Figure 2.** Glycerol metabolism during anaerobic fermentation (adapted from [10])

produces pyruvate, before conversion to different metabolites and H2 [10].

The production of PD is achieved through a reductive pathway in the anaerobic fermentation of glycerol. However, production of H2 and other metabolites (ethanol, butanol, acetone, acetate, butyrate and lactate) compete with the production of PD by oxidative pathways [24].

However pyruvate formed during the conversion of glycerol (using the oxidative route) may be employed in various ways by microorganisms. The pyruvate is responsible for the forma‐ tion of numerous metabolites such as lactate, ethanol, acetone, butanol and butyrate, as demonstrated during glucose metabolism (Figure 3). A similar metabolism using glycerol first

In many bacteria there are two biochemical pathways for glycerol metabolism: an oxidative pathway, where H2 is generated and a reductive pathway leading to PD generation. When both pathways exist in the same microorganism PD production is preferential against H2 [10].

an intermediate.

482 Biofuels - Status and Perspective

The biotechnological production of H2, ethanol and PD from glycerol has been demonstrated by several bacteria species. The species of anaerobic bacteria as *Klebsiella* sp., *Enterobacter* sp., *Citrobacter* sp., *Lactobacillus* sp., *Bacillus* sp. [32] and some *Clostridium* sp., have demonstrated the ability to ferment glycerol or mixtures of glycerol and sugars [9]. In addition to the anaerobic microorganisms, nutrients are required in the reaction medium. These allow growth and fermentation of organic substrates leading H2 production. Complex compounds used as nutrients include: peptone, tryptone, polypeptone, yeast extract, vitamin solutions, among others. Such bacterial species may generate H2 from various carbon sources, particularly sugars [33] and glycerol.

Table 2 shows some studies about the bioconversion of glycerol using pure cultures or mixed microbial consortia to generate H2 and other byproducts.

PD and H2 are the two major products which can be obtained by bioconversion of crude glycerol. A co-culture of anaerobic bacteria, which can simultaneously use PD and produce H2 via glycerol fermentation, may be a suitable option for crude glycerol bioconversion [10]. However, a combined production process of hydrogen and ethanol provides higher energy yield when compared with hydrogen or ethanol alone [40].


**Table 2.** Bioconversion of glycerol to H2 and other products

A range of reactor types has been used in hydrogen production that utilizes organic waste materials such as crude glycerol or pure glycerol. They may be simple serum bottles [33], laboratory scale fermenters, pack-bed or up flow reactors [41] (Figure 4). However, it should be noted that most of the studies were performed in batch anaerobic reactors on laboratory scale (Table 3). To the best of our knowledge, there are no studies with pilot scale reactors, this suggests that the research into the bio-production of hydrogen using glycerol is currently in a preliminary phase.

**Figure 4.** Anaerobic batch reactors applied on laboratory scale


**Table 3.** Different bioreactors applied for bioconversion of glycerol

However, a combined production process of hydrogen and ethanol provides higher energy

Microorganisms Hydrogen Yield Other by products Ref. *Enterobacter aerogenes* HU-101 63 mmol H2 l-1 h-1 0.85 mol ethanol mol-1 glycerol [34] *Klebsiella pneumoniae* ATCC 25955 - PD [35] Mixed micro-flora of organic waste or soil 11.5-38.1 ml H2 g-1 COD PD [36] Mixed culture 0.31 mol H2 mol-1 glycerol 0.59 mol PD mol-1 glycerol [37] Anaerobic digested sludge 0.41 mol H2 mol-1 glycerol 0.784± 0.063 L CO2L-1 media [38] *Halanaerobium saccharolyticum* 0.62 mol H2 mol-1 glycerol PD, butyrate, ethanol [39]

A range of reactor types has been used in hydrogen production that utilizes organic waste materials such as crude glycerol or pure glycerol. They may be simple serum bottles [33], laboratory scale fermenters, pack-bed or up flow reactors [41] (Figure 4). However, it should be noted that most of the studies were performed in batch anaerobic reactors on laboratory scale (Table 3). To the best of our knowledge, there are no studies with pilot scale reactors, this suggests that the research into the bio-production of hydrogen using glycerol is currently in a

1.11 mol CO2 mol-1 glycerol,

[39]

acetate

yield when compared with hydrogen or ethanol alone [40].

*Halanaerobium saccharolyticum* 1.61 mol H2 mol-1 glycerol

COD- Chemical Oxygen Demand; PD - Propanediol

preliminary phase.

484 Biofuels - Status and Perspective

**Table 2.** Bioconversion of glycerol to H2 and other products

**Figure 4.** Anaerobic batch reactors applied on laboratory scale

However there are many promising results of hydrogen generation using different configu‐ rations of anaerobic reactors fed with industrial wastewater, sugars, starch and others. The configurations of anaerobic reactors applied for biological H2 production are AFBR (Anaerobic Fluidized-Bed Reactor), CSTR (continuously stirred tank-reactor), EGBS (Expanded granular sludge bed reactor) and UASB (Up-Flow Anaerobic Sludge Blanket reactor) (Table 4).


**Table 4.** Different configurations of anaerobic reactors applied for H2 production

The UASB reactor is a single tank process where the wastewater enters from the bottom and flow upward (Figure 5). A suspended sludge blanket filters treats the wastewater flows through it and bacteria living in the sludge break down organic matter by anaerobic digestion, transforming it into biogas. Some advantages of this configuration are: the conversion of the organic matter in all reactor areas (bed and sludge blanket); the microorganisms can grow close to the bottom of the reactor in the form of flocks or granules (1 to 5 mm); the mixing of the system is promoted by the upward flow of wastewater and gas bubbles [49,50].

**Figure 5.** Schematic representation of UASB reactor (Adapted from [50])

The EGBS reactor has a cylindrical structure, packed with inert particles (0.3 to 3.0 mm of diameter) as support for microorganisms to form the biofilm (Figure 6). Several types of materials may be used as support mediums such as sand, coal, PVC, resins, ground tire and PET [51], etc. The biofilm may develop on the particles surface [49].

**Figure 6.** Schematic representation of EGBS reactor (Adapted from [50])

AFBR has the same operating principles of the EGBS reactors, except the particles size (0.5 to 0.7 mm) of the support medium and the expansion rates (Figure 7). The upward velocity of the liquid must be enough high to fluidize the bed until it reaches the point at which the gravitational force is equaled by the upward drag force. A high recirculation rate is necessary and the particles do not stay a fixed position inside the bed [49].

**Figure 7.** Schematic representation of AFBR reactor (Adapted from [50])

**Figure 5.** Schematic representation of UASB reactor (Adapted from [50])

486 Biofuels - Status and Perspective

**Figure 6.** Schematic representation of EGBS reactor (Adapted from [50])

PET [51], etc. The biofilm may develop on the particles surface [49].

The EGBS reactor has a cylindrical structure, packed with inert particles (0.3 to 3.0 mm of diameter) as support for microorganisms to form the biofilm (Figure 6). Several types of materials may be used as support mediums such as sand, coal, PVC, resins, ground tire and

AFBR has the same operating principles of the EGBS reactors, except the particles size (0.5 to 0.7 mm) of the support medium and the expansion rates (Figure 7). The upward velocity of CSTR is known as a mix batch reactor and is an ideal type reactor in chemical engineering, for studies on laboratory scale [50]. CSTR can provide continuous or intermittent flow and comprises the follow steps: (1) filling (input of organic matter and microorganisms); (2) reaction (organic matter come into contact with microorganisms and they will degraded it); (3) sedimentation (settling of anaerobic sludge) and (4) emptying (removal of treated effluent) (Figure 8).

Studies on anaerobic fermentation of glycerol present major advances in pure cultures, such as with *Enterobacter aerogenes*. However, pure cultures do not represent real situations, such as those found in industrial waste [43]. To address this, research has been conducted onto hydrogen generation with mixed cultures obtained from anaerobic microorganisms present in biological treatment system sludge [39]. However, H2-generating bacteria may be present in addition to bacteria that consume this gas, such as methanogenic archaea.

The fermentative production of hydrogen can be facilitated with methanogen inhibition, since methanogenic archaea use hydrogen in anaerobic biological processes [25]. To inhibit this methane formation process, reagents can be introduced, such as 2-brometanosulfonic acid (BES) and acetylene [2]. Additionally, pH control and heat treatment may provide other effective ways to prevent methanogens [25]. These methods may promote hydrogen produc‐ tion and encourage the growth of endospore-forming bacteria which are tolerant to high temperatures and adverse environmental conditions [21].

**Figure 8.** Schematic representation of CSTR reactor: (1) filling; (2) reaction; (3) sedimentation and (4) emptying (Adapt‐ ed from [50])

When the pH is controlled, organic acids that favor microbial selection and the consequent production of hydrogen gas are formed. Other methods for elimination hydrogen consumers utilize ultra-sonication, acidification, sterilization and freezing/thawing [10]. Sá et al. [52] studied the biological hydrogen production using anaerobic sludge of the sewage treatment system of Rio de Janeiro city, Brazil. The authors (op cit.) applied heat treatment (120 °C for 1 h) upon the sewage to inhibit methanogenesis. Tests in anaerobic batch reactors using glycerin for H2 production were obtained of 0.80 mol-H2.mol glycerine-1. Therefore, all such methods need to be verified for crude glycerol fermentation and hydrogen production efficiency.
