International Biodeterioration & Biodegradation 87 (2014) 34e43

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier.com/locate/ibiod

Review

Antimicrobial colorants in molasses distillery wastewater and their

removal technologies
Milton M. Arimi a, Yongjun Zhang a, *, Gesine Götz a, Kirimi Kiriamiti b, Sven-Uwe Geißen a
a
Technische Universität Berlin, Department of Environmental Technology, Chair of Environmental Process Engineering, Secr. KF 2, Straße des 17. Juni 135,
D-10623 Berlin, Germany
b
Moi University Main Campus, Faculty of Technology, P.O. Box 3900, Eldoret, Kenya

a r t i c l e i n f o a b s t r a c t

Article history: Molasses is a widely used feedstock in the bioethanol distilleries, which generate the dark colored
Received 6 September 2013 wastewater known as molasses distillery wastewater (MDWW). This type of wastewater leads to
Received in revised form pollution problems in the local environment where it is disposed of due to the high content of pollutants,
4 November 2013
among which colorants are of great concern. The main MDWW colorants are polyphenols, melanoidin,
Accepted 4 November 2013
Available online 23 November 2013
alkaline degradation products of hexoses, and caramels whose formation, concentration and antimi-
crobial effects are summarized in this review. A lot of efforts have been made in the community to
remove the colorants. Effective treatment methods are discussed, including biological treatment, enzy-
Keywords:
Melanoidin
matic treatment, chemical oxidation, and coagulation. These technologies could also be applied to
Caramel remove the colorants as a final treatment step after the anaerobic digestion.
Polyphenol Ó 2013 Elsevier Ltd. All rights reserved.
Inhibition

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35
2. Cane MDWW generation and characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35
3. MDWW colorants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.1. Melanoidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.1.1. Formation of melanoidin and its structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.1.2. Antimicrobial effects of melanoidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.2. Polyphenol colorants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.2.1. Occurrence of polyphenols in molasses and molasses wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.2.2. Inhibitory effects of polyphenols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.3. Alkaline degradation products of hexoses (ADPH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.4. Caramels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
4. Colorants removal from MDWW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
4.1. Anaerobic digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
4.2. Aerobic post biotreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
4.3. Enzymatic treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.4. Chemical oxidation processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.5. Adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.6. Coagulation and flocculants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

* Corresponding author. Tel.: þ49 (0)30 31425298; fax: þ49 (0)30 31425487.
E-mail addresses: [email protected], [email protected]
(Y. Zhang).

0964-8305/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ibiod.2013.11.002
 M.M. Arimi et al. / International Biodeterioration & Biodegradation 87 (2014) 34e43 35

1. Introduction are applied. On the other hand, MDWW also contains a lot of nu-
trients: w1000 mg/l total nitrogen, w100 mg/l total phosphorus,
Sugar molasses is a viscous dark brown liquid by-product which and >4000 mg/l potassium (Espana-Gamboa et al., 2011), which
is generated in the sugar production process with sugarcane, grapes might point to advantages of MDWW being used as an agricultural
or sugar beets. The main application of molasses is the bioethanol fertilizer. However, this application is questionable due to the acidic
production, which in turn produces a lot of wastewater, called pH, strong dark color and other chemical substances which may
molasses distillery wastewater (MDWW) or vinasse. Bioethanol is lead to groundwater contamination and soil compaction (Gemtos
increasingly used as an alternative fuel because of the high global et al., 1999). In addition, molasses has also been applied as a
fuel demand and the need to reduce greenhouse gas emissions. The cheap carbon source in white biotechnology, e.g., the production of
worldwide production of bioethanol was above 50 billion litres in yeast (Ferrari et al., 2001), lactic acid (Nandasana and Kumar 2008),
2007 and over 60 billion litres in 2008 which represents almost 4% pharmaceutical intermediates (Olbrich, 1963; Küçükaşik et al.,
of world gasoline consumption (Sanchez and Cardona, 2008; Balat 2011), and enzymes (Chapla et al., 2010). These production pro-
and Balat, 2009; Mussatto et al., 2010). The demand for bioethanol cesses generate wastewater with similar characteristics to MDWW
is projected to rise beyond 120,000 million litres in 2020 (Mutlu et al., 2002). As a result, the effort to tackle the problem of
(Demirbas, 2007). Assuming that 40% bioethanol came from sug- MDWW will be beneficial to these industries as well.
arcane and sugar beet sources (Mussatto et al., 2010) and a 1:10 This review will discuss the properties of the main MDWW
ratio of bioethanol to vinasse in the production, the world sugar- colorants, including their formation, concentration and antimicro-
cane/beet MDWW can be estimated to be over 200 million tons in bial effects. Finally, some technologies for the removal of those
year 2007. India, the fourth largest world producer of bioethanol, colorants will also be discussed.
exclusively uses sugar molasses as raw material for bioethanol
production. The same applies to most bioethanol producing coun- 2. Cane MDWW generation and characteristics
tries in the tropics (Sanchez and Cardona, 2008). In the Mediter-
ranean region and in parts of Europe, sugar beet and beet molasses The entire process of MDWW generation from raw cane reeds is
are also used to produce bioethanol. Recent advances in technology shown in Fig. 1 where the data is calculated from publications
like the application of surface-engineered yeast and whole cell (Olbrich, 1963; Wilkie et al., 2000; Sirianuntapiboon and
biocatalysis are expected to boost bioethanol production in the Prasertsong, 2008; Espana-Gamboa et al., 2011; Yadav and Chan-
future (Fukuda et al., 2009). dra, 2012). First, the sugarcane is milled and sugar is extracted with
The disposal of MDWW is problematic due to its high content of water. The solid semi-cellulose by-product called bagasse is used as
pollutants: 50e100 g/l chemical oxygen demand (COD), >50 g/l boiler fuel or treated for fermentation. The sugar extract is heated
total dissolved solid (TDS), and strong acidity (pH 4e6). In addition, and most of the water evaporates. The supersaturated solution is
it has a strong odour and a dark brown color. All these character- passed through a crystallizer where the sugar is crystallized and
istics, plus the high daily production volume, could cause potential recovered. The effluent after a maximum of three crystallization
environmental problems unless appropriate treatment processes steps is called molasses, which contains a high content of sugars

Fig. 1. The general process from the sugar extraction to the MDWW generation.
36 M.M. Arimi et al. / International Biodeterioration & Biodegradation 87 (2014) 34e43

(>50%). The molasses is diluted and brought to a fermentation The melanoidin formation involves 3 stages as summarized in
plant to produce bioethanol with the addition of microbes (yeast). Fig. 2. It starts with the formation of Amadori products by the
The fermentation broth is filtered and then distilled to concentrate condensation of hexose molecules and amino acids. The second
bioethanol. The final effluent after the distillation of bioethanol is a step involves the degradation of Amadori products at pH > 7 to
viscous, dark colored liquid, called MDWW. produce either furfurals or reductones intermediate compounds
In a typical molasses distillery plant, the daily MDWW pro- which undergo a polymerization reaction in the third step to form
duction is as high as 10 L per litre of bioethanol produced (Gough melanoidin. The process also incorporates other reactions including
et al., 1998). A medium sized alcohol distillery can produce cyclisation, dehydration, and retroaldolisations.
approximately 1,000,000 L of ethanol per year (Wilkie et al., 2000); The structure and biological properties of melanoidins have
this approximates to 10 million litres of MDWW annually. It is been reviewed and documented elsewhere (Chandra et al., 2008;
important to note that the composition of MDWW depends on the Wang et al., 2011). There is no exact structure of melanoidins and
source material (beet or sugar) and the geographical location where their size may range from small molecules to very large polymers
beet or cane are cultivated. (le et al., 1998; Yaylayan and Kaminsky, 1998; Wang et al., 2011;
For the treatment of MDWW, the anaerobic digestion is mostly Wagner et al., 2002). A study on glycine-glucose melanoidins
used to recover energy in the form of biogas. However, despite this concluded that all melanoidins had a similar degree of hydration of
reduction the effluent from anaerobic digestion is still dark colored about 3 water molecules per melanoidin (Wedzicha and Kaputo,
and has some remnants of BOD and COD. To meet the stringent 1992). Another study involving synthetic glucose/glycine mela-
environmental discharge standards, further treatment of MDWW is noidin isolated three different types of polymers (Yaylayan and
necessary to reduce these parameters. In particular the MDWW Kaminsky, 1998). The first polymer had nitrogen in its molecule
color is of great concern because some colorants are not only and its empirical formulae was C7H11NO4 and the other two mel-
recalcitrant to biodegradation but also inhibitory to biological ac- anoidin components were non-nitrogenous polymers with an
tivities. Consequently, a high dilution rate is normally applied for empirical formula [CH2O]n (Yaylayan and Kaminsky, 1998). The
the anaerobic digestion which consumes a lot of fresh water. It is of structure of Maillard reaction products depends on several factors
great interest to design an effective treatment process to remove including: pH, temperature, solvent, reaction time, reactants type
the antimicrobial effects of MDWW but it is necessary to identify and their concentrations (Yaylayan and Kaminsky, 1998). The basic
the main source of the antimicrobial effects first. structure of glucose derived melanoidin was suggested as Fig. 3
(Naik et al., 2008). Melanoidins have metal chelating ability,
3. MDWW colorants acting as anionic hydrophilic polymers that can form complexes
with metals (Cämmerer et al., 2012).
The color of MDWW is dark brownish due to the presence of Melanoidins are difficult to characterize because they occur at
natural colorants. The occurrence of synthetic colorants in the different sizes and vary according to the type of sugar and amino acid
molasses is unlikely because there are no inorganic pigment ma- involved in the reaction. Model melanoidin can be prepared by
terials added. Natural colorants are of a wide variety and have been heating amino acids with sugar in presence of sodium hydrogen
well documented including: carotenoids, chlorophyll, heme pig- carbonate at temperatures above 100  C for a few of hours. Pure
ments, anthocyanins, betalains, riboflavins, quinones pigments, melanoidin can be extracted with ultrafiltration from the synthesized
caramels, melanoidins, alkaline degradation of hexoses (ADPH) and model melanoidin (le et al., 1998) or by combination of diafiltration
melanins (Delgado-Vargas et al., 2000; Dai and Mumper, 2010). and ultrafiltration steps (Rufian-Henares and Morales, 2007b).
Among these colorants, melanoidins, plant polyphenols, ADPH, and
caramels have a high potential of occurrence in MDWW. 3.1.2. Antimicrobial effects of melanoidins
Melanoidins from different sources have shown some antimi-
3.1. Melanoidins crobial activities against various microorganisms (Rufian-Henares

Melanoidin and Maillard reaction products (MRP) highly

contribute to MDWW color (Onyango et al., 2011) and account for
over 2% (m/v) of MDWW composition (Yadav and Chandra, 2012).
Melanoidins are brown colored complex polymeric compounds
(Wedzicha and Kaputo, 1992; Yaylayan and Kaminsky, 1998). They
are formed during the last stage of the Maillard reaction, which
occurs non-enzymatically as a result of heating amines and sugars
(Rufian-Henares and Morales, 2007a). Melanoidin formation in
sugar-making processes depends on the nitrogen content, espe-
cially the amino acids. Beet juice has a higher nitrogen content than
cane juice which implies a higher content of melanoidin in the
former (Coca et al., 2004). Some researchers have attributed
MDWW color and its antimicrobial effects to melanoidins (Mohana
et al., 2007; Chandra et al., 2008; Naik et al., 2010).

3.1.1. Formation of melanoidin and its structure

Melanoidin are formed in sugar-making processes through the
Maillard reaction which takes place during the purification step.
This reaction occurs with the presence of sugars and amino acids,
which is favored by the high pH (11e12) and temperature (>50  C)
conditions. Formation of melanoidin mostly occurs during the
evaporation step where the temperature is raised above 100  C
(Coca et al., 2004). Fig. 2. The formation of melanoidin via the Maillard reaction.
 M.M. Arimi et al. / International Biodeterioration & Biodegradation 87 (2014) 34e43 37

(Taylor et al., 2003). Some studies have indicated that the possible
genotoxicity of melanoidin resulted from the complexation of
melanoidin-metal, e.g. with Cu2þ. The radical of reduced metals in
the complex can tear the DNA strands, hence resulting in the
genotoxicity (Cämmerer et al., 2012). The antimicrobial activities of
melanoidins are summarized in Table 1. The antimicrobial effect of
melanoidins can be found in hundreds of mg/l which is much lower
than their typical concentrations nearly 2% (w20 g/L) of MDWW
(Krishna Prasad and Srivastava, 2009). The calculation in Table 1 is
based on the minimum inhibition concentration of Oxytetracyclin
at 16 mg/l which was the maximum value attained for several
gram-negative bacteria (Krishna Prasad and Srivastava, 2009).
Therefore, melanoidins could be one of the main sources of the
antimicrobial effects of MDWW.

3.2. Polyphenol colorants

Polyphenols are natural antioxidants containing phenol

group(s) in their structure and have attracted lots of interest in
recent times due to their special properties including anti-oxidant,
anti-microbial and anti-carcinogenic activities (Tripoli et al., 2005;
Jimoh et al., 2008; Dai and Mumper, 2010). They are categorized
into three broad classes: phenolic acids, flavonoids, and tannins.
Phenolic acids have been detected in MDWW, including benzoic
acid and its derivatives (e.g., gallic acid), cinnamic acid and its de-
Fig. 3. Basic structure of glycine-glucose melanoidin as proposed by Cämmerer et al.
rivatives (e.g., coumaric acid, caffeic acid, chlorogenic acid and
(2002). ferulic acid) (Payet et al., 2005, 2006; Incedayi et al., 2010). During
the extraction of juice, phenolic and its acid derivatives can be
released in the milling processes which are used to break the cane’s
and Morales, 2007a; Ibarz et al., 2008). A study with melanoidin cell membrane and hemicellose components. They are initially
derived from glucose and four different amino acids indicated that clear but can be oxidised to a brownish-yellow color with the
glucose-lysine melanoidin had the highest antimicrobial activity exposure to air. The presence of flavonoids and tannins in MDWW
with the minimum inhibitory concentration of less than 600 mg/l has not been widely reported in literature.
on Geobacillus sterothermophylus (Rufian-Henares and Morales,
2007a). Melanoidins were reported to cause the inhibition of en- 3.2.1. Occurrence of polyphenols in molasses and molasses
zymes involved in the breakdown of protein (Ibarz et al., 2008) as wastewater
well as to possess antioxidant properties (Borrelli et al., 2002; The occurrence of polyphenols in molasses wastewater is
Wagner et al., 2002; Xu et al., 2007; Rufian-Henares and Morales, dependent on both the sources of molasses and the sugar con-
2007b; Kim and Lee, 2009). It was also observed that the soluble centrations in the feed flow (Table 2). In general, polyphenols in
fraction of a model melanoidin had a much higher antioxidant ef- most molasses wastewater are in the range of dozens to hundreds
fect than the insoluble fraction (Wagner et al., 2002). Moreover, it of mg/l (Palla, 1983; Martın et al., 2002; Martin et al., 2003; Jimenez
was also found that the high-molecular-weight melanoidin con- et al., 2004; Bustamante et al., 2005). The type of polyphenols in
tributes more to melanoidin color than the low-molecular-weight cane sugar products is summarized in Table 3 in which the con-
component (le et al., 1998). centration of individual polyphenols is calculated by factoring 14 L
On other hand, the volatile products associated with Maillard of MDWW per litre ethanol and 4.4 L molasses per litre ethanol
reactions have been observed to induce some genotoxic effects production (Nandy et al., 2002). Polyphenols have been identified
when ingested at very high concentrations; these effects were in all cane products including juice, syrup, massecuite, molasses
however not found in both light and heavy fractions of melanoidin and sugar with different compositions. The molasses polyphenol

Table 1
Antimicrobial activities of melanoidins.

Melanoidin type Microorganism Minimum inhibition Reference

concentration

Glucose-glycine model Geobacillus stearothermophylus 400 mg/l MIC (Rufian-Henares and Morales, 2007a)
Glucose-histidine model 200 mg/l MIC
Glucose-alanine model 200 mg/l MIC
Glucose-lysine 100 mg/l MIC
Coffee melanoidin-medium S. aureas 13 g/l MIC Calculated from (Rufian-Henares and Morales, 2008)
roasted (2 g/l)
Coffee melanoidin E. coli 10e45 g/l MIC (Rurián-Henares and Morales, 2008)
Biscuit melanoidin E. coli 7.5e50 g/l MIC
Glucose-asparagine model Peptidases enzyme 420 mg/l and 770 mg/l (Ibarz et al., 2008)
Coffee Melanoidin B. cereus 2.5 g/l MIC (Rufian-Henares and de la Cueva, 2009)
E. coli 5 g/l MIC
S. aeruginosa 4 g/l MIC
Salmonella sp 4.5 g/l MIC
38 M.M. Arimi et al. / International Biodeterioration & Biodegradation 87 (2014) 34e43

Table 2 shown in Table 2. Therefore, the phenolic compounds only

Polyphenols in molasses and molasses wastewater. contribute a minor part into the antimicrobial effects of MDWW.
Molasses type Polyphenol Reference
concentration 3.3. Alkaline degradation products of hexoses (ADPH)
Beet molasses e liquid 450 mg/l (Jimenez et al., 2004)
Cane molasses e dry 400 mg/kg (Palla, 1983) Alkaline degradation products of hexoses are formed when
Vinasse e liquid 477 mg/l (Martın et al., 2002) hexoses are degraded in alkaline media at high temperatures
Wine distillery e liquid 29e474 mg/l (Bustamante et al., 2005)
(above 80  C). Some of the derivatives of degraded hexoses were
Vinasse e liquid 65e766 mg/l (Bustamante et al., 2005)
Pomegranate molasses e dry 551.6e9695.2 mg (Incedayi et al., 2010) detected in cane vinasse, such as 2,2-bifuran-5-carboxylic acid, 2-
GAE/kg nitroacetophenone, P-chloroanisole, 2,3-dimethylpyrazone, 2-
Cane sugar A-molasses e dry 13.88 mg GAE/kg (Payet et al., 2006) methylhexane, methylbenzene, 2-3dihydro-5 methylfuran, 3-
Cane sugar B-molasses e dry 22.34 mg GAE/kg (Payet et al., 2006)
pyrroline and acetic acid which can be transformed by bacteria to
Cane sugar C-molasses e dry 27.21 mg GAE/kg (Payet et al., 2006)
Cane MDWW e liquid 660 mg GAE/kg (Martin et al., 2003)
other products including: 2-nitroacetophenone, p-chloroanisole,
2,2-bifulan, indole, 2 methylhexane and 2,3-dihydro-5-methyl-
GAE ¼ gallic acid equivalent.
furan (Bharagava and Chandra, 2010). The degradability of ADPH by
microorganisms is dependent on the temperature and pH of the
content given in Table 3 represents the highest possible average medium.
value calculated for the three types of molasses: A-molasses (after The antimicrobial effect of APDH is however not significant. The
one crystallization step), B-molasses (after two crystallization ADPH with a carbon chain <C6 are generally colorless with mini-
steps) and C-molasses (after three crystallization steps). Grade C mal antimicrobial effects (Yang and Montgomery, 2007). Those
molasses was reported to exhibit the highest polyphenol concen- with a longer chain >C6 have different compositions depending on
tration (Payet et al., 2006), which can be explained by the fact that the temperature, pH, metal alkali, and sugar used in the process.
more water has been evaporated and more sugar recovered in C Long chain APDH products may polymerize to form colored prod-
molasses than in A and B molasses. ucts (Coca et al., 2004). It was reported that 75% of MDWW color,
The above calculation assumed that the polyphenols in molasses including most of tested ADPH color compounds, can be removed
remained constant throughout bioethanol formation and were all with a consortium of bacteria (Yadav and Chandra, 2012).
passed into MDWW. However, new polyphenols can be formed
during the fermentation process and some may be transformed 3.4. Caramels
from one form to another during different phases of fermentation
process (Romero et al., 2004). This explains why the presence of Caramel is a brownish-black viscous liquid or a hygroscopic
benzoic acid can be found in massecuites but not in the clear juice powder with a high molecular weight (>10 kDa). Caramels from
and syrups during sugar manufacturing. Nevertheless, aerobic and sucrose have been used commercially as food colorants for decades
anaerobic studies of the natural fermentation of black olives indi- and are so far still the most popular in the food industry (Myers and
cated that individual polyphenol underwent both increasing and Howell, 1992). They are formed in a complex series of reactions
decreasing phases during fermentation but the sum of total poly- involving the dehydration of carbohydrates (predominantly
phenols slightly increased during the process (Romero et al., 2004). monosaccharides) to reactive species, which then polymerize
It was also reported that the composition of phenol compounds in together. The caramelization reaction occurs when sugars are
the culture media can influence the formation of polyphenols subjected to high temperatures and the process can be catalyzed by
during the fermentation of wine by lactic acid bacteria (Silva et al., acid and bases. In the process of sugar production caramels are
2011). formed when sucrose syrup is raised above 210  C. This occurs
mainly in the crystallizer where sugar crystals come into contact
3.2.2. Inhibitory effects of polyphenols with hot surfaces. At present, there is no evidence that caramel has
Studies have shown that polyphenols can compromise the a significant antimicrobial effect.
integrity of bacteria’s membrane and thus cause the cell contents,
nucleus, proteins and inorganic ions to diffuse out of the bacteria. 4. Colorants removal from MDWW
The effect is attributable to the structure and lipophilicity of poly-
phenols (Campos et al., 2009). Some phenolic compounds found in Even after anaerobic digestion of MDWW, the effluent still
different wines and in MDWW have been reported to negatively contains high COD and a dark color which does not meet envi-
affect microbial growth (FitzGibbon et al., 1998; Payet et al., 2006; ronmental regulations and standards and thus must be further
Vaquero et al., 2007). The presence of phenolics above 1 g/l can treated. Moreover, the antimicrobial colorants in MDWW could
inhibit methanogesis (Fedorak and Hrudey, 1984). This negatively inhibit the digestion and other biotreatment. As discussed above,
influences the process kinetics like the COD removal and the melanoidins are the main antimicrobial sources of MDWW while
methane production. Phenolics can also inhibit the aerobic degra- polyphenols contribute less to the antimicrobial effect. Various
dation of MDWW (Jimenez et al., 2003). The study of microbial treatment technologies have been employed to remove both
effects of polyphenols with Escherichia coli and Pseudomonas groups of colorants, as discussed below.
putinda in microtitre plates found that polyphenols in the culture
media can interfere with the formation of bacterial biofilm as well 4.1. Anaerobic digestion
as the quorum sensing mechanism (Huber et al., 2003). The
inhibitory effects of polyphenols are also widely observed in olive Anaerobic digestion involves obligate or facultative bacteria in
mill wastewater (OMW) (Hamdi, 1996). However, the polyphenol the absence of oxygen and with organic carbon as an energy source.
content in OMW is much higher (3e6 g/l) (Bertin et al., 2011) than Raw MDWW has a high COD which can be tapped for the recovery
in MDWW. The inhibitory levels of polyphenols on different mi- of energy with the formation of biogas. Anaerobic digestion is thus
croorganisms are summarized in Table 4. Generally, the inhibition the first step of MDWW treatment. The most widely used anaerobic
effects of polyphenols occur at the concentration of several hun- process is the up-flow anaerobic sludge blanket (UASB) (Wilkie
dred mg/l which is the upper limit of the value found in MDWW as et al., 2000; Mohana et al., 2007; Satyawali and Balakrishnan,
 M.M. Arimi et al. / International Biodeterioration & Biodegradation 87 (2014) 34e43 39

Table 3
Polyphenols in sugarcane products (mg/kg) and calculation of their concentration in MDWW (mg/l).

Polyphenol type Clear juice Syrups Massecuites Sugar Reference Molassesa MDWW

Protocatechuic acid 6.3 8.5 19.4 0.4 (Payet et al., 2006) 71.1 22.2
r-hydroxybenzoic acid 15.8 16.4 27.6 _ 73.77 23.05
Chlorogenic acid e e e e 11.83 3.70
Vanillic acid e 2.2 7.5 e 25 7.81
Cafeic acid 8.3 57 25.2 0.6 147.7 46.16
Syringic acid 5.0 11.1 13.5 e 54.8 17.12
Vanillin 8.1 10.3 14.5 e 44.2 13.81
r-coumaric acid 135.8 140.9 144.7 10.2 424.6 132.69
Ferulic acid 69.8 114.4 71.3 11.2 255.1 79.72
Benzoic acid e e 2.1 e 128.4 40.13
Phenyl glucosides e e e e e e
Phenylpropanetriol glucosides e e e e (Palla, 1983) e e
a
The average value of the three types of molasses (A, B & C).

2008). The anaerobic process can reduce the BOD content by over volatile polyphenols but can be degraded by a group of bacteria
80%, of which almost 90% is recovered as biogas. The superiority of called Pseudomonas (Liu et al., 2012). It was also observed that
UASB lies in its ability to retain high biomass in the reactor. The Pseudomonas fluorescens could degrade highly chlorinated phenols
process can also be modified with the application of granular bed, as a sole carbon source up to 480 mg/l of phenol (Lin et al., 2008).
fixed bed, filter, etc. Other innovations include the involvement of Supplementation of carbon source to the culture media can
mesophile culture and a two-phase system, which separates the enhance the bacterial growth and consequently improve the
acidogenic and methanogenic steps. More information on the phenol degradation (Lin et al., 2008). It was also reported that
applicable anaerobic processes can be found in other reviews Ochrobactarium sp isolated from industrial wastewater could
(Wilkie et al., 2000; Satyawali and Balakrishnan, 2008). degrade phenol with the addition of molasses substrate (Kılıç,
One of the limitations to MDWW anaerobic treatment is the 2009). The ability of some bacteria species to breakdown MDWW
requirements of high dilutions due to the antimicrobial compo- colorants is facilitated by some enzymes which they produce. A
nents as discussed above (Jimenez et al., 2004; Bharagava and consortium of four bacteria was reported to excrete enzymes
Chandra, 2010). Despite the high COD removal with diluted (manganese peroxidase and laccase) which were shown to be able
MDWW, the inhibiting chemicals still remain in effluent after to reduce the molasses melanoidin by over 70% (Yadav and
anaerobic digestion. Therefore, a further treatment step is required Chandra, 2012). Another study indicated that some yeast strains
to remove the remaining dark color and COD. Another strategy is to could substantially reduce the melanoidin contents in MDWW
pre-treat MDWW before aerobic digestion, e.g., with ozonation, UV (Sirianuntapiboon et al., 2004). A consortium of two Bacillus sp. and
light plus titanium dioxide in order to improve the efficiency of one Alcaligenes sp was reported to remove 70% color from vinasse
MDWW anaerobic digestion (Martın et al., 2002; Jimenez et al., with nutrient supplements (Bharagava and Chandra, 2010). Though
2004). However most of the input energy in these processes goes Bacillus sp has a high capability of degrading distillery colorants, the
to eliminate the organic COD which could be recovered as biogas. optimization of culture conditions is necessary to achieve good
This makes the pretreatment of MDWW untenable. It is thus results (Kaushik and Thakur, 2009).
preferable to treat the MDWW anaerobically first and then with Other than bacteria, white-rot fungi also have a great capability
further treatment steps. to remove polyphenol/melanoidin from wastewater. The fungi are
capable of degrading recalcitrants and are also tolerant to toxic
4.2. Aerobic post biotreatment environment, which makes them robust and versatile in bioreme-
diation of toxic chemicals. In MDWW, it has been widely shown
Aerobic biotreatment mostly applies either as a pure strain or as that the fungi can effectively remove MDWW color, thanks to their
a consortium of microorganisms in the presence of air or oxygen. extracellular enzymes including manganese peroxidase, lignin
This process step is ordinarily a post-treatment step after anaerobic peroxidase and phenol oxidase (laccase) (Raghukumar et al., 2004;
digestion. According to a report by Li et al. (2009), a mixed culture Asgher et al., 2008). One limitation of applying fungi to remove
from activated sludge can remove tannic acid polyphenol under melanoidin in MDWW is the requirement of very high dilution
aerobic condition. Phenol and chlorophenols are more toxic than rates and the addition of food supplements (Raghukumar et al.,

Table 4
Antimicrobial activities of polyphenols.

Microorganism Polyphenol/phenol Concentrations Effects Reference

S. putinda & S. sobrinus Molasses polyphenols 4000 mg/l MIC Bacterial growth inhibition (Takara et al., 2007)
Salmonella 10 types Avg MICs, 1519 mg/l Growth inhibition (Taguri et al., 2004)
E. coli Avg MICs, 795 mg/l Growth inhibition
B. stearothermophilus Tea polyphenols 200 mg/l Vegetative growth inhibition (Sakanaka et al., 2000)
500 mg/l Spores growth inhibition
S. mutans & S. sobrinus Cranberry polyphenol 100 mg/l Biofilm formation (Yamanaka-Okada et al., 2008)
500 mg/l Growth inhibition
Bacteria and yeast Hemicellulose plant 100e500 mg/l MIC Growth inhibition (Cruz et al., 2001)
E.coli Plant polyphenols 2500 mg/l MIC Growth inhibition (Gohar et al., 2003)
S.aureus 3160 mg/l MIC Growth inhibition
Candida albicas 1990 mg/l MIC Growth inhibition
Methanogens bacteria OMW 50e1000 mg/l Methanogenesis inhibition (Borja et al., 1997)
40 M.M. Arimi et al. / International Biodeterioration & Biodegradation 87 (2014) 34e43

2004). This would make these processes not economically viable consume a lot of non-selective free radicals. On the contrary, ozone
considering the very high MDWW volume in a typical bioethanol can selectively attack the double bonds (e.g. C]C, N]C) and
plant. Another disadvantage of fungi is their slow growth rate, functional groups (e.g. eOCH3, eOH, and eCH3) in acid or neutral
especially when faced with competitors. It is important to note that conditions with limited concentrations (Mantzavinos and Psillakis,
these fungal processes are still in laboratory scale and are not yet 2004). Polyphenols, melanoidins and other MDWW contaminants
optimized for large-scale applications. Culture contamination have double bonds (C]N & C]C) and other functional groups in
common in wastewater treatment remains a major challenge in the their structure can be selectively broken down by ozone without
application of fungi for the color removal especially when large the extensive oxidation of the vast organic matters (Naik et al.,
quantities of wastewater are involved and sterilization is not 2010). The selectivity of ozone was clearly demonstrated in the
feasible. study of Martin et al. (2003): 80% of phenolics were removed after
In addition, it was also found that the toxicity of MDWW was ozonation of MDWW at an acid condition while only a 16% removal
increased after the treatment with fungus Pleurotus sp (Tsioulpas of the organic load occurred.
et al., 2002). This was probably due to the transformation of poly- However, some contradictory results have been reported on
phenols to less colored but more toxic products. A consortium of color removal by ozonation. It was found that ozonation was able to
bacteria using different nutrients was shown to reduce the color increase anaerobic biodegradability of diluted MDWW from zero to
compounds in vinasse by 70% but some compounds were not 33% (Battimelli et al., 2010). Another study observed no improve-
biodegraded and some ADPH were transformed to other colored ment of the biodegradability of r-coumaric acid polyphenol by
compounds (Bharagava and Chandra, 2010). However, a different pretreatment with ozone (Otal et al., 2004). In a study with the
consortium of bacteria was observed to reduce 75% MDWW color diluted cherry stillage, ozonation was able to increase phenolic
with a removal of most of all ADPH color compounds tested (Yadav removal after an aerobic digestion by more than two folds (Beltrán
and Chandra, 2012). The limitations of aerobic digestion method et al., 2001).
include the energy costs required for aeration and the high volume The main drawback of using the above-mentioned oxidation
of sludge formed. The process also requires nutrient supplemen- processes is their high costs. However it is still interesting to study
tation and operates at high dilution rates. the chemical pretreatment (partial oxidation) to increase microbial
biodegradability of MDWW colorants to find the optimal balance of
4.3. Enzymatic treatment cost and performance.

The most studied enzymes for the breakdown of colored pol- 4.5. Adsorption
lutants in wastewater treatment include manganese peroxidase,
lignin peroxidases and laccases (Bibi et al., 2011). For instance, lac- Adsorption occurs when substances (adsorbate) in gas or liquid
cases from Pleurotus sajor-caju fungi were able to remove over 60% of are physically or chemically bonded to a surface of a solid (adsor-
initial phenol concentrations in OMW (Massadeh and Modallal, bent). Adsorption process has been successfully used to recover the
2007). Commercial laccase was also found to be able to reduce polyphenols from wastewater using common solvents and adsor-
polyphenol up to over 40% of the initial concentration of 2.5 g/l in bents (Pendyal et al., 1999; Agalias et al., 2007; Bertin et al., 2011;
OMW (Tsioulpas et al., 2002). The application of commercial en- Soto et al., 2011). A complete removal of polyphenol was ach-
zymes could be useful in systems like MDWW where it is difficult to ieved with a process involving the successive filtration and
cultivate specific fungi or bacteria due to the competition from other adsorption steps with commercial adsorbent resins (XAD16,
microorganisms. However, the cost of commercial enzymes at the XAD7HP) followed by the recovery of solvents (Agalias et al., 2007).
moment is high and would make the process not cost effective. In The high cost of commercial adsorbents is their main drawback
addition, some technologies are required to keep the enzyme in a for application, which leads to a search for cheap alternative ad-
reactor, e.g., by immobilization or membrane separation, which may sorbents, e.g. from solid waste. Granular activated carbon (GAC)
further increase the investment and operational cost. from sugarcane bagasse was found to adsorb polyphenol more
efficiently than GAC made from several other agricultural products
4.4. Chemical oxidation processes (Pendyal et al., 1999). Elsewhere, adsorbents like activated carbon
from bagasse bottom ash showed promising results in the mela-
The most popularly studied chemical oxidation processes in noidins removal (Simaratanamongkol and Thiravetyan, 2010).
pollution remediation are the advanced oxidation processes (AOP) Despite their low costs compared to activated carbon, natural ad-
which can oxidize a broad range of organic compounds with the aid sorbents and modified plant materials have the disadvantage of a
of free radicals. Various research groups have reported applications low adsorption capacity. In addition, it would be very interesting to
of AOP to reduce polyphenol in wastewater (Flouri et al., 1996; study how to selectively adsorb MDWW colorants and thus to
Dwyer et al., 2008, 2009; Afify et al., 2009). The efficiency of AOP improve the biodegradability of MDWW.
on colorants removal varies with the method chosen. A study re- However, the adsorbed colorants are mostly not destroyed and
ported an increase of biodegradability of OMW more than three therefore their disposal remains a problem afterwards. It was re-
folds after Fenton treatment (Kallel et al., 2009). However, ported that a process involving the simultaneous adsorption and
hydrogen peroxide alone at 20 g/l showed a color reduction up to biodegradation of melanoidin via the biofilm on activated carbon
50% of initial value in another study (Flouri et al., 1996). A combi- can largely improve the color removal in comparison to the biofilm
nation of AOP (UV/H2O2) followed by biological treatment of syn- or activated carbon alone (Onyango et al., 2012). Melanoidins are
thetic melanoidin was also reported to produce a higher color useful ingredients in food industry particularly in beer making
removal than a treatment with AOP only (Dwyer et al., 2008). where they are used as melanoidin malt because of their texture
Ozonation is another oxidation process which has attracted and flavor (Morales et al., 2012), which makes their recovery after
great interest in colorant removal, e.g. polyphenols from waste- adsorption useful. Adsorption followed by desorption steps can be
water (Battimelli et al., 2010). The method has advantages over AOP used to recover melanoidins from wastewater for potential appli-
since it can be used in either the pretreatment step (Battimelli et al., cations. However, the value of melanoidins is not as high as that of
2010) or in the final treatment step (Beltrán et al., 2001). AOP are polyphenols whose recovery from wastewater has attracted a lot of
mostly used in the final treatment because other organics will interest.
 M.M. Arimi et al. / International Biodeterioration & Biodegradation 87 (2014) 34e43 41

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