Accepted Manuscript

Biotechnological revalorization of Tequila waste and by-product streams for

cleaner production – A review from bio-refinery perspective

Gibrán S. Alemán-Nava, Ilaria Alessandra Gatti, Roberto Parra-Saldivar, Jean-

Francois Dallemand, Bruce Ritmann, Hafiz M.N. Iqbal

PII: S0959-6526(17)31581-0

DOI: 10.1016/j.jclepro.2017.07.134

Reference: JCLP 10137

To appear in: Journal of Cleaner Production

Received Date: 28 June 2016

Revised Date: 17 July 2017

Accepted Date: 17 July 2017

Please cite this article as: Gibrán S. Alemán-Nava, Ilaria Alessandra Gatti, Roberto Parra-Saldivar,
Jean-Francois Dallemand, Bruce Ritmann, Hafiz M.N. Iqbal, Biotechnological revalorization of
Tequila waste and by-product streams for cleaner production – A review from bio-refinery
perspective, Journal of Cleaner Production (2017), doi: 10.1016/j.jclepro.2017.07.134

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 ACCEPTED MANUSCRIPT

(Wordcount=8205)

Biotechnological revalorization of Tequila waste and by-product streams for cleaner

production – A review from bio-refinery perspective

Gibrán S. Alemán-Navaa, Ilaria Alessandra Gattia, Roberto Parra-Saldivara,*, Jean-Francois

Dallemandb, Bruce Ritmanna,c, and Hafiz M. N. Iqbala,*

aTecnologico de Monterrey, School of Engineering and Science, Campus Monterrey, Ave.

Eugenio Garza Sada 2501, Monterrey, N.L., CP 64849, Mexico; bEuropean Commission,

Joint Research Centre, Institute for Energy, Via E. Fermi 2749, TP 450, 21027 Ispra (Va),

Italy; cSwette Center for Environmental Biotechnology, The Biodesign Institute, Arizona

State University, Tempe, AZ, USA.

*Corresponding author: Tel.: +52 (81) 8358 2000 Ext. 5561 Sub-ext. 115; Email

addresses: r.parra@itesm.mx (R. Parra-Saldivar); hafiz.iqbal@my.westminster.ac.uk and

hafiz.iqbal@itesm.mx (H.M.N. Iqbal).

Abstract

In this paper, industrial processing and biotechnological revalorization of Tequila waste and

by-product streams have been reviewed. Tequila production process generates different

kind of waste and by-products with a huge potential to produce value-added products.

Therefore, many research projects, scientific investigations and innovative utilizations of

such waste and by-product streams have been the object of recent studies. Moreover, the

bio-based transformation from a petrochemical-based economy to a bio-based economy

necessitates the novel exploitation of cost-effective natural materials for both future

biorefinery development and a range of value-added products of interests. The present

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review article focuses on an area not comprehensively reviewed previously, the potential of

utilizing waste and by-product streams from current Tequila industry activities. The first

part of this review focused on various processing aspects and prospects on the fermentative

production of Tequila. In the second and third parts, statistical and sustainable aspects and

the generation of waste and by-product streams of Tequila industry are critically reviewed,

respectively. In the fourth part, various industrial and biotechnological applications of

Tequila’s wastes are comprehensively discussed from a bio-refinery perspective. In

conclusion, it is evident that biotransformation of waste and by-product streams has great

potential and significant prospects for wider industrial and biotechnological applications.

Keywords: Agave; Bagasse; Green biotechnology; Vinasses; Waste valorization

1. Introduction

Tequila is a Mexican alcoholic beverage, both production and commercialization are

verified and certified by the Mexican Tequila Regulatory Council (CRT) (Lo´pez-Alvarez

et al., 2012; Nava-Cruz et al., 2015). According to the norm, Tequila has to be prepared

from the heads of the Agave Tequilana Weber var. azul, hydrolyzed or cooked, and then

subjected to alcoholic fermentations with yeast. With 219 producers and 1648 brand names,

Tequila was registered from the CRT in 2014. Tequila industry signifies an important

economic boost for the 180 municipalities within the Denomination of Origin territory

(CRT, 2014). The protected territories by the tequila denomination of origin, Jalisco,

Nayarit, Guanajuato, and Tamaulipas states with 8, 6, 29 and 12 municipalities,

respectively, are shown in Figure 1 (Iñiguez-Covarrubias et al., 2001a). The number of

direct employees in this sector in 2013 was 29,360, 5,360 of which in the industry and

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24,000 on the field. The sector generated 997.0 million USD in export, and the value of the

national market was estimated to be around 12,500 million MXN (CNIT, 2014). Production

of Tequila increased massively between 1995 and 2008, going from 104.3 millions of liters

(40% Alc. Vol.) in 1995 to 312.1 millions of liters in 2008 (CNIT, 2009). The complex

cultivation of agave has always complicated supply and demand patterns. The fact that the

plant takes 6 to 10 years to mature after being planted has repeatedly caused cycles of

shortage and surplus of agave, as can be noticed in Figure 2. This kind of trend has

progressively made it difficult for supply chain actors to successfully coordinate the supply

of agave with the demand for Tequila. After 2009, Tequila production increased slightly

until 2011, when it began decreasing once again due to an agave shortage that made the

prices boost up to 500%. In 2013, Tequila production reached 226.5 millions of liters, with

a similar agave demand of 756.9 thousands of tons (CNIT, 2014).

The agave waste from the Tequila industry has a great potential for biotransformation into

value-added products. A wider spectrum of high-added value products with industrial

interests e.g. platform chemicals including enzymes, fuels, and electricity, bioactive

molecules for nutraceutical, pharmaceutical, biomedical and biotechnological sectors of the

modern world, can be obtained using naturally abundant materials such as agave and other

lignocellulosic-based materials (Bilal et al., 2017; Arevalo-Gallegos et al., 2017).

Therefore, biomass waste (re)-valorization has recently emerged. There have been many

contributions in utilizing waste biomass both from plant and animal wastes and reviewed

elsewhere (Iqbal et al., 2013; Liguori et al., 2013; Santibañez-Aguilar et al., 2014;

Sukumara et al., 2014; Xu et al., 2014; Evcan Tari, 2015; Fernández-Rodríguez et al., 2016;

Gallegos et al., 2016; Masran et al., 2016; Narron et al., 2016; Putro et al., 2016; Rouches

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et al., 2016; Zhang et al., 2016). However, the present review mainly focuses on an area not

comprehensively reviewed previously, the potential of utilizing waste and by-product

streams from current Tequila industry activities. The part of the review focused on various

processing aspects and prospects industrial and biotechnological applications of Tequila’s

wastes from a bio-refinery perspective.

2. Tequila production process

The Tequila production process starts with the harvesting of the agave plants considered to

be mature enough: the sugar content is at a maximum when the plant is approximately 10

years old. Plants who are not deemed to be ready yet are left to complete the growth. The

harvesting consists in bluntly cutting the leaves from the agave, to be able to extract the

head (also known as “piña”) from the ground. The heads are then transported to the

distillery, where a sample of each head is taken and analyzed to determine reducing sugar

content (an indicator of the amount of inulin), along with many other critical parameters

e.g. moisture content, ash content, processing temperature and pH. Moreover, to further

facilitate a uniform cooking and handling during the production process, the head parts of

the agave plant are sliced into small pieces. Agave leaves represent the 46% of the whole

plant weight and usually left in the field during harvesting process (Cedeño Cruz and

Alvarez Jacobs, 1991; Iñiguez-Covarrubias et al., 2001; Corbin et al., 2015).

The next phase is the cooking, which hydrolyzes the complex carbohydrates (inulin) of the

agave heads into simple sugars (fructose and sucrose) which are suitable for fermentation

processes. It also softens the heads to make the process of sugar extraction easier. The

traditional cooking takes place in brick or stone ovens, and it is a process that lasts from 50

to 72 h and involves steam injection. More modern versions of this process usually take

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place in autoclaves of several tons capacity: the higher sealed capability of these tanks

allows the required time for this process to be reduced to 14 or even 8 h. After a stipulated

cooking time, a sweet liquid i.e. mainly fructose is collected and used later as a source of

free sugars.

Fermentation follows the sugar extraction via traditional extraction method. Fermentation

time varies depending on environmental temperature, but it usually lasts many hours and

differs among factories due to different standards applied. Once the fermentation process is

concluded, and the must have reached an alcoholic content of 5 to 7%, it can be left to settle

a few hours before moving onto the distillation stage. The fermented must was then used to

separate further and concentrate the alcohol contents via process distillation. Tequila is

usually distilled twice. The first distillation is known as “destrozamiento”: it takes a couple

of hours, and it yields the “Tequila Ordinario”, meaning a liquid with an alcohol level of

around 20%. The second distillation, also known as “rectificación”, takes between 3 to 4 h.

The result of this process is a liquid with an alcohol level near 55%, also known as “Tequila

Blanco”. This product can be sold, or it can be aged to become another type of Tequila.

Distillation phase generates a liquid waste called vinasses, which remains in the bottom of

the still. In a typical Tequila distillery, 7 to 10 liters of this effluent are produced per liter of

Tequila. Due to their low pH, high temperature, and high organic loads, they represent a

significant disposal problem. Figure 3 illustrates a schematic diagram of the Tequila

production process.

3. Sustainability of Tequila Industry

3.1. Economic and Social Sustainability

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In the 1990s, a fungal infection and an early winter frost, along with the usual cycles of

surplus and shortage, caused a decrease of 50.7% in the blue agave population in Jalisco

(González, 2002; Bowen and Zapata, 2009; Figueroa-Castro et al., 2013). The shortage

consequences were amplified by the increased popularity of Tequila in domestic and

international markets and pushed many small Tequila companies out of the market (Bowen

and Zapata, 2009). This crisis led to several changes in the production relations of the

Tequila industry:

• Agave production expanded into new areas, where there was no traditional agave

cultivation (Macías, 2001)

• The main tequila producing sectors became more self-sufficient in their agave supply, to

avoid and tackle the associated risks of surplus and shortage (Bowen, 2008)

• Tequila industry became more concentrated, with major firms controlling the majority of

the market

• Formerly Mexican firms were bought by multinational companies (Casas, 2006).

As a result, without a proper intervention, local Mexican traditions, and culture connected

to the Tequila production process risk to be prejudiced irreparably.

3.2. Environmental Sustainability

From the environmental point of view, the explosion of the Tequila market started in the

1990s has caused the need for intensifying and expanding agave cultivations. This situation,

along with federal government restrictions on the variety of agave to be used to produce

Tequila has an adverse effect on other varieties of agave progressively used previously for

the same purpose, leading to a reduction of the biodiversity in the region. The Agave

Tequilana Weber var. azul is a semelparous plant, meaning that once it flowers, it dies. Its

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reproduction can be sexual, by seeds, or asexual, by offshoots from rhizomes or bulbils

from the inflorescence. For Tequila production purposes, rather than allowing the plant to

grow the flower to attract pollinating birds, bats, and moths that feed on the nectar, the

flower stalk is cut as soon as it begins to sprout, interrupting in this way the flowering

process. The reason is that during the flowering process the plant consumes its reserve of

carbohydrates and then dies. The nineties explosion of the Tequila market encouraged

growers to continue the traditional practice of removing the inflorescence before

fertilization and seed formation occurs and using the vegetation propagation (asexual

reproduction) exclusively for producing new plants. This overuse of asexual reproduction

has in time created whole fields of genetically identical blue agaves. The agave shortage

between 1999 and 2003, for instance, was caused by a massive epidemic that lowered

agave stem production from 780 to 413 thousand tons (CRT, 2004). The cultivation of a

single, preferred genotype is a very common practice in commercial agriculture. However,

it is usually available a base of genetic variation for the crop species that can be exploited if

needed. Recent studies have shown that it is a very different case for the Agave Tequilana

Weber var. azul: one of the lowest levels of polymorphism to date was detected for this

species. It, therefore, appears to be no readily available base of genetic variation for this

variety of agave (Vega et al., 2001).

The Agave Tequilana has been considered to have a minimal impact on soil fertility due to

its unusually long harvesting cycle. The shallow rooting system and succulent morphology

also facilitate its environmental adaptation in desert regions. This is mainly due to its

crassulacean acid metabolism (CAM photosynthesis), which allows a carbon fixation

pathway with minimum loss of water. These factors have contributed to the success of the

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cultivation of the Agave used in Tequila production. In the past 20 years, though, the rising

global consumption of Tequila and the consequent increase in the cultivation of agave have

newly prompted questions regarding its impact on soil composition and characteristics.

Another common practice is to allow livestock to graze on mature fields, as it is supposed

to be beneficial for controlling weeds and manure distribution over the fields as a fertilizer.

A third common practice is to treat the field with the distillery effluent, vinasses. The

disposal of this effluent had become difficult and expensive due to new and stricter

regulations. As a result, Tequila companies started to use it as an organic fertilizer. It also

appeared that it brought elevated soil calcium and foliar phosphorus and magnesium

(Monroy Reyes, 1999), which was considered to be beneficial for the agave cultivation.

Recent studies have examined the effects of the common agricultural practices involved in

the blue agave cultivation on soil fertility (Gobeille et al., 2006). Practices of tillage,

livestock grazing and field amendment with distillery effluent were studied. The addition of

distillery effluent was found to increase mean levels of soil phosphorus, potassium,

calcium, and boron, as well as CEC (cation exchange capacity). These results show that

blue agave cultivation stimulates the depletion of soil nutrients and alters soil physical

properties. Whether a lower availability of soil nutrients is associated with reduced

productivity or not is yet to be ascertained.

4. Generation of wastes – problematic issue

The production process of Tequila generates a considerable amount of waste. Figure 4

shows the amount of waste generated per liter of Tequila produced. As these numbers want

to prove, the disposal of Tequila industry waste has increasingly become a problem for

Tequila producers, due to the growing market.

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Agave Tequilana Weber var. azul leaves typically are 90 to 120 cm in length and 8 to 12

cm in width, firm fibrous, rigid, acuminate and concave, generally blue or gray-green in

color (Gentry, 2004; Chattopadhyay and Khan, 2012; Hulle et al., 2015)). The parts of the

leaves are cut off from the base of the plant during the harvesting and are left in the field to

recycle nutrients or amassed in wide spaces, unused. It was found that the wet agave head

represents the 54% of the plant in mass, while the remaining 46% is made of leaves,

meaning that almost half of the plant usually remains unused (Iñiguez-Covarrubias et al.,

2001).

The residual material left behind after the harvesting and cooking of head part following

mining and washing for sugar extraction is usually termed as agave bagasse, which is

fibrous in nature. It is composed of thick-walled and long fiber (approximately 5-10 cm)

and pith (Iñiguez-Covarrubias et al., 2001). Table 1 shows physical composition and

chemical composition of dry agave bagasse at 5% water content (Iñiguez-Covarrubias et

al., 2014). Moreover, most of the bagasse is not utilized at all, causing illegal dumping or

improper applications on agricultural land, along with pollution by leachates, odor

generation and habitat for pests and diseases (Rodriguez, 2001). The solution to the

problem of final disposal of agave bagasse is to find an appropriate technology for indirect

assessment and treatment, so that it can be integrated into the environment (manure,

compost), within the food chain (forage, fodder yeast), or in industrial processes of

economic interest (paper pulp, agglomerated materials).

Vinasses represent significant disposal and treatment problems due to their complex

physicochemical composition. Table 2 shows the physio-chemical characteristics of

Tequila vinasses in detail. They are usually toxic in natural, particularly for micro-

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organisms, along with some anti-oxidant features and are among potential candidates for

waste management (López-López et al., 2010). With ever increasing scientific knowledge,

ecological awareness and demands from the legislative authorities, an eco-friendly

remediation treatment is an absolute requirement to save the current ecological system.

Figure 5 illustrates a tentative pilot plant scheme for the treatment of Tequila vinasses.

5. Biorefinery from Tequila’s Waste

5.1. Agave Leaves as Fiber Source for Paper Products

The paper is considered a ubiquitous product, in nature, and used for many applications in

our daily lives (Manda et al., 2012). The pulp & paper industry processes huge quantities of

agricultural-based lignocellulosic materials every year which is harvested either directly or

indirectly from various sources. Owing to the cost-effective ratio, improvement in the

processing modalities along with an ever increasing demand (Singh et al. 2012; Iqbal et al.,

2013), pulp & paper can be made from lignocellulosic-based materials including agave

leaves. Three main processing steps, i.e., (1) pulping, (2) bleaching, and (3) production are

potentially involved in the paper manufacturing process (Iqbal et al., 2013). Based on the

processing mechanism there is three main types of pulping i.e. (i) mechanical pulping, (ii)

chemical pulping and lastly (iii) chemical and mechanical pulping (combination pulping)

(Iqbal et al., 2013; Gallegos et al., 2016).

Recent studies have tried to revalorize the agave leaves, a troublesome waste left behind the

field, by their fiber content and quality (Iñiguez-Covarrubias et al., 2001). Many

researchers have tried to investigate the potential of agave leaf as a source of fibers for

paper production. Iñiguez-Covarrubias and co-workers (2001) have compared the fibrous

characteristics of the agave leaves with two major types of woods i.e. (i) softwood e.g. pine

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and (ii) hardwood e.g. eucalyptus, which is particularly being used in papermaking. In the

same study, they have also found that agave fibers have higher cellulose content than either

type of the woods mentioned above. Owing to this high cellulosic contents, agave leaves

have the remarkable potential to provide a pulp with higher yields. As compared to the

softwood e.g. pine and hardwood e.g. eucalyptus, agave leaves have lower lignin contents

which are also an important property to avoid and reduce chemical consumption during the

entire pulping process. In summary, the agave-based fibrous material has a large

perspective that could be potentially utilized for many paper-based products e.g. handicraft,

geotextiles, filters, packaging and composites (Iñiguez-Covarrubias et al., 2001).

5.2. Agave Leaves as a Substrate for the Production of Enzymes

Enzyme production is a growing field of biotechnology and has become an integral part of

the current biotechnological sector from the modern world. One of the most appropriate

approaches to produce cost-effective, competent, highly active and novel enzymes for

multifunctional purposes is to capitalize the lignocellulosic-based waste materials e.g.

agave leaves (Huitron et al., 2008). Such potential materials contain noteworthy

concentrations of soluble carbohydrates and potent inducers of enzyme synthesis ensuring

efficient production of enzymes (Reddy et al., 2003, Moldes et al., 2004; Elisashvili et al.,

2006; Iqbal et al., 2011a, b; Asgher and Iqbal, 2011; Asgher et al., 2012a, b, c). In recent

years, many potent technologies ranging from flask shake to large scale have been

developed and well documented in the literature for various enzymes ligninases e.g. lignin

peroxidase, manganese peroxidase, versatile peroxidase, manganese independent

peroxidase, phenol oxidoreductase i.e. laccases along with many other like cellulytic

mainly cellulases production (Moldes et al., 2004; Elisashvili et al., 2006).

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Enzymes like ligninases, cellulases, xylanases, pectinases and inulinases are nowadays used

in a variety of sectors, from food and beverage manufacturing to biomass conversion and

waste treatment. The potential use of Agave Tequilana Weber var. azul has been exploited

as a fermentative substrate for enzyme production. In an earlier study conducted by Huitron

and co-workers (2007), two filamentous fungal strains were found able to simultaneously

secrete endopectinases, and exopectinases, along with xylanases, and inulinases. In this

context, agave wastes particularly agave leaves have gained exceptional consideration from

both the academic and industrial researchers because of its potential as an inexpensive

carbon and energy sources for the production of ligninolytic and lignocellulolytic enzymes.

Likewise, many other research investigations have also proved the potential of using Agave

Tequilana waste as a prospective candidate material for many biotechnological applications

(Huitron et al., 2007; Nava-Cruz et al., 2015; Yang et al., 2015). Ligninolytic, cellulases

and hemicellulases are important industrial enzymes having numerous applications and

biotechnological potential for various industries including chemicals, fuel, food, brewery

and wine, animal feed, textile and laundry, pulp and paper and agriculture (Couto and

Sanromán 2006; Levin et al., 2008; Oberoi et al., 2010; Asgher and Iqbal 2011; Iqbal et al.,

2011a; Iqbal et al., 2013).

5.3. Agave Bagasse for Animal Feeding

In a ruminant’s diet, cellulose is considered a primary carbon and energy source. In this

context, agave bagasse utilization for animal feeding could have a remarkable influence on

livestock management. However, the presence of lignin limits the digestibility of cellulose

thus requires some preferential delignification treatment before use for animal feeding. To

enhance the pre-digestibility and nutritional value, the concept of preferential

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delignification of lignocellulosic materials has been applied (Iqbal et al., 2013). Moreover,

to further facilitate this kind of use, it is, therefore, necessary to physically separate the

more digestible part of the bagasse, the pith, from the low digestibility fibers. In particular,

bagasse that came from shredded agave heads before cooking and sugars extraction in

pressing mills yielded the higher percentage of recovered pith (56%). Feeding trials have

been conducted on Pelibuey crossbred male sheep, testing 3 comparative diets: 79.3% (on

dry basis) ground corn-based diet, 63.2% agave bagasse pith based diet and 63.2% ground

corn stubble based diet. It was found that daily feed consumption and average weight gains

were statistically the same for the three diets (Iñiguez-Covarrubias et al., 2001). The study

also calculated that a Tequila factory is producing 68 ton/day of agave bagasse, with 36%

pith recovered, could feed 24,480 animals daily if using a proper agave bagasse pith

balanced diet. Finally, it is important to underline that agave bagasse could potentially have

an economic value with an advantage of being available all year around (Iñiguez-

Covarrubias et al., 2001).

Another study investigated the chemical and physical composition of agave bagasse, along

with its potential for ruminants feeding material (Ramírez-Cortina et al., 2012). The authors

underlined the high content of lignin being a problem for digestibility, but also stated the

benefit of the high proportion of free sugars (22% in dry basis) for direct use in ruminant

food portions. Digestibility value of agave bagasse was compared with sunflower’s and

straw’s and located between the two. An alternative suitable technology to increase its

digestibility was also proposed. Alkaline pretreatments, besides of saponifying waxes and

cuticles, also perform solubilization of lignins and hemicellulose, increasing digestibility of

lignocellulosic residues. Therefore, in this case, the high content of lignin was decreased

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using a calcium hydroxide treatment. Moreover, the treatment prevents drying, responsible

for the decrease of free sugars. Furthermore, Ca(OH)2 concentration in treatment provides

an important contribution of calcium, which is beneficial for the composition of animal

feeding (Ramírez-Cortina et al., 2012).

5.4. Agave bagasse for fiberboard production

From the last few decades, there has been increasing research interests towards the

utilization of lignocellulosic-based materials and by-products as composite materials, in

particular, for thermal insulation, false ceilings, fiberboards and packaging materials.

Recent articles have investigated the potential of agave bagasse to be used for these

purposes. In the same study on the use of agave bagasse as feeding material, the agave

bagasse fibers separated from the pith were tested for fiberboard production (Iñiguez-

Covarrubias et al., 2001). A range of medium density and high-density boards samples

were prepared and tested by Iñiguez-Covarrubias and co-workers 2001, using short and

long agave bagasse fibers. Medium specific gravity fiberboards were found to have similar

moisture and mechanical properties, as compared to similar fiberboards made from aspen

fiber. High specific gravity boards were stronger in bending tests than the ANSI standards

(American National Standards Institute) for hard boards. The comparable properties and the

better bending resistance of fiberboards made of agave bagasse fibers show that this could

be a possibility of cost-efficient use of this low-value waste by-product of the Tequila

industry (Iñiguez-Covarrubias et al., 2001).

5.5. Agave Bagasse for Agricultural Purposes

Another potential use for agave bagasse has also been proposed for agricultural purposes as

a substrate. A recent study investigated the composting process of agave bagasse from two

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different Tequila companies, using ammonium nitrate as a nitrogen source to adjust the

agave bagasse C: N ratio to 25:1, the recommended value for an excellent biodegradability

(Iñiguez-Covarrubias et al., 2011). Furthermore, a field study was conducted, using the

agave bagasse composts as substrates for tomato production. Comparisons with commercial

substrates were also carried out. The different composts used did not show statistically

significant differences at the end of the 21 days of composting: total grams of tomatoes

produced per plant and their quality (color, diameter, deformations) were found to be

similar. These results show the potential of the use of agave bagasse as a substrate for

agriculture cultivations.

Agave bagasse based compost has also been used as a substrate for new-born agave plants.

As it has already been mentioned, agave reproduction and propagation is conducted

asexually, which guarantees more control, but weakens the new plantations and makes

them more sensitive to pests and infestations. This procedure involves seedling and

micropropagation in vitro. After that, micro-propagated agave plants must pass a period of

adaptation in the nursery, for nine to twelve months. Only after they can be taken to the

fields. This period of acclimatization is usually carried out in a greenhouse, with proper

treatments. Canadian peat and coconut powder substrates are commonly used for this

purpose, although they are expensive. On this matter, agave bagasse based substrate could

represent a potential cheap substitute. A recent study investigated this potential. In

particular, the response of micro-propagated seedlings of agave in four mixtures of agave

bagasse compost was analyzed. A comparison with coconut powder, Canadian peat, and the

commercial substrate was also carried out, by evaluating the following parameters:

diameter of the cone and stem diameter, the length of longest leaf, the width of the longest

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leaf and number of leaves (González et al., 2013). It was found that treatments based on

agave bagasse compost led to better values of the morphological parameters if compared to

the coconut powder and the peat results. These results show the possibility of substitute the

commercial substrate with the compost substrates, particularly with the mixtures 30, 50 and

70%. By making this substitution, a huge waste of the Tequila industry would be used

(bagasse) through composting, avoiding environmental pollution, and reducing production

costs, since the compost can be made locally (González et al., 2013).

5.6. Agave Bagasse for Biosolids Treatment and Vinasses Disposal

Agave bagasse can also potentially be used to solve the problem of vinasses treatment

partially. In particular, recent studies investigated the use of bagasse as a final disposal for

vinasses. Composting of agave bagasse was once again involved, but this time addition of

nutrients was not participating in the degradation of agave bagasse (Iñiguez-Covarrubias et

al., 2005). Only urea and vinasses were added to the composting process to maintain

moisture. Four bagasse piles were considered. During the composting process two of them

were irrigated with vinasses and the other two with water. One water wetted pile, and one

vinasse wetted pile was added with urea at the beginning of the process, to adjust the C: N

ratio to the optimum 25:1. Every two months the piles were moved to facilitate aeration and

the addition of water and vinasses. Samples were taken for the analysis of pH, organic

matter, and cellulose. At the end of the composting time, composts were subjected to

analysis of ash, total organic carbon, N, P, K, and conductivity, along with some

phytotoxicity analysis. The test period for the piles with urea was 228 days. During this

time, 0.912 L of vinasses were added per kg of wet bagasse. It was also found that the

obtained compost had similar characteristics to the garden soil without problems of

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phytotoxicity: composts from the four treatments had no adverse effects on seedling

emergence, relative growth, germination and root elongation of cucumber seeds (Iñiguez-

Covarrubias et al., 2005).

5.7. Agave Bagasse for Slaughterhouses and Tanneries Waste Disposal

Tanneries waste are classified as hazardous residues and mainly come from the preparation

of the hide for hardening and are composed of hair and flesh material. Due to their organic

composition and previous experiences in the biodegradation of animal waste, the agave

bagasse residues could potentially be used to compost slaughterhouses and tanneries waste.

Since this is an aerobic process of degradation, in some cases it is necessary to facilitate the

passage of air by adding some material that increases the pore structure of the residue

susceptible to degradation. A recent study investigated the use of agave bagasse as a

promoter of the air passage in the composting process of tanneries waste, as well as the

potential use of the final product for cultivation purposes (Íñiguez et al., 2003).

Temperature changes in the thread material were daily monitored during biodegradation

process. Microbial and chemical analyses were performed on the compost, as well as

germination studies. From the microbial analyses, the presence of Escherichia coli was

found.

5.8. Agave Bagasse for Bioplastic Production

There has been considerable interest in agricultural-based materials like agave bagasse in

recent years due to their potential as an alternative source of the traditional petroleum-based

synthetic materials. Another possible use for agave bagasse could be for the production of

bioplastics. A recent study investigated the ability of Saccharophagus degradans to

degrade the major components of plant cell walls and to synthesize polyhydroxyalkanoates

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(PHAs, also known as bioplastics) (Alva Munoz and Riley, 2008). Biopolymers-based

bioplastic such as PHAs generated from renewable natural sources by micro-organisms are

often biodegradable, biocompatible and non-toxic in nature (Iqbal et al., 2014a; 2015).

Therefore, the development of bioplastics using one or more individual biopolymers are

among the routes to improve some of the properties of biodegradable polymers (Miao and

Hamad, 2013; Hooshmand et al., 2014; Iqbal et al., 2014a,b). PHAs belongs to a family of

bio-polyesters produced by microbes under limited nutritional conditions (e.g. nitrogen or

phosphate) (Sudesh et al., 2000, Chen and Wu, 2005), or excess carbon source (Ojumu et

al., 2004; Keshavarz and Roy, 2010). The unbalanced nutritional supply causes the bacteria

to accumulate PHAs in the form of granules as an internal energy storage, as shown in

Figure 6. The main members of the PHAs family are presented in Table 3 based on the

generic structural formula for the PHAs where x is 1 or higher, and R can be either

hydrogen or hydrocarbon chains of up to C16 in length (Iqbal, 2015). PHAs offer a broad

range of mechanical properties, are biodegradable and could potentially substitute oil based

plastics and polymers. It has already been demonstrated that S. degradans can attach to

cellulosic fibers and degrade the cellulose to use it as a primary carbon source. Its use is

attractive also due to its non-pathogenesis, its capacity of decomposing and metabolize a

wide variety of insoluble complex polysaccharides, and its production of three key enzymes

for PHA synthesis (Ekborg et al., 2005). Over the past several years, owing to the above

discussed characteristics, many scientists, around the globe, have directed their research

interests into the development of engineered constructs using PHAs for various applications

including bio-medical, pharmaceutical, drug delivery, antibacterial packaging or sanitary

materials, and household items (Fillat et al., 2012; Iqbal et al., 2014a; 2015a,b).

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6. Conclusions

This work presents various alternatives for the disposal of Tequila industry byproducts,

following the concept of the biorefinery. Agave leaves, mainly composed of cellulose,

hemicellulose, and lignin could potentially be used as a sugar source for animal feed or

alcohol production, as a fiber source for paper products or as a substrate for enzymes

production. Agave bagasse, a fibrous solid residual, was found to be suitable for animal

feeding or fiberboard production, as well as composting. It was also considered as an

efficient way for slaughterhouses and tanneries waste disposal and bioplastic production.

Recent studies also investigate the use of Agave bagasse for ethanol production. Vinasses,

the residual generated during distillation, represent significant disposal and treatment

problems due to their complex physiochemical composition. Their high biological oxygen

demand, dissolved salts content and low pH make them a recalcitrant waste that is hardly

decomposed by the usual biological processes. New anaerobic treatments for biogas

production are being considered and evaluated at laboratory scale. It is firmly believed by

the author that, due to the large amounts of waste produced by the Tequila industry, the best

solution for the disposal problem presented in this paper does not lie in the choice and

implementation of a single treatment. On the contrary, a mix of some of the alternative

treatments presented would probably represent the most efficient option, from both an

economical and environmental point of view.

Acknowledgement

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This paper was supported by the Emerging Technologies Research Group and the

Environmental Bioprocesses of Tecnologico de Monterrey, Mexico. The authors would like

to thank Tecnologico de Monterrey, Mexico for providing literature facilities.

Declaration of interest

The authors report no declarations of interest in any capacity, i.e., competing or financial.

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Table 1 Physio-chemical composition of dry agave bagasse at 5% water content

(Reproduced from Iñiguez-Covarrubias et al., 2014, with permission from Elsevier).

Parameter Characteristic or percent value

Physical composition
Texture Not very rigid
Color Brown-yellow
Fiber length 5-10 cm
Diameter 0.3-0.1 mm
Chemical composition
Cellulose 43%
Hemicellulose 19%
Lignin 15%
Nitrogen 3.0%
Pectins 1.0%
Fats 1.0%
Reducing sugar 5.0%
Ash 6.0%
Others 2.0%

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Table 2 Physio-chemical characteristics of Tequila vinasses.

Parameter Value
pH 3.4-4.5
Oils and fats (mg/L) 10-100
Total COD (mg/L) 60,000-100,000
Soluble COD (mg/L) 40,000-80,000
Total BOD (mg/L) 35,000-60,000
Soluble BOD (mg/L) 25,000-50,000
Total solids (mg/L) 25,000-50,000
Total suspended solids (mg/L) 2,000-8,000
Fixed suspended solids (mg/L) 10-500
Volatile suspended solids (mg/L) 1,990-7,500
Total dissolved solids (mg/L) 23,000-42,000
Settle-able solids (mg/L) 10-900
Total alkalinity (mg/L) < 6.00
Total acidity (mg/L) 1,500-6,000
Fixed acidity (mg/L) 1,480-5,800
Volatile acidity (mg/L) 20-200
Ca (mg/L) 200-1,100
Mg (mg/L) 100-300
K (mg/L) 150-650
Phosphates (mg/L) 100-700
Total nitrogen (mg/L) 20-50
Organic nitrogen (mg/L) 5.0-10
Total reducing sugars (%w) 0.5-2.0
Direct sugars (%w) 0.4-1.0
Cu (mg/L) < 3.0
Fe (mg/L) < 45
Ni (mg/L) < 0.02
Zn (mg/L) < 1.0

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Table 3 Main PHAs structures based on the general structure (Reproduced with permission
from Iqbal, 2015).
Name Abbreviation x value R group
Poly(3-hydroxypropionate) P(3HP) 1 Hydrogen
Poly(3-hydroxybutyrate) P(3HB) 1 Methyl
Poly(3-hydroxyvalerate) P(3HV) 1 Ethyl
Poly(3-hydroxyhexanoate) P(3HHx) 1 Propyl
Poly(3-hydroxyheptanoate) P(3HHp) 1 Butyl
Poly (3-hydroxyoctanoate) P(3HO) 1 Pentyl
Poly (3-hydroxynonanoate) P(3HN) - Hexyl
Poly(3-hydroxydecanoate) P(3HD) 1 Heptyl
Poly(3-hydroxyundecanoate) P(3HUD) 1 Octyl
Poly(3-hydroxydodecanoate) P(3HDD) 1 Nonyl
Poly(3-hydroxyoctadecanoate) P(3HOD) 1 Pentadecanoyl
Poly(4-hydroxybutyrate) P(4HB) 2 Hydrogen
Poly(5-hydroxybutyrate) P(5HB) 2 Methyl
Poly(5-hydroxyvalerate) P(5HV) 3 Hydrogen

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Figure captions

Figure 1 Protected territories by the tequila denomination of origin, Jalisco and Nayarit,

Guanajuato, and Tamaulipas states with 8, 6, 29 and 12 municipalities, respectively

(Reproduced from Iñiguez-Covarrubias et al., 2001a, with permission from Elsevier).

Figure 2 Tequila production and agave demand from 1995 to 2013.

Figure 3 A schematic illustration of Tequila production process using agave as a potent

substrate.

Figure 4 Waste generation per liter of Tequila produced.

Figure 5 A tentative pilot plant scheme for the treatment of Tequila vinasses.

Figure 6 PHAs granules (A) and schematic of a PHA granule (B). The core consists of

PHA polymer that is enwrapped by a phospholipid monolayer and proteins on the outside.

The proteins consist of PHA polymerase, PHA depolymerase, structural proteins, and

proteins of unknown function (Reproduced with permission from Iqbal, 2015).

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List of Figures

Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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