Review

Valorization of whey using a biorefinery

Jessica L. Sebastián-Nicolás , Luis G. González-Olivares, Gabriela A. Vázquez-Rodríguez,
Carlos A. Lucho-Constatino, Araceli Castañeda-Ovando, Centro de Investigaciones Químicas,
Universidad Autónoma del Estado de Hidalgo, Pachuca, Hidalgo, Mexico
Alma E. Cruz-Guerrero, Departamento de Biotecnología, Universidad Autónoma Metropolitana,
Mexico City, Mexico

Received August 27 2019; Revised March 14 2020; Accepted March 18 2020;

View online at Wiley Online Library (wileyonlinelibrary.com);
DOI: 10.1002/bbb.2100; Biofuels, Bioprod. Bioref. (2020)

Abstract: In Mexico, whey is discarded into drains without treatment, which represents an environmental
problem. It is known that whey is a mixture of nutrients that increase biochemical demand (BOD)
and chemical oxygen demand (COD), exceeding the limits established by national and international
standards. Some uses of whey have been proposed to reduce the negative impact it produces on
the environment. Some of these uses are the production of high-value raw materials, separation of
macromolecules of industrial interest, and the generation of energy, among others. The integral use of
waste under a biorefinery scheme represents a viable alternative for the use of this waste. In this way,
the chemical composition of the waste can be used to recover compounds with high added value. The
main objective of this review is to propose a dairy whey biorefinery to use components of commercial
interest. This biorefinery process involves three stages: the first is the separation of lactoferrin using a
polyacrylamide matrix (PAM) impregnated with copper; the second is lactic fermentation using the residue
resulting from the separation of lactoferrin, from which lactic acid, hydrolyzed proteins, and probiotics
will be obtained. Finally, in the third stage, the residue from the previous stage can be used to generate
biogas. © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd

Key words: biorefinery; milk whey; pollution; bioactive peptides and lactic acid

Introduction the technology to recover value-added compounds from

MW. In developing countries, such as Mexico, 30% of the

T
he dairy industry is an important source of income
worldwide. In 2015, milk production was 650 million
tons and cheese production was 20 million tons.
This generated 180 million tons of milk whey (MW).1 A
total of 40 × 106 tons/year of whey is produced in the EU
and 557 000 tons is exported globally, especially to Asian
countries.2 This exported whey represents around of 30%
of the total and it is used to produce different products,
principally dry whey, lactose, and whey protein concentrate
(WPC). However, only in industrialized countries is there
MW generated is used mainly for feeding animals and
obtaining ricotta cheese. The remaining volume is discarded
to phreatic mantles and soils, representing an environmental
problem in these countries.3
An alternative treatment of whey for the generation of
biogas has therefore been sought, producing methane (CH4)
and hydrogen (H2) in an efficient manner. However, this has
only been carried out at a laboratory level with small volumes
of whey, or diluted whey to reduce the retention time of the
liquid in the production chambers. This process does not

Correspondence to: Alma E. Cruz-Guerrero, Departamento de Biotecnología, Universdidad Autónoma Metropolitana,

Mexico City, Mexico. E-mail: aec@xanum.uam.mx

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd 1
 J Sebastián-Nicolás et al.
allow the recovery of the high value-added products present
in it, such as proteins, lactose, and salts.4, 5
The proteins found in the MW have been studied due
to their bioactive properties. During the hydrolysis of
these proteins, bioactive peptides are released, which, after
being ingested by humans, are used as adjuvants to reduce
chronic degenerative diseases.6 The search for alternatives
to separate and purify these compounds using biological
activity has generated interest in their incorporation into
food and food supplements.7 It is known that the peptides
released from lactic fermentation have biological activity as
anticancer, antimicrobial, antihypertensive, antithrombotic,
and metal carrying. This activity is related to the sequence of
amino acids that are part of the structure of oligopeptides.8
These compounds can be produced by chemical and
biotechnological synthesis. Chemical synthesis is a process
with high costs, but biotechnological processes lead to lactic
fermentation with microorganisms of different genera,
producing various products of commercial value such as
bioactive peptides, probiotics, organic acids, hydrolyzed
proteins, and amino acids.9–12 Lactoferrin is also present in
MW, which has a biological function that depends on the
presence of metal ions in its structure.13
The objective of the separation of proteins from MW is
to use them as nutritional supplements and in some cases
as bioactive substances in the preparation of functional
foods.14–16 Milk whey is commonly used as livestock
food and sometimes in the production of lactose or
protein concentrates through an ultrafiltration process.17
Ultrafiltration has also been used to reduce the contamination
Proteins
Ultrafiltration process
Proteins
Emulsifier Fermentation
Hydrolyzed
Proteins
Food and
Pharmaceutical
Industry
Figure 1. Potential useful components available in milk whey (MW). Source: Mollea et al. (2013).21
2 © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2100
Review: Valorization of whey milk through plan biorefinery
that this waste emits into the ambient environment. Proteins
or lactose have been recovered with this separation technique;
however, the cost is very high.18, 19
Biorefinery processes have been used as an alternative to
reduce the negative impact of agro-industrial waste on the
environment. A biorefinery aims to recover value-added
products through the integration of chemical, physical, and
biological techniques. Currently, with these processes, the
time that the pollutant remains in the environment is shorter
and industries generate ‘zero waste’. For this reason, the
proposed process for biorefining MW will take advantage
of the value-added components found in it. The whey
biorefinery integrates chemical, physical, and biological
techniques for the recovery of lactoferrin, lactic acid,
probiotics, hydrolyzed proteins, and bioactive peptides as
well as the characterization of the final residues to generate
biogas. For this reason, the aim of this review is to propose
a biorefinery taking advantage of the whey’s composition
and technological value. This biorefinery could be integrated
taking the most value components reaching a treated waste
biotechnologically.
Whey composition
To illustrate the importance of the components of MW, Fig. 1
shows its chemical composition, its health benefits, and the
industrial importance of each of the nutrients.20
Cheese making can be carried out by enzymatic treatment
or through the addition of organic acids. The first treatment
produces sweet whey. It involves adding proteases such
Cheese Whey
Lactose Minerals Water
Salts
Permeate
Chemical and
Agricultural
Industry
Lactic Acid
and Organic
Acids
Polymer and
Biotechnological
Industry
 Review: Valorization of whey milk through plan biorefinery J Sebastián-Nicolás et al.

as chymosin or from a microbial source. These exhibit Whey proteins

high coagulant activity, producing good-quality cheese at
Proteins are the most studied component of whey. Their
lower cost. Novel specific peptidases for milk coagulation
importance relates to some of their biological processes,
have been obtained from various fungal genera, such as
which lead to the production of proteins or peptides that
Mucor, Rhizomucor, Aspergillus, Endothia, Rhizopus, and
are used in the food and pharmaceutical industries.28 Whey
Phanerochaete.22
proteins represent 20% of the proteins present in bovine
The second treatment is carried out in the presence of
milk.21 They are divided according their solubility, molecular
organic acids, so that acid whey is obtained. Although
weight, and biological properties (Table 2).29, 30 Due to the
the manufacturing processes are different, the chemical
nutritional value and characteristics of these proteins, various
composition of whey is similar regarding the content of
purification methods have been used to obtain them. Several
protein, lactose, and minerals, as shown in Table 1.
lines of research have therefore been developed during the
Some products could be manufactured in order to
last decade.
valorize cheese whey (CW). Process such as condensation,
ultrafiltration, and demineralization are used to process the
whey into different commercial products, increasing the
Lactose
value of this waste. These kinds of processes take advantage of Lactose is the most abundant carbohydrate with respect
whey’s composition (Fig. 2). to the other nutrients present in MW. It is found in

Table 1. Chemical whey composition (concentration expressed as g L−1).

Component Sweet whey Acid whey Component Sweet whey Acid whey
Total solid 63.0–70.0 63.0–70.0 Phosphate (PO43−) 1.0–3.0 2.0–4.5
Lactose 46.0–52.0 44.0–46.0 Lactate 2.0 6.4
Protein 6.0–10.0 6.0–8.0 Chloride (Cl−) 1.1 1.1
+
Calcium (Ca) 0.4–0-6 1.2–1.6 Sodium (Na ) 3.1 0.21
Source: Yadav et al.,23 Jelen et al.,24 Kosseva et al.,25 and Nishanthi et al.26

Cheese Whey

Condensed or Demineralized Ultrafiltration Unutilized

powdered whey whey process disposed as waste

Human food Water

Infant food
Whey protein
Animal feed Permeate
concentrate (WPC)

Purified lactose Purified proteins

Lactitol Lactose Fermentation
Lactulose

Unicelular protein
Probiotic production
Bacteriocins
Enzymes
Organic acids
Exopolysacharides (EPS)
Bioplastics

Figure 2. Final products obtained from milk whey (MW) by different process. Source: Mollea et al. (2013)21 and Marwaha and
Kennedy (1988).27

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2100 3
4
Table 2. Proteins and properties from cheese whey (CW).
Protein General characteristics Concentration Separation or purification Biological functions References
gL−1 techniques
31–35
β-lactoglobulin (β-lg) Chain of 162 amino acids and molecular 3.2–3.6 Ultrafiltration and ion exchange Fat-soluble vitamins and
weight of around 18.4 kDa. Different (diethylaminoethyl cellulose) cholesterol transport.
genetic variants and the most abundant Regulator in the mammary
are A and B gland to carry out
J Sebastián-Nicolás et al.

phosphorus metabolism
Synthesis of glutathione
stimulation
33, 34, 36, 37
α-lactoalbúmin (α-la) Chain of 123 amino acids and molecular 1.2–1.6 Ultrafiltration, precipitation, Synthesis of lactose
weight around 14.4 kDa. Structure with and ion exchange
wo domains, one formed by an α helix (sepharoseiminodiacetate-Cu+2)
and the other by a β sheet
38–41
Albumin of the bovine Chain of 582 amino acids and molecular 0.4–0.5 Ultrafiltration, precipitation and ion Transport of low molecular
serum (BSA) weight of 69 kDa. The structure is similar exchange (MAG polyethyleneimine anion weight fatty acids
to blood serum albumin exchange)
42–44
Immunoglobulins (Igl) Group of proteins synthetized as 0.7–1.0 Ion exchange (sepharose 6B-organic Antimicrobial and antiviral
antybodies. Molecular weight between ligands Cu2+)43 activity. Lower cholesterol
22 and 69 kDa. The Igl have very similar and blood pressure
structures and are linked together by
disulfide bonds
45–47
Glycomacropeptide (GMP) Chain with 64 amino acid residues and a 1.2–1.3 Ultrafiltration with some modifications Effect over phenylketonuria
molecular weight of 6.8 kDa. Absence of (membrane 5000 Da) illness
aromatic amino acids Ultrafiltration combined with ion-
exchange chromatography
48–53
Lactoferrin (Lf) Chain around 690 amino acid residues 0.1–0.2 Hydrophobic interaction. Affinity Biological effect as anti-
and a molecular weight of 80 kDa. separation (superparamagnetic inflammatory, antifungal,
Fe3+ atoms form four binders with polyglycidyl) antiviral, antibacterial, and
two tyrosines, one aspartate and Methacrylate particles coupled with anticancer
one histidine which are united in the heparin. Ion exchange
presence of carbonate ions (CO32)
53–55
Proteose-peptone (Pp) Chain of 311 fractions from β-casein, as 0.6–1.8 Hydrophobic interaction and Action as bifidogenic factor
a result of the activity of plasmin chromatography with a dual salt system

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2100
55–57
Lactoperoxidase (Lap) Natural milk enzyme with a chain of Traces Ion exchange (sepharose 6B-reactive Association with 17
612 amino acid residues. It contains 15 red 4 dye and cryogel embedded with antimicrobial activities
cysteine residues, a heme group and cellulose beads)
carbohydrate residues.
Review: Valorization of whey milk through plan biorefinery
 Review: Valorization of whey milk through plan biorefinery J Sebastián-Nicolás et al.

concentrations of 5–6% and is commonly recovered for WPC

use as a food additive and as a carbon source in microbial
culture media. This nutrient has been recovered from MW
by ultrafiltration, diafiltration, and reverse osmosis. During
these processes, larger particles such as proteins are retained
by the use of membranes. The recovery efficiency of pure
lactose with these methods is up to 98%; however, applying
membranes in a purification process has a high cost, so only
some industries that have this type of technology give added
value to MW.58–60
Whey minerals
Milk whey retains some minerals present in milk. Among
the most abundant minerals are calcium (Ca), sodium (Na),
phosphorus (P), magnesium (Mg), potassium (K), zinc
(Zn) in low concentrations, iron (Fe), and copper (Cu).
Manganese (Mn) can be found at between 40–130 mg L−1
and the other minerals are below 40 mg L−1. The presence of
Ca, Mg, and P, is important in milk due to their association
with the retention of casein micelles in the curd during the
manufacture of cheese.61, 62
Technological use of whey
Animal feeding
Industries that make handmade cheese generate volumes of
MW lower than 500 L per day. This whey is regularly used
as a food source for pigs, sheep, and cattle. This reduces
food costs because this product increases the animal’s
weight. However, for this to be carried out, farmers must
collect fresh MW and transport it, avoiding any type of
microbial contamination that could affect the animal’s
health. Although this activity is associated with small
industries, not all the MW produced is used as livestock
feed, especially in times of high temperatures and during
the rainy season.63
In the middle of the 20th century, the production of protein
concentrates was made from large volumes of MW. This was
possible through the application of ultrafiltration processes
with membranes allowing the production of WPC, which
varied from 34% to 80% of the protein.64, 65 This type of
product has a high demand, mainly from athletes, as it
increases muscle mass.66 The WPC is produced with different
concentrations of protein because its chemical composition is
variable (Table 3).8, 67
Proteins are produced commercially by ultrafiltration to this
day. The process starts with the pretreatment of whey (6% of
protein) using skimming, bactofugation, and pasteurization,
and then reverse osmosis to eliminate ions and salts. After
pretreatment, a concentrate whey, with 20% protein, is
obtained. This is the basis for the production of WPC with
different concentrations of protein. The general process is
shown in Fig. 3.68
However, there is an economic disadvantage due to the
use of membranes. The lifetime of a membrane used for
these purposes is affected by physicochemical factors such
as pH, temperature, and the presence of ions. That is why,
currently, before starting the concentration process, the CW
is pretreated, involving measures such as the elimination of
fat, increasing the pH, and the elimination of calcium, leading
to an improvement in the yield from the process.69
Figure 4 shows a WPC developed by Barba et al.70 The
mass balance is developed in each part of the process. It is
starts with acidification of the whey with H2SO4 (0.1 N) and a
diafiltration process is carried out to obtain WPC powder.
Process to obtain lactoferrin from whey
Over time, the purification of whey proteins has been a
subject of research for pharmaceutical and nutritional
purposes due to their biological functions. Proteins can
be recovered by chromatographic methods. Affinity

Table 3. General composition of different whey protein concentrates (WPC).

Product Protein Lactose Fat Ash Moisture
%
Whey powder 11–14.5 63–75 1–1.5 8.2–8.8 3.5–5.0
Reduced lactose whey powder 18–24 52–58 1–4 11–22 3–4
Demineralized whey powder 11–15 70–80 0.5–1.8 1–7 3–4
Whey protein concentrate-34 34–36 48–52 3–4.5 6.5–8.0 3–4.5
Whey protein concentrate-50 50–52 33–37 5–6 7.5–8.5 3.5–4.5
Whey protein concentrate-80 80–82 4–8 4–8 3–4 3.5–4.5
Whey protein isolate 90–92 0.5–1.0 0.5–1.0 2–3 4.5

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2100 5
 J Sebastián-Nicolás et al. Review: Valorization of whey milk through plan biorefinery

Figure 3. Schematization of the two scenarios considered (BS = baseline, AS = alternative) Bacenetti et al.87

8
5 6 9

1 3
11
Acidif DRYING
2 UF DF 1° DF 2nd
10
4 7

Volume Proteins Lactose Ashes Fats Proteins

Stream Fluid
L g L-1 g g L-1 g g L-1 g g L-1 g %
1 Whey 47 8.6 405 42 2000 5.8 270 0.5 23 15
2 H2SO4 0.1 N 0.05 - - - - - - - - -
3 Acid whey 47 8.6 405 42.8 200 5.8 270 0.5 23 15
4 Permeate UF 21 2.4 49 42.8 900 5.8 1120 0 0 -
5 Retentate UF 26 13.6 356 42.8 1100 5.8 150 0.9 23 22
6 Ret.DF first stage 26 11 290 4.7 123 1.0 26 0.9 23 63
7 Per. DF first stage 73 2.7 198 15 1100 2 150 0 0 -
8 Water 73 - - - - - - - - -
9 Ret. DF second stage 26 6 160 0.35 9 0.03 0.82 0.9 23 83
10 Per.. DF second stage 73 1.8 135 1.6 114 0.34 25 0 0 -
11 WPC poder - - 160 - 9 - 0.82 23 83

Figure 4. Diafiltration process and mass balance during production of WPC. Source: Barba et al.70

6 © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2100
 Review: Valorization of whey milk through plan biorefinery J Sebastián-Nicolás et al.

chromatography has been used for the separation and
purification of proteins according to specific functional
groups.71
Lf was isolated for the first time in the secretions of the
mammary gland by Sörensen et al.72 Later, it was identified
that this protein had Fe3+ ions in its structure, providing
various biological functions, with antimicrobial and
anticancer activity being studied the most at present.73–76
Currently, different methods have been used to separate and
purify lactoferrin (Table 4).
Carvalho et al.52 used a functionalized cryogel for the
separation of lactoferrin from whey and Sebastián et al.77
reported a polyacrylamide matrix (PAM) with Cu (II) to
carry out the separation and enrichment of lactoferrin from
MW, obtaining 82.5% recovery of this protein. The use of
chromatographic methods for the purification of lactoferrin
has increased its efficiency due to modifications that are
made to the supports. These modifications allow high yields
regardless of the volume of whey.50, 78–83
Lactic acid production through
fermentation
Recent research has focused on the lactic fermentation of
residues containing simple carbohydrates for the production
of lactic acid. Whey is an optimal substrate for the growth of
different lactic acid bacteria (LAB), yielding 70–90% lactic
acid.84, 85 The production of lactic acid by fermentation
has disadvantages, especially the high cost of recovering
it by conventional methods such as distillation or simple
extraction.86
Conventionally, lactic acid could be recovered by different
methods87 (Fig. 5) – for example by final crystallization after
a long and difficult process using sulfuric acid and activated
carbon. This method is expensive and unfriendly to the
environment. To reduce the cost, many studies have been
conducted to examine the recovery of lactic acid. Methods
such as diffusion dialysis, solvent extraction, distillation,
membrane extraction of liquid surfactant, adsorption,
chromatographic methods, ultrafiltration, reverse osmosis,
drying, conventional electrodialysis, and microfiltration have
been used.88–91
Although some methods are cheaper and more effective
than electrodialysis or adsorption, it is these last two that offer
a better balance between effectiveness and cost.92, 93
Bioactive peptides production
During lactic fermentation, LAB has an important role,
not only in the production of organic acids but also in
the generation of peptides. These fractions are released
by the proteolytic system of the same bacteria, as they
have an efficient proteolytic system to reduce proteins
to oligopeptides or small chains of amino acids. These
released fractions are the nitrogen source necessary for cell
growth incorporating amino acids into their metabolism.94
This system consists of three main steps: 1. proteolysis by
proteases bound to the cell wall to form a large number of
peptides; 2. transport of peptides to the interior of cells by
a transport system of peptide fractions, specific for each
microorganism; 3. degradation of peptides by a diverse
group of peptidases to form free amino acids, which are
metabolized or expelled to the medium and used for the
synthesis of new proteins.95–97
The biological activity of bioactive peptides depends
largely on the amino acids that constitute them. That is why,
in recent years, there have been many efforts to study the
different beneficial activities that these peptides can have
on humans, including their antihypertensive, antimicrobial,
opioid, immunostimulatory, mineral transporting, and
antithrombotic activity.98 These molecules are of great
interest, mainly for the industrial sector (food and drugs)

Table 4. Separation and purification methods of lactoferrin from cheese whey (CW).
Separation and purification method Characteristics Yield References
52, 77
Ion exchange and solid-phase separation Copper immobilized in polyacrylamide cryogel 82.5%
78
Dye-affinity chromatography Dye-affinity chromatography with yellow HE-4R
attached to chitosan mini-spheres
79
Cation exchange Cryogel monoliths for extraction of minor proteins
from milk by cation exchange
52
Supermacroporous column of polyacrylamide cryogel
with copper ions
80
Adsorptive membrane chromatography
81
Cation-exchange Expanded bed adsorption
50
Mixed-mode (hydroxyapatite) chromatography
method for one-column fractionation

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2100 7
 J Sebastián-Nicolás et al.
Diluate
Retentate
Bipolar
NaOH
electrodialysis
Concentrated salt
solution HCl
Monopolar
Salt-containing
electrodialysis fraction
Figure 5. Scheme of a co-product free fermentation and downstream process. Dashed lines illustrate further suggestions for
process improvements. Source: Pleissner et al.87
because they can be the basis for the production of food and /
or medicines.99
Whey as a source of minerals
The MW is demineralized because of the negative effects
on food products obtained from it, like ice cream, drinks,
deserts, or cooked products.100 However, the minerals from
whey are generally not recovered because of the high cost of
the process. But there are some studies related to the recovery
of minerals like Na, Ca, and P, which are relevant to the
production of food, fertilizers, and plant supplements.101, 102
Environmental impacts of whey
Whey has a higher polluting potential with high contents of
biological and chemical oxygen demand (COD) (20–60 g L−1
and 50–102 g L−1, respectively). Organic load is mainly due
to lactose, proteins, and fats (Table 1). Although this organic
matter is 99% biodegradable, it is considered to be highly
polluting – 60 to 80 times more than domestic wastewater.
If whey is discharged into water streams without treatment,
several serious problems can arise. The organic matter it
contains is easily biodegraded in environmental conditions,
leading to dissolved oxygen depletion. Along with septic
conditions, strong odors are released, and the aquatic
medium turns into a breeding place for pests such as malaria-
8 © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2100
Review: Valorization of whey milk through plan biorefinery
Lactic acid
fermentation
Retentate
Fermentation broth
Microfiltration
Permeate
Cromatography
Lactic acid containing
fraction
Concentration
Lactic acid
causing mosquitoes.103 Lactose, a major whey constituent,
has been reported as a growth promoter of sewage fungus
in waterways, which are slimy plumes that are aesthetically
undesirable and affect the normal river biota.104 Another
important impact of whey in aquatic media is due to its
high nutrient (nitrogen and phosphorus) content and its
potential to act as eutrophication enhancer. Lastly, colloidal
and particulate matter in whey alter the color and turbidity of
the receiving water bodies, affecting the light penetration and
primary productivity of these ecosystems.103
Land application of whey has been practiced for decades
in the USA, Canada, and Australia, among other countries,
as a slow-release nitrogen and phosphorus fertilizer.105
This practice can be seen as sustainable because whey
has been reported to provide an adequate substitute for
potassic superphosphate, while some soil properties
are improved.106–110 However, adverse changes in soil
properties, as well as depressed plant growth, can result
at high application rates. It has been estimated that the
irrigation of 1 mm3 of whey results in about 400–600 kg of
total salt added per hectare, leading to high salinity levels
and minor crop yields.105 To overcome this salinity problem
and to obtain an acceptable irrigation water quality, a 1: 20
dilution of whey with clean water has been suggested.
Sodium accumulation following high rate whey application
is likely to occur at greater soil depths, ultimately affecting
future land uses.
 Review: Valorization of whey milk through plan biorefinery J Sebastián-Nicolás et al.

Whey nitrogen is subject to biological transformations
such as aerobic nitrification, which produces highly
leachable nitrites and nitrates that can pollute groundwater.
Denitrification (the microbial reduction of nitrates to N2O
and N2) occurs in anoxic zones and when a suitable carbon
source is available, and leads to losses (about 5–20%) of the
whey nitrogen applied.107 As N2O is an important greenhouse
gas, it accounts for additional deleterious environmental
effects of land whey application.
Biorefinery CW: Valorization
At present, technologies for the treatment of agro-industrial
waste aim to reduce the pollution that they cause to the
environment (Table 5).111 That is why the use of whey in
biorefinery schemes has been considered a topic of great
importance due to the contamination that it causes when
is discharged to rivers and soils without treatment. Three
types of biorefineries, known as phase I, II, and III, have
been described by Kamm and Kamm112 and by Van Dyne
et al.113 A phase I biorefinery produces a single final product.
A process where more than two different products are
obtained is considered to be a phase II biorefinery. Phase III
biorefineries combine different technologies to produce their
own energy.
Németh and Kaleta114 studied the production of ergoesterol
from whey under a biorefinery scheme. The residues
resulting from their design were used for the production
of propionic acid and vitamin B12. This study revealed that
during this process there is an appropriate relationship
with the ‘zero waste’ theory and the value-added products
obtained. Other studies like this developed by Domingos
et al.115 show the feasibility of producing high concentrations
of polyhydroxyalkanoates (PHAs) through the anaerobic
fermentation of CW. These plastics are biodegradable and are
used in the production of biopolymers.

Table 5. Type of milk whey (MW) biorefineries.

Process description Products Disadvantage Type Reference
114
Two batch fermentations. First with Ergosterol, propionic acid High cost of process. Not optimized I
S. cerevisiae and the second with and whey powder process. Batch process.
Propionicbacterium acidipropionici
(DSM20273). At the final process the whey
powder was obtain through a spray dry
technique.
115
Anaerobic fermentation of cheese whey (CW). Production of There is no application for the final I
An electrodialysis (ED) step was proposed to polyhydroxyalkanoates waste
obtain a concentrate acidic stream, suitable (PHAs)
for being employed as feeding solution in the
consecutive fed-batch culture system for PHA
production
Ricotta cheese whey (RCW) as a source Lactobionic acid There is no application for the final I 117
during fermentation in aerobic conditions with waste
Pseudomonas taetrolens
CW including in a multi-feed biorefinery Bioethanol. Combination of different wastes I 118
design as from lignocellulosic residues and increasing the resident time during
tequila vinasses. The process is carried out in fermentation. But there are different
anaerobic conditions. beneficial products at the end of the
process
Batch fermentations with Kluyveromyces Recovery of bioethanol Only one product is produced and II 116
lactis CBS2359 of CW permeate and biomass rich in β- there is no application for the final
galactosidase activity, at waste
the same time
Two-step bioprocess using Saccharomyces Bioethanol and galactonic There is no application for the final II 119
cerevisiae and Gluconobacter oxydans. First, acid waste
the lactose in CW powder was pretreated
with β-galactosidase to obtain glucose
and galactose. Subsequently, the glucose
is selectively fermented to ethanol by S.
cerevisiae to enable G. oxydans-mediated
bio-oxidation of galactose to galactonic acid.

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2100 9
 J Sebastián-Nicolás et al.
The use of fermentation to obtain products with a high
commercial value has been proposed in different biorefining
process. Sampaio et al.116 evaluated the production potential
of bioethanol from whey using Kluyveromyces lactis as an
alternative to provide a treatment for this residue from the
dairy industry and to increase its value. De Giorgi et al.117
evaluated the preliminary conditions for the industrial use
of whey as a substrate for the development of Pseudomonas
taetrolens as a possible biotechnological treatment of this
residue.
Sánchez et al.118 developed a multi-feed biorefinery
design as a solution for the production of bioethanol from
lignocellulosic waste and at the same time for treating
agro-industrial waste (CW and tequila vinasse). This kind
of process is more complex than the simplest conventional
lignocellulosic biofuel biorefineries. However, this design
improves overall energy productivity and achieves
environmental and economic sustainability values similar
to those of a single raw material biorefinery that produces
lignocellulosic ethanol and electricity. Zhou et al.119
proposed a bioprocess that involved two stages using two
microorganisms (Saccharomyces cerevisiae and Gluconobacter
oxydans) for the generation of ethanol and galactonic acid
using whey as a substrate. During this process the whey was
pretreated with β-galactosidase.
Biorefineries that are currently proposed using agro-
industrial wastes integrate both, the advantage of the
chemical composition and, the economical and the
environmental sustainability evaluation. In general, the
biorefining of whey is focused on obtaining a main product
using physical, chemical, or biotechnological processes,
Figure 6. Environmental impact of milk whey (MW) and industrial opportunities.
10 © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2100
Review: Valorization of whey milk through plan biorefinery
and then using the rest to produce energy. Due to the
composition of whey, many products could be obtained by
different continuous integrated processes using chemical and
biotechnological techniques. Such proposals integrate the
production of high-value products with efficient treatment in
a ‘zero waste process’.
Proposed whey biorefinery
Due to the little importance that has been given to whey
as a raw material for compounds with high added value,
mainly those with biological activity, this has become an
environmental problem in cheese-producing regions. (Fig. 6).
Despite several possibilities for CW utilization, which
were mentioned in the previous sections, a major fraction
of the world’s whey production is currently discarded.120
Biorefineries are being proposed as a way to readdress the old
linear schemes of cheese production into circular and holistic
approaches. In the whey biorefineries found in the literature,
biotechnological processes are often included, mostly based
on lactic acid bacteria.120 The goal is to generate several high
value-added products in a multipurpose plant and, at the
same time, to minimize waste. For instance, whey has been
proposed as a substrate for the production of d-lactic acid and
polylactic acid, coupled with the production of biodegradable
biopolymers such as PHAs and lactulose by Caulobacter
segnis.121
Pinto et al.122 conceived a biorefinery where whey is
microfiltered and ultrafiltered to separate lactose and
proteins. Lactose undergoes enzymatic hydrolysis while the
protein concentrate can be dried or submitted to sequentially
 Review: Valorization of whey milk through plan biorefinery
controlled proteolysis to produce peptides for use as
supplements for parenteral feeding. This biorefinery was
economically evaluated, simulated, and monitored through
web-based remote software.122 By using process simulation
(i.e. flowsheeting), a biorefinery was proposed in which whey
lactose would be transformed into ergosterol (previtamin
D2), propionic acid, probiotics, and vitamin B12.114 In
another study, the production of PHA was considered for a
biorefinery based on the culture of recombinant Escherichia
coli (a strain modified to express the genes of Cupriavidus
necator encoding to metabolize lactose) on whey.123
The biorefinery proposed in this review involves different
physical, chemical, and biological techniques for the recovery
of value-added products from whey obtained from the
manufacture of cheese. This process is designed taking into
account the chemical composition of whey, and represents an
alternative for the recovery of biologically active compounds
of commercial interest to the food, pharmaceutical, and
polymer industries.
The purpose of the proposed dairy whey biorefinery is
to recover lactoferrin, lactic acid, probiotics, hydrolyzed
proteins, and bioactive peptides, in a continuous process
divided into three stages as shown in Fig. 7. Fat and casein
fractions should be removed from the MW before the process
starts, as a preparative phase.
In the first stage (S1) of the process, the separation and
recovery of lactoferrin is carried out through the use of
a PAM with Cu (II). During this separation, a residue is
obtained with imidazole and proteins (residue without Lf)
that were not retained in the matrix, as well as lactose, water,
and minerals.
Figure 7. Proposal of milk whey biorefinery.
© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2100
J Sebastián-Nicolás et al.
In stage two (S2), the residue without Lf is recovered
for the organic matter, rich in nitrogen and carbon, that
it contains. It will be used as a suitable substrate for the
growth of LAB homofermentative probiotics. Due to the
chemical composition of this substrate, LAB can develop
in the medium, initiating the fermentation process.
Through this, products of commercial interest such as
lactic acid, hydrolyzed proteins, bioactive peptides, and
probiotics can be obtained. During this process, low
molecular-weight peptides between 15 and 5 kDa will be
released, which have an impact on the pharmaceutical
industry. On the other hand, the probiotics could be
used as precursors for new food products, while lactic
acid would serve for the production of biodegradable
polymers.124 The importance of hydrolyzed proteins is
due to their ability to have greater bioavailability and
absorption in the digestive system, so this product would
be beneficial to people with deficiencies in the absorption
of protein fractions – mainly those patients who have some
essential amino acid deficiency.59
During the fermentation, the LAB transforming the
lactose present in the waste produces lactic acid. The lactic
acid obtained in this stage is intended to be separated by an
electrodialysis process, this method being the most feasible
to avoid an increase in contamination with chemical agents
for its purification, such as lactic acid esters. Once the
lactic acid has been separated from the fermentation broth
it can be used in the food industry as a preservative or to
generate biodegradable polymers such as PLA. At present,
biodegradable polymers are used to reduce the pollution that
has been emitted by conventional polymers for decades.125
11
 J Sebastián-Nicolás et al.
After the separation of the products generated in the lactic
fermentation process, the final residue (R4) can be used
for different purposes as it will contain minerals, galactose,
glucose, amino acids, and lactose, which are not transformed
by the LAB.126–130
The R4 residue can be used to purify the remaining
compounds, or can be discarded to the sewer as it meets
Mexican standards for this purpose. Finally, R 1, 2, and 3
could be mixed to generate biogas or biofertilizer (S3).131–136
These proposals would benefit the regions where this
biorefinery process is used as it would be taking advantage
of a waste with excellent potential for recovering different
value-added products. The biotransformation of MW and
the recovery of compounds of industrial interest are an
alternative for the treatment of this dairy industry residue.
Additional to the biorefinery proposed, 1 ton of pretreated
whey was taken as the basis of material balance during the
process.
In stage 1 (S1) the lactoferrin content of the whey is up to
0.02%.48–53 With the method proposed at this stage, 82.5% of
this protein could be recovered.52, 77 So, 165 g of lactoferrin
would be obtained from this stage. Approximately 96% of the
serum is recovered as a residue without lactoferrin. That is,
from each ton of serum, 960 kg of residue is obtained, which
will then be used for the fermentation stage.
In stage 2 (S2), the probiotic inoculated into the fermenter
uses only the glucose in the lactose molecule for the generation
of lactic acid with a yield of 47.5%.96 From 44 to 50 kg of
lactose, contained in 1 ton of residue without lactoferrin,
it is obtained 20.90 to 23.75 kg of lactic acid. The efficiency
of protein hydrolysis during fermentation is 80%. The
ultrafiltration process will recover 95% of hydrolyzed proteins.
The microorganism hydrolyzes approximately 80% of the
proteins but 10% of the hydrolyzed protein is lost due to the
metabolism and development of the probiotic.98 Thus, between
60 kg and 100 kg of protein is obtained per ton of residue
without lactoferrin. About 51 to 85 kg of a protein mixture
with hydrolized protein is obtained . The biomass recovered
at this stage depends on the probiotic’s metabolism and is
considered as a product of high extra value to the process.
In stage 3 (S3) the remaining compounds such as glucose,
lactose, minerals, amino acids, and water could be used for
the generation of biogas, which might be used to feed the
process.
Costing
It is difficult to estimate the cost of the proposed biorefinery
due to the absence of detailed costs for each part of
the process, equipment designed for the process, and
information about the process for obtaining the different
12 © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2100
Review: Valorization of whey milk through plan biorefinery
valuable products. However, the use of whey which, is not
used to obtain sub-products with high value, increase the
expectation to design a cheap process taking advantages of its
composition. Therefore, biorefineries process could be built
in order to produce new resources to obtain energy in self-
sustainable technologies at low cost.
Challenges and opportunities in
whey valorization
The use of whey has changed. In the 17th to 19th centuries it
was a medicinal liquid. Today it is a source of a wide range of
useful and valuable components. From the mid-20th century
it has been considered to be a source of environmental
pollution, but scientific knowledge about whey’s components,
their characteristics, and their useful properties has increased.
Whey biorefineries could be parallel process to the dairy
industry transforming a waste in high value products.
Advances in processing technology allow cost-effective
process implementation and efficient manufacture of
products with whey components as ingredients.
Whey and its associated components (proteins, peptides,
lactose, and lipids) have been used for commercial
applications in foods, specialized formulations, bio-medical
products, pharmaceuticals, and cosmetics in a marketplace
that craves the nutritional, functional, and biological traits of
whey ingredients. Notably, proteins are used in a vast array of
applications.
The whey industry currently faces new challenges and
opportunities. An example is the expansion of the dairy
industry, of which whey production is part. Other examples
include the production of acid whey, decreasing costs of
manufacturing whey ingredients, market competition from
other ingredients, and possibilities in the area of functional
whey peptides. The integration of different procedures
to obtain new constituents is the main challenge to the
treatment of this kind of waste. Biorefining could be the
solution to a worldwide problem. The valorization of the
waste would end in the complete use of the waste and the
generation of energy through a sustainable process. These
looming challenges will be addressed and the opportunities
will be captured using science and technology to make a
strong dairy industry.
Conclusion
Where whey is an environmental problem, the biorefinery
process can enable the problem to be solved by recovering
value-added products. This review has discussed some types
 Review: Valorization of whey milk through plan biorefinery
of biorefineries that take advantage of the composition of
MW. It has proposed a biorefinery that uses simple processes
and cheap technology.The products from these biorefineries
can be used as raw materials to increase the nutritional
value of some foods and to elaborate new products such as
biodegradable polymers. The consumption of functional
foods with bioactive compounds will bring health benefits to
consumers, while the production of biodegradable polymers
will have a direct positive effect on the environment.
It is necessary to establish the relationship between the
valorization of industrial food wastes and their economic value
to explore the viability of biorefineries in depth. However, new
challenges and opportunities abound in environmental science
and process engineering – for example the optimization of
processes to obtain products with a high commercial value. The
looming challenges will be addressed and the opportunities
captured using technological options, making a strong industry
that increases the value of the biorefinery process.
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biofuels and biobased products: from lab to pilot plant.

Butanext Workshop, October 17th, Pamplona, Spain (2017) Jessica L. Sebastián-Nicolás
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engineering for a cheese whey biorefinery: An Internet-
includes the application of green
based application for process design, economic analysis,
processes to agro-industrial waste.
monitoring, and control of multiple plant sites. Bioprocess
Biosyst Eng 32(1):69–78 (2009). She has a PhD in environmental sci-
123. Pagliano G, Ventorino V, Panico A and Pepe O, Integrated ence from Universidad Autónoma del
systems for biopolymers and bioenergy production from Estado de Hidalgo, Mexico.
organic waste and by-products: A review of microbial
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126. Bastidas OJR, Fang C, Almardeai S, Javid U, Yousuf ma Metropolitana and a researcher-
A and Schmidt JE, Waste biorefinery in arid/semi-arid professor in the biotechnology
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and Kulkarni BD, Valorization of potato peel: A biorefinery biotechnology.
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Reddy G, Single step fermentation of starch to L (+) lactic Gabriela Vázquez-Rodríguez is chem-
acid by Lactobacillus amylophilus GV6 in SSF using ical engineer and professor of envi-
inexpensive nitrogen sources to replace peptone and yeast ronmental sciences at the Chemistry
extract–optimization by RSM. Process Biochem 41(2):465– Department of Universidad Autónoma
472 (2006). https://doi.org/10.1016/j.procbio.2005.07.011. del Estado de Hidalgo. Her research
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Technological tools to include whey proteins in cheese: for urban water management and the
Current status and perspectives. Trends Food Sci Technol biological treatment of water and wastewater. She has
64:102–114 (2007). https://doi.org/10.1016/j.tifs.2017.04.007.
a PhD in biotechnology from Institut National des Sci-
132. De Jesús CS, Ruth VG, Daniel SF and Sharma A,
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Food Science, Technology, and Engineering, Vol. 2, ed. by Hui Carlos A. Lucho-Constatino has
HY. Taylor & Francis Group, New York, NY, pp. 61–68 (2006). a master’s degree and a PhD in
135. Alais C and Jollès P, Étude comparée des caséino- biotechnology and currently works
glycopeptides formés par action de la présure sur les at the Chemical Research Center,
caséines de vache, de brebis et de chèvre: II. Étude de la Autonomous University of Hidalgo,
partie non-peptidique. Biochim Biophys Acta 5:315–322 Mexico. His research interests include
(1961). https://doi.org/10.1016/0006-3002(61)90172-X.
research in environmental science,
136. Janczukowicz W, Zielin´ ski M and De˛  bowski M,
agricultural crop science and irrigation, water man-
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selected sections of dairy production. Bioresour Technol agement and the valorization of biomass and indus-
99(10):4199–4205 (2008). https://doi.org/10.1016/j.biort trial wastes in biorefineries.
ech.2007.08.077.

© 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2100 17
 J Sebastián-Nicolás et al. Review: Valorization of whey milk through plan biorefinery

Araceli Castañeda-Ovando Alma E. Cruz-Guerrero

Araceli Castañeda Ovando is
Chemist and professor of Food and
Analytical Chemistry at the Chem-
istry Department of Universidad
Autónoma del Estado de Hidalgo.
Her research includes studies about
food properties and use of by-prod-
ucts of the food industry. She has a PhD in Chem-
istry from the Universidad Autónoma del Estado de
Hidalgo, Mexico.
Alma Elizabeth Cruz-Guerrero is a
food engineer and researcher-pro-
fessor in the Biotechnology Depart-
ment of the Universidad Autónoma
Metropolitana. Her research includes
food enzymes, probiotics, prebiot-
ics, the metabolism of lactic acid
bacteria, and alternative uses of milk, whey, and their
derivatives. She has a master’s degree and a PhD
in biotechnology from the Universidad Autónoma
Metropolitana.

18 © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd | Biofuels, Bioprod. Bioref. (2020); DOI: 10.1002/bbb.2100