Journal of Cleaner Production 143 (2017) 814e823

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Journal of Cleaner Production

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Review

Comprehensive utilization of shrimp waste based on biotechnological

methods: A review
Xiangzhao Mao*, 1, Na Guo 1, Jianan Sun, Changhu Xue
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China

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

Article history: Shrimp has constituted a major part of crustacean consumption in recent years. Solid wastes, including
Received 27 October 2016 the head, shell, and tail portions, accumulate owing to shrimp processing. The accumulated biowastes
Received in revised form without appropriate utilization have resulted in a squander of sources and problems of waste disposal
8 December 2016
and environmental pollution. Proper shrimp waste processing is an approach to recover biomaterials
Accepted 10 December 2016
Available online 10 December 2016
such as chitin, protein, lipids, astaxanthin, avor compounds, and calcium carbonate. These active
components have large-scale applications in biology as well as in food, pharmaceutical, agricultural,
cosmetic, pulp, and textile industries. Traditionally, methods applied for the utilization of shrimp waste
Keywords:
Shrimp waste
are chemical procedures using corrosive or hazardous reagents (such as HCl and NaOH), which are
Comprehensive utilization known to cause environmental pollution and resource wastage (or incomprehensive utilization) and
Chitin increase the associated costs. However, new environment-friendly and clean technologies are emerging.
Protein In this review, we briey analyze the bioactive compounds recovered from shrimp waste. A concise
Astaxanthin overview of the comprehensive utilization of shrimp waste in recent years has been included.
Biotechnological method Biotechnological methods such as biocatalysis and biotransformation with enzymes and microorganisms
have also been described. The rapid development of corresponding biotechnology enables a simple, fast,
effective, clean, economic, and controllable bioprocess for the comprehensive utilization of crustacean
waste.
2016 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 815
2. Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 815
3. Bioactive compounds of shrimp waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 815
3.1. Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 815
3.2. Chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 816
3.3. Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 816
4. Bioprocesses for isolating protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 816
5. Isolating pure chitin and derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 817
5.1. Process for pure chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 817
5.2. DA and hydrolysis of chitin/chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 818
6. The process for astaxanthin recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819
7. Future challenges and trends in comprehensive utilization of shrimp byproducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 820
8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 820
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 820
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 821
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 821

* Corresponding author.
E-mail address: xzhmao@ouc.edu.cn (X. Mao).
1
Co-rst authors: Xiangzhao Mao and Na Guo contributed equally to this paper.

http://dx.doi.org/10.1016/j.jclepro.2016.12.042
0959-6526/ 2016 Elsevier Ltd. All rights reserved.
 X. Mao et al. / Journal of Cleaner Production 143 (2017) 814e823 815

1. Introduction

It is reported that approximately 6e8 million tons of crustacean

waste is produced worldwide every year (Organization, 2014). With
the development of aquaculture in recent years, the culture of
shrimp has become widespread. In particular, the shrimp produc-
tion in Asia increased fast these years, and accounted for over 80%
of the worldwide yield (Organization, 2014). Moreover, approxi-
mately 45% of processed seafood comprises shrimp; thus, pro-
cessing industries have contributed to the accumulation of plenty
of shrimp waste (Chen et al., 2015; Holanda and Netto, 2006). In
industries, shrimp is usually processed as meat, remained head,
shell, and tail portions as processing byproducts (Knorr, 1991).
Frozen shelled shrimp is the major export product of the shrimp
processing industry. In general, approximately 45%e60% of whole
shrimp turns to be byproducts (head and hard carapace) after
processing, which varies among species and processing methods
(Sachindra and Mahendrakar, 2005; Sila et al., 2012).
An increase in shrimp waste is unavoidable owing to the
increased amount of consumption. Discarding shrimp waste,
particularly together with shery waste, poses a critical environ-
mental and economic problem because valuable biomaterials are
squandered (Kandasamy et al., 2012; Morgan and Chuenpagdee,
2003). Biowaste is recognized as an abundant resource of biolog-
ical compounds, including chitin, protein, lipids, pigments, avor
compounds, and calcium carbonate (Bueno-Solano et al., 2009;
Shahidi and Brown, 1998; Yan and Chen, 2015). Sun-drying was
previously used to process the waste into feeding material for
veterinary practice and aquaculture. However, the processing
conditions were unhygienic for animal feed. Besides, shrimp waste
was dried on the beaches, which caused an unpleasant smell, sur-
face pollution, and other environmental pollution (Nwanna et al.,
2004).
Chemical treatments (strong alkali and acid) have been used to
recover chitin, protein, and carotenoids. This results in a high cost
and production of harmful efuent wastewater that is inappro-
priate with respect to economic and environmental considerations
(Mahmoud et al., 2007). Thus, the application of appropriate
technology to convert biomaterials of shrimp waste into value-
added products is urgently required. In the present review, we
address the recent developments toward the complete use of
shrimp waste for the efcient recovery of biomaterials. We also
discuss existing environment-friendly biotechnological methods
for exploiting shrimp waste, including biocatalysis and biotrans-
formation using enzymes and microorganisms. Finally, we discuss
emerging biotechnologies for the comprehensive extraction and
utilization of shrimp waste that may facilitate cleaner industriali-
zation. The comprehensive utilization of shrimp waste is summa-
rized in Fig. 1. In our previous studies, we successfully
demonstrated the feasibility of biotechnology for the comprehen-
sive utilization of shrimp waste. Although there are existing re-
views on the utilization of shrimp waste (Kaur and Dhillon, 2015;
Kandra et al., 2011), here we provide the rst thorough and in-
depth review of emerging biotechnologies, including biocatalysis
and biotransformation, for the efcient isolation of major bioactive
compounds (protein, chitin, and carotenoids) from shrimp waste.
2. Methodology
The data presented in this review were collected from articles
published in Science Citation Index Expanded journals and con-
ference proceedings using strict and reliable selection criteria.
Selected studies were then examined for originality and scientic
veracity. The key words crustacean, shrimp waste, chitin, protein,
fermentation, and enzymes were used to search the Web of
Fig. 1. The comprehensive utilization of shrimp waste.
Science, Elsevier, and Springer databases. Almost all articles
retrieved were published during the period from 1995 to 2016.
Finally, 120 articles describing biotechnological methods applicable
to shrimp waste processing were reviewed.
The accepted literature is grouped based on the specic tech-
nologies and bioactive products recovered in the comprehensive
utilization of shrimp waste. The major products isolated from
shrimp waste are chitin and its derivatives, proteins, and caroten-
oids. Although chemical treatment is the traditional method used
for recovering bioactive compounds from shrimp waste, we focus
on technologies that are environment friendly, cleanly and sus-
tainably extract products using processes such as biocatalysis and
biotransformation with enzymes and microorganisms.
Articles describing particularly promising technologies and
important products are selected for a more in-depth review based
on methodological criteria, clarity of presentation, and success in
efcient recovery of bioactive compounds. Limitations and poten-
tial improvements to these technologies are then suggested based
on other relevant literature. Therefore, this paper reviews the
development, advantage and challenge of the biotechnologies to
recover bioactive compounds from shrimp and crustacean waste.
3. Bioactive compounds of shrimp waste
Shrimp waste is considered to be a potential source of animal
protein together with important biomaterials such as chitin, lipids
(glyceride, phospholipids, and carotenoids), and calcium carbonate
(Shahidi and Synowiecki, 1991). A previous report suggested that
the dry weight of shrimp comprises 18% chitin, 43% protein, 29%
ash, and 10% fat (Synowiecki and Al-Khateeb, 2000). In addition,
shrimp waste contains a wide range of bioactive components such
as unsaturated fatty acids, carotenoid pigments, free amino acid,
and trace elements. Therefore, efcient recovery of those bioactive
components from shrimp waste has broad industrial and scientic
applications.
3.1. Protein
Protein, a large bioactive molecule, is one of the six essential
nutrients in living organisms. Notably, shrimp waste is rich in
proteins and provides essential amino acids for animal feed
816 X. Mao et al. / Journal of Cleaner Production 143 (2017) 814e823
supplements or human nutrition (Cah et al., 2012; Cheong et al.,
2014). The protein content in shrimp waste comprises approxi-
mately 35%e65% of its dry weight, and the amounts depend on the
processing modes and species (Mizani et al., 2005), whose essential
amino acids and non-essential amino acids are 56.8% and 43.2%,
respectively (Narayan et al., 2010). The protein existing in shrimp
waste is closely associated with chitin and minerals. Similar to
chitin, the process of protein recovery from shrimp waste is
through deproteinization (DP).
Proteins extracted from the proteinechitineminerals complex
have diverse applications. As reported, protein hydrolysates from
shrimp waste can be used in various industries, including phar-
maceuticals, human nutrition, animal nutrition, and cosmetics.
Moreover, protein hydrolysates are good nitrogen source in the
growth media for microorganisms (Quitain et al., 2001; Holanda
and Netto, 2006).
3.2. Chitin
As the second richest polysaccharide in the world, chitin is
abundant in crustacean shell (shrimp, crabs, etc.), fungi, insects,
algae, and mushrooms (Arcidiacono and Kaplan, 1992). Chitin, a
polysaccharide polymer, is a cellulose-like polymer that mainly
comprises unbranched chains of N-acetyl-D-glucosamine. After
deacetylation (DA), chitin can be transformed to chitosan, which
comprises D-glucosamine chains (Yeul and Rayalu, 2012). The
structures of chitin and chitosan are shown in Fig. 2.
The annual worldwide industrial production of crustacean was
over 10,700,000 tons in 2008 (Organization, 2010). Chitin and
chitosan are important bioactive materials, with many highly
potent activities such as immune function, hemostasis and wound
healing, antioxidant action, antimicrobial activity, and heavy metal
and other pollutant removal (Feisal and Montarop, 2010). There-
fore, as renewable resources, chitin and its derivatives have a wide
range of applications in food and nutrition (Limam et al., 2011),
pharmaceutical (Kato et al., 2003), biotechnological (Kim and
Mendis, 2006), cosmetic (Muzzarelli et al., 2012), packaging
(Leceta et al., 2013), textile, waste water treatment (Bhatnagar and
Sillanpa
a, 2009), and agricultural (Jin et al., 2005) industries.
3.3. Carotenoids
Carotenoids in lipids of shrimp waste are the most widely
distributed pigments, ranging from red to yellow in plants and
animals. Astaxanthin is a valuable redeorange carotenoid initially
discovered in several birds and sh and is mainly found in inver-
tebrate crustaceans such as shrimp, crabs, and lobsters (Armenta-
pez et al., 2002). Astaxanthin comprises 3 stereoisomers in a
Lo
racemic mixture, forming a complex with a protein that
Fig. 2. The structures of chitin and chitosan.
accumulates in the crustacean exoskeleton (Schiedt et al., 1993). It
can be green, purple, or blue in living organisms, turning red after
heat treatment (Hendry and Houghton, 1996).
Astaxanthin is dened as a super oxidant because of its strong
antioxidant ability, which is 10 and 500 times higher than that of
other carotenoids and vitamin E, respectively (Shmidzu et al., 1996).
Owing to its various bioactivities, such as antioxidative, anticancer,
immunomodulating, antidiabetic, and antiinammatory effects
(Ikeuchi et al., 2006), astaxanthin is widely applied as a coloring
agent in diets in aquaculture as well as in cosmetic and pharma-
ceutical industries.
Shrimp waste is thought to contain high levels of good-quality
astaxanthin. Because of its particular binding characteristics,
astaxanthin mainly exists in shrimp waste in association with other
compounds. The pigment forms a chemical complex with protein
(carotenoproteins) or lipoproteins (carotenolipoproteins) (Higuera-
Ciapara et al., 2006).
4. Bioprocesses for isolating protein
Shrimp protein can be separated by DP in the form of protein
hydrolysate liquor. Moreover, it can be processed into powder,
paste, and liquid, which can be used as supplement with shrimp
avor in feed. The extraction efciency of protein products of
shrimp waste varies depending on the processing methods. Instead
of strong alkali (4% NaOH) treatment methods, biotechnological
methods, mainly containing biocatalysis and fermentation, have
been used to isolate proteins from shrimp waste. Certain enzymes
such as papain, trypsin (Bougatef, 2013), pepsin (Chakrabarti,
2002), and alcalase (Gildberg and Stenberg, 2001) have been used
for DP, to control hydrolysis, and to minimize undesirable reactions
(Bueno-Solano et al., 2009). In addition, crude (Bkhairia et al., 2015)
and endogenous (Cah et al., 2012) enzymes extracted from shrimp
heads are used to prepare protein hydrolysates from shrimp waste.
Digestive alkaline proteinases from golden grey mullet (Liza aurata)
were extracted and characterized, which showed optimal activity
at pH 8.0 and 60
C. Moreover, the crude proteases were veried to
be effective in the DP of shrimp wastes with the protein recovery
reaching about 76% after 3 h at 45
C with an enzyme/substrate (E/
S) ratio of 10 U/mg protein (Bkhairia et al., 2015). The enzymatic
methods for preparing protein hydrolysates are summarized in
Table 1.
Fermentation is an eco-friendly and cost-effective alternative
process for DP (Kim and Park, 2015). It works through proteolytic
enzymes generated by microorganisms such as Lactobacillus,
Pseudomonas aeruginosa (Wang and Chio, 1998), P. maltophilia
(Wang and Chio, 1998), Bacillus subtilis (Yang et al., 2000), and
B. licheniformis (Mao et al., 2013a). Thus, chitin in solid fraction and
liquor with soluble protein hydrolysates, peptides, free amino acids,
 X. Mao et al. / Journal of Cleaner Production 143 (2017) 814e823 817

Table 1
Summary of enzymatic method of shrimp waste for protein production (DH: degree of hydrolysis, DP: deproteinization, NA: Not available).

Products Shrimp waste sources Enzymes Conditions DH/DP (%) References

pH Temperature (
C) Time(h)

Flavor protein Penaeus chinensis Dispase 6.5 57 3 57.6 Guo et al., 2014
hydrolysate
Protein powder Penaeus.semisulcatus Alcalase 8 40 1 65.1 Mizani et al., 2005
Antioxidant protein Metapenaeus dobsoni Alcalase 8.2 45.4 3 42.4 Gunasekaran et al., 2014
hydrolysates
Protein hydrolysates Litopenaeus vannamei Acid stable protease 3.5 40 6 95 (DP) Baron et al., 2015
Protein hydrolysates Penaeus monodon Pepsin 2 40 22 33.5 Randriamahatody et al., 2011
Antioxidant protein Farfantepenaeus Alcalase 8 55 1.7 66.8 (DP) Vieira et al., 2015
hydrolysates
Enzymatic protein Jasus edwardsii Alcalase 8 55 1.5 85.8 (DP) Nguyen et al., 2016
hydrolysate
Protein hydrolysate Penaeus monodon Alcalase 8.2 59.4 1.4 33.1 Dey and Dora, 2014
Protein hydrolysate Penaeus kerathurus Trypsin 7.9 50 1 NA Limam et al., 2008
Protein hydrolysate Penaens vannamei Endogenous enzyme 7.8 50 3 45.1 Cao et al., 2008
Protein hydrolysate Penaens vannamei Endogenous enzyme 9 50 2 92.1 (DP) Cao et al., 2014
Protein hydrolysate Penaens vannamei Endogenous enzyme 8 50 0.4 87.5 (DP) rdova-Murueta et al., 2013
Co
Antioxidant protein Shrimp by-products Alcalase 7.2 NA NA NA Zhao et al., 2013
hydrolysates
Protein hydrolysates Parapenaeus longirostris Alcalase 8 50 2 13 Sila et al., 2014
Antioxidant protein NA Digestive alkaline 8 45 3 76 (DP) Bkhairia et al., 2015
hydrolysates proteinases from Liza
aurata

pigments, phenolics, and antioxidant compounds can be recovered
(Kan et al., 2014; Sun et al., 2014). Protein hydrolysates were pre-
pared through lactic acid fermentation of shrimp by-products. And
the protein-rich liquid hydrolysates were further processed into a
concentrated paste via vacuum evaporation or were also processed
into a dry powder using a spray drying method. The protein content
of the three hydrolysates have been compared, ranging from
8.43 0.22 to 46.73 1.29 g/100 g of wet mass (Bueno-Solano et al.,
2009). In addition, The Antarctic krill juice fermented by Saccha-
romyces cerevisiae was produced with the best avor and the most
abundant nutrition. It showed that the amino acid content and the
scavenging activity of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radi-
cals were up to 1368.3 mg/100 mL and 71.3%, respectively.
Furthermore, there was a variety of amino acids in this fermented
liquor, which contained all eight of the essential amino acids not
manufactured by the human body (Kan et al., 2014). In our previous
research, we analyzed the fermentation broth of Antarctic krill with
B. subtilis OKF04 and found various bioactive substances, including
polypeptides (9.5 g/L), free amino acids (3469.4 mg/L), phenols
(1.1 g/L), and polysaccharides (200.8 mg/L) (Sun and Mao, 2016). A
summary of fermentation processes for shrimp waste recovery is
presented in Table 2.
5. Isolating pure chitin and derivatives
In general, chitin in shrimp waste is tightly associated with
proteins and minerals (calcium carbonate). Several chemical
methods to treat shrimp waste with strong alkali and acid for DP
and demineralization (DM) have been previously reported (Aye and
Stevens, 2004). However, these traditional processes can result in
depolymerization, affecting the molecular weight, viscosity, and
acetylation of the polymer. In addition, these chemical methods are
hazardous and energy consuming and lead to environmental
pollution (Gortari and Hours, 2013). Moreover, protein and mineral
(calcium carbonate) components of shrimp waste are rendered
useless during the processes of DP and DM (Sachindra et al., 2005).
Along with stricter regulations, there is an increased demand to
utilize shrimp waste in a more appropriate and efcient manner. As
an alternative to chemical methods, different technologies for
chitin recovery have been exploited, including enzyme catalysis
and microbial fermentation.
5.1. Process for pure chitin
Fermentation of shrimp waste with protease- or organic acid-
producing bacteria leads to the isolation of the solid fraction of
chitin from liquor comprising shrimp proteins, minerals, pigments,
and other components (Prameela et al., 2010a; Rao and Stevens,
2005). In addition, the remained solid fraction after DP is tradi-
tionally subjected to strong acid (HCl) treatment for removing
minerals and yielding pure chitin. Proteases produced by Erwinia
chrysanthemi and HCl for the extraction of pure chitin from lobster
waste has been previously reported. DP of the protein reaches 95%
with the proteases produced by E. chrysanthemi; this is higher than
that of commercial protease. In addition, the concentration of cal-
cium decreased by more than 99% after treatment with 1.5 N HCl
(Giyose et al., 2010).
However, considering the drawbacks of using chemical reagents

Table 2
Summary of fermentation of shrimp waste for protein production (DH: degree of hydrolysis, DP: deproteinization).

Products Shrimp sources Microorganisms DH/DP (%) References

Protein hydrolysates Penaeus japonicas Pseudomonas aeruginosa 82 (DP) Wang and Chio, 1998
Protein liquid and powder Penaeus spp. Commercial lactic inocula NA Bueno-Solano et al., 2009
Protein feedstuff Macrobrachium vollenhoveni Lactobacillus plantarum NA Fagbenro and Bello-Olusoji, 1997
Protein powder Penaeus mondon Pediococcus acidolactici NA Sachindra and Bhaskar, 2008
CFR2182
Protein liquid Litopenaeus vannamei B. licheniformis OPL-007 85.3 (DP) Mao et al., 2013a
818 X. Mao et al. / Journal of Cleaner Production 143 (2017) 814e823

such as strong acids, the trend toward clean and effective
biotechnological applications for exploiting shrimp waste has been
highlighted. Similar with the addition of organic acids for DM
(Lurdes et al., 1998), organic acid-producing microbial strains can
be used for DM in combination with DP by fermentation (co-
fermentation or two-stage fermentation) (Xu et al., 2008). Suc-
cessive co-fermentation was conducted to biologically extract
chitin from shrimp waste in combination with a protease-
producing bacterium, Bacillus licheniformis 21886 (B. 21886), and
an acid-producing bacterium, Gluconobacter oxydans DSM-2003 (G.
2003). When the waste was fermented with B. licheniformis 21886
followed by G. oxydans DSM-2003, the chitin content was up to
90.8%, with the DP and DM efciencies as 87 and 93.5%, respectively
(Liu et al., 2014). Studies on biological treatments for the pure chitin
recovery are summarized in Table 3.
5.2. DA and hydrolysis of chitin/chitosan
Chitosan, a polysaccharide comprising b-(1, 4)-linked D-
glucosamine, is prepared from chitin by DA. In general, it is soluble
in aqueous acid when the DA degree of chitin is over 50%. Treat-
ment with 40%e50% alkali at 100
Ce160
C for several hours can
achieve nearly 95% DA, resulting in the production of chitosan
(Honarkar and Barikani, 2009).
In recent years, the use of chitin deacetylases (CDA) has become
an alternative method to obtain chitosan from chitin because of the
maintenance of the compounds properties and environment-
friendly processing (Beaney et al., 2007). It is rst reported in
1975 that CDA was found in the supernatant fraction of Mucor rouxii
(Araki and Ito, 1975). Subsequently, CDA was also found in Colle-
totrichum lindemuthianum (Kauss and Bauch, 1988), Absidia
coerulea (Gao et al., 1995), Aspergillus nidulans (Alfonso et al., 1995),
and Metarhizium anisopliae (Nahar et al., 2004) as well as marine
bacteria and insects (Zhao et al., 2010).
However, there are some limitations in developing the enzy-
matic DA reaction because of the crystallinity, insolubility, and high
molecular weight of chitin (Pareek et al., 2012). Thus, further at-
tempts, such as modication, chemical treatment, steam-explosion
and supercritical technology have been made to optimize the pa-
rameters of the enzymatic process for maximal DA (Yang et al.,
2015).
Chitin was modied to improve the efciency of the enzymatic
DA process for the production with biological properties (Beaney
et al., 2007). Modied chitin was then treated for 24 h with
fungal enzymes extracted from C. lindemuthianum. It turned out
that the modication of chitin by the solvent and drying methods
had a signicant impact on the efciency of the enzymatic DA
process. The most successful modication method was to freeze
dry a colloidal chitin suspension, which increased the degree of
enzymatic DA by 20 fold. In another study, chitin deacetylase from
Penicillium oxalicum SAEM-51 was used for the bioconversion of
chitin to chitosan in a two-stage chemical and enzymatic process.
Chemical treatment was used to improve the morphology, crys-
tallinity, and thermal properties of chitin. Then, pretreatment with
chitin deacetylase from P. oxalicum SAEM-51 to produce chitosan
yielded a maximum DA of 79.5% (Pareek et al., 2012). Besides, in
order to circumvent the drawback of the relatively high crystalline
structure of chitin, chitin was subjected to a pretreatment based on
suspension in supercritical 1,1,1,2-tetrauoroethane (scR134a),
which possesses a critical temperature of 101
C and pressure of
40 bar, followed by rapid depressurization to atmospheric pressure
and further brillation. This method was compared to chitins

Table 3
Summary of biological treatments for pure chitin recovery (DP: deproteinization, DM: demineralization).

Products Shrimp waste sources Treatment DP (%) DM(%) References

Chitin Shells of crab, shrimp, prawn, krill and Crude enzymatic DP (with Bacillus 84 94 Pachapur et al., 2015
lobster licheniformis) followed by a 1.28 M HCl
DM process
Chitin Metapeneaus monoceros Fermented with Pseudomonas 90 92 Ghorbel-Bellaaj et al., 2012
aeruginosa A2
Chitin Chionoecetes japonicas Fermented with L. paracasei KCTC-3074 90.5 97.2 Jung et al., 2006
and S. marcescens FS-3
Chitin Metapenaeus monoceros Crude protease from Bacillus cereus SV1 89.6 NA Manni et al., 2010
Chitin Metapenaeopsis dobsoni Fermented with Bacillus subtilis 84 72 Sini et al., 2007
Chitin and chitosan Metapenaeus monoceros Balistes capriscus proteases and Bacillus 78 NA Younes et al., 2014
mojavensis A21
Chitin Penaeus monodon and Crangon crangon Fermented with Bacterium HP1 98 99 Xu et al., 2008
followed by Lactobacillus casei MRS1
Chitin Penaeus vannamei Fermented with Serratia marcescens 94.5 93 Zhang et al., 2012
B742 followed by Lactobacillus
plantarum ATCC 8014
Chitin Penaeus vannamei Fermented with Lactobacillus 79.6 88.6 Wahyuntari et al., 2011
acidophilus FNCC116 followed by
Lactobacillus acidophilus FNCC116
Chitin Litopenaeus vannamei Fermented with Streptococcus 92 93.6 Mao et al., 2013b
thermophilus
Chitin Litopenaeus vannamei Acid stable protease 95 99 Baron et al., 2015
Chitin Litopenaeus vannamei Protease from Streptomyces griseus and 91.1 98.6 Hongkulsup et al., 2015
acetic
Chitin and chitosan Penaeus semisulcatus Lactobacillus plantarum, Lactobacillus NA NA Khanafari et al., 2008
crystalline powder acidophilus,Lactobacillus rhamnosus
Chitin Litopenaeus vannamei Fermented with Bacillus licheniformis 87 93.5 Liu et al., 2014
21886 and Gluconobacter oxydans DSM-
2003
Chitin Palinurus sp. Proteases produced by Erwinia 75 80 Giyose et al., 2010
chrysanthemi
Chitin Penaeus monodon 1 M NaOH followed by mixed organic 91.4 86 Charoenvuttitham et al., 2006
acids
Chitin Penaeus vannamei autolysis at pH 2 NA NA Sjaifullah and Santoso, 2016
 X. Mao et al. / Journal of Cleaner Production 143 (2017) 814e823 819

without treatment and subjected to steam explosion demon-
strating improved production of enzymatic hydrolysis (Villa-Lerma
et al., 2016).
Chitooligosaccharides and N-acetyl glucosamine are the prod-
ucts of chitosan and chitin hydrolysis, respectively. Similar to chi-
tosan polymers, these products have diverse biological
characteristics and potential applications in medical, agricultural,
and food industries as well as environmental protection.
Hydrolysis of chitosan for producing chitooligosaccharides can
be achieved using different physical and chemical methods such as
hydrothermal methods (Sato et al., 2003), microwave processing
(Xing et al., 2005), ultrasound (Wu et al., 2008), and chemical
methods using acid. Acid hydrolysis was traditionally the most
widely used method, despite its suboptimal features, including
hard to control, low quality of production, and environmental
pollution.
In recent years, more gentle and controllable enzymatic hy-
drolysis of chitin and chitosan has been developed as an alternative
method. Chitosan depolymerized by HCl hydrolysis and commer-
cial enzymatic degradation have been previously compared
(Cabrera and Cutsem, 2005). The fragments produced by the two
methods differed in the degree of polymerization and acetylation,
as detected using mass spectrometry. The results indicated that the
enzymatic procedure produced smaller molecules with a higher
proportion of fully deacetylated chitooligomers, although acid hy-
drolysis led to fragments with a degree of polymerization of up to
16 and more monoacetylated residues.
Chitinase hydrolyzes the b-(1, 4) bonds of the chitosan or chitin
chain down to the N-acetyl-D-glucosamine dimer (Jolle s and
Muzzarelli, 1999). In addition, diverse enzymes have been used to
produce chitooligosaccharides such as pepsin, cellulase, amylase,
pectinase, and crude enzymes. Chitooligosaccharides were pre-
pared from C. bilineata larvae skin by DM, DP, DA and hydrolysis
using commercial a-amylase under the optimal hydrolysis condi-
tions of pH 5.5, 55
C, 4 h and 40 mg/(g chitosan) of enzyme
amount. The Fourier transform infrared spectra indicated that
chitooligosaccharides with a degree of polymerization in the range
of 2e8 were the main component of the nal product, and the
chitooligosaccharide content and yield were up to 95.8% and 96.2%
(w/w), respectively (Wu, 2012). Biochemicals from chitooligo-
saccharides produced by various enzymes are shown in Table 4.
6. The process for astaxanthin recovery
Conventional chemical treatment of shrimp waste by DP and
DM will damage the structure of biomolecules such as astaxanthin,
rendering them useless (Gortari and Hours, 2013). Thus, acid
ensilage with milder organic acids has been reported to stabilize
carotenoids and to enable their further recovery (Sachindra et al.,
2007). Nevertheless, the astaxanthin recovery method relies on
solvent extraction (including acetone, ethyl acetate, hexane, iso-
propanol, methanol, methylethyl ketone, and ethanol) from natural
materials and is considered hazardous, expensive, and time
consuming and causes environmental pollution. Moreover, the
stability of astaxanthin decreases and its structure is damaged by
 pez et al.,
the large number of organic solvents (Han et al., 2012; Lo
2004). Supercritical uid extraction, such as using supercritical
carbon dioxide, is employed to recover astaxanthin (Valderrama
and Perrut, 2003).
Increasing concerns about the environmental hazards and

Table 4
Biochemicals from chitooligosaccharides by enzymes.

Products Chitin source Enzymes Condition Yield (%) References

pH Temperature Time(h)
(
C)

N,N-diacetylchitobiose Chitin Vihrio anyurllarum strain E-383a 7 30 48 40.3 Takiguchi and Shimahara, 1988
Chitooligosaccharide Chitosan Chitinases from Bacillus cereus TKU027 6 50 NA NA Wang et al., 2012
Chitooligosaccharide Chitosan Enzyme from Paenibacillus chitinolyticus 7 NA 60 99.2 Arajo et al., 2013
and Paenibacillus ehimensis
Chitooligosaccharide Chitosan a-amylase 5.5 55 4 96.2 Wu, 2012
N-acetylglucosamine, Chitin Chitosanase from Aspergillus sp. 5.5 40 0.5 NA Sinha et al., 2014
glucosamine,
Chitooligosaccharides
Chitoligosaccharides Chitosan Mixture of cellulase, a-amylase, and 5.6 40 0.7 NA Zhang et al., 1999
proteinase
Chitoligosaccharides Chitosan Pectinase from Aspergillus niger 3.0 38 6 86.0 Kittur et al., 2005
N-acetylglucosamine Chitin Chitinase C and 5 55 8 90 Nguyen-Thi and Doucet, 2016
N-acetylhexosaminidase from
Streptomyces coelicolor A3 (2)
Chitoligosaccharides Chitosan Cellulase 5.3 45 6 79.8 Dong et al., 2015
Chitoligosaccharides Chitosan Chitosanase from Ficus awkeotsang 4.5 50 NA 94 Chang et al., 2016
Makino
Chitooligosaccharides and Chitosan Chitosanase from Streptomyces sp. 6.8 40 2 40 Sinha et al., 2012
Glucosamine
Beta-glucosamin Chitosan Exochitosanase from Penicillium 5.6 42 NA NA Nidheesh et al., 2015a
decumbens CFRNT15
Chitooligosaccharides Chitin and Chitinase from Pyrococcus furiosus and 5.0 40 24 67 Mahata et al., 2014
chitosan Trichoderma viride
Chitosan oligomers Chitosan Papain 4.0 45 24 NA Lin et al., 2002
N-acetyl-b-D-glucosamine Chitin Chitinase 4.5 45 25 92.6 Fu et al., 2014
Chitooligosaccharide Chitosan Chitin deacetylase from Colletotrichum NA 55 12 NA Kang et al., 2014
lindemuthianum
Chitooligosaccharide Chitosan Glycosyltransferase 5.0 50 NA NA Montilla et al., 2013
Chitooligomers Chitosan Chitosanase from Purpureocillium 5.6 32 96 NA Nidheesh et al., 2015b
lilacinum CFRNT12
Chitooligosaccharide Chitosan Cellulase from Aspergillus niger 5.0 50 24 NA Xie et al., 2009
(GlcNAc)2 Chitin Chitinase from Paenibacillus barengoltzii 5.5 55 12 89.5 Yang et al., 2016
Chitooligosaccharide Chitosan Pectinex Ultra Spl 5.5 37 24 NA Cabrera and Cutsem, 2005
820 X. Mao et al. / Journal of Cleaner Production 143 (2017) 814e823

Table 5
Different treatments for carotenoids (astaxanthin) extraction by biotechnologies.

Products Shrimp waste sources Treatment enzymes/microorganisms sources carotenoids recovery (%) References

Carotenoid brown shrimp Enzymatic hydrolysis Trypsin 55 Chakrabarti, 2002

pigment (Metapenaeus monoceros)
Carotenoids Penaeus indicus Enzymatic hydrolysis Alcalase 82.5 Sowmya et al., 2014
Carotenoids Penaeus monodon Fermentation Natural probiotic 72.6 Prameela et al., 2010b
Carotenoids Penaeus monodon Fermentation Pediococcus acidolactici CFR2182 >75 Bhaskar et al., 2007
Astaxanthin rock lobster(Jasus lalandii) Enzymatic hydrolysis Papain 54 mg/g Auerswald and Ga de, 2008
Free astaxanthin Litopenaeus vannamei Fermentation Lactobacillus plantarum 2400 mg/g Pacheco et al., 2010
Astaxanthin X. kroyeri Enzymatic hydrolysis Alcalase and pancreatin 47-57 mg/g (dry waste) Holanda and Netto, 2006
Carotenoids Litopenaeus vannamei Autolysis Endogenous enzymes 826 mg/g Cah et al., 2012
Carotenoids Penaeus monodon Autolysis Endogenous enzymes 63.4 Sowmya et al., 2011
Carotenoids Penaeus semisulcatus Enzymatic hydrolysis Trypsin NA Jafari et al., 2012
and alkaline treatment
Astaxanthin Pandalus borealis Enzymatic hydrolysis Proteolytic enzymes 91.9 Lee et al., 1999
and reddish top layer
Xanthophylls Parapenaeus longirostris Enzymatic hydrolysis Barbel and bovine trypsins 80 mg/g Sila et al., 2012
Carotenoids Litopenaeus vannamei Fermentation and Pediococcus pentosaceus, 66000 mg/g Armenta and
enzymatic Savinase and Lipolase Guerrero-Legarreta, 2009
hydrolysis
Carotenoids Penaeus indicus Fermentation Lactobacillus plantarum B 4496 31.3 mg/g Sachindra et al., 2007
Astaxanthin Penaeus sp. Fermentation and Lactobacillus bacterials,savinase, 46000 mg/g Armenta-Lopez et al., 2002
enzymatic neutrase, alcalase and esperase
hydrolysis
Free astaxanthin Litopenaeus vannamei, Fermentation Lactobacillus plantarum 115 mg/g Gimeno et al., 2007
stylirostris and setiferus

economic effects related to the applications of these physical
technologies promoted the search for a simple, fast, and efcient
alternative method for extracting astaxanthin from shrimp waste.
Enzymatic hydrolysis using commercial, crude, and endogenous
enzymes has been explored as an alternative method to extract
astaxanthin ester from shrimp waste. The use of a mixture of two
enzymes, barbel and bovine trypsin, to recover astaxanthin has
offered promising results with good levels of xanthophylls
(93.2 mg/g) and total protein (7.4 mg/g) (Sila et al., 2012). Car-
otenoproteins from shrimp waste were hydrolyzed with protease
(Savinase) and lipase (Lipolase), and 900 mg/g soluble protein and
66 mg/g total carotenoids were achieved by a combination of 15
proteolytic units together with 10 lipolytic units. In addition, a
relatively protein-free form of astaxanthin derived from shrimp
waste carotenoproteins was used in natural health products and
cosmetics (Armenta and Guerrero-Legarreta, 2009).
Fermentation can also be used for stabilizing and extracting
pigments. According to previous reports, lactic fermentation has
been employed to extract carotenoids, particularly astaxanthin,
stabilizing the residues by producing acids. The results were as
good as those obtained using the fermentation broth (Armenta-
pez et al., 2002). The different treatments for carotenoid (astax-
Lo
anthin) extraction by biotechnological processes are summarized in
Table 5.
7. Future challenges and trends in comprehensive utilization
of shrimp byproducts
The biotechnologies described in this review are promising
methods for efcient shrimp waste recovery with lower cost,
reduced labor, and greater safety than traditional methods (such as
acid hydrolysis). However, to date, there are limitations and
outstanding challenges in developing biotechnologies for the
comprehensive utilization of shrimp byproducts. The technical
bottlenecks of biocatalysis, biotransformation, and fermentation
methods include limited reuse and stability of enzymes, difculty
in sustaining continuous reactions, and small total sample capac-
ities. Thus, further research on rational enzyme design, enzyme
immobilization, solid-state fermentation, and continuous mixing
bioreactor systems are required to make biocatalyst reactions with
high activity, stability, specicity, and total yield.
Siegel et al. (2010) speculated that improving the reaction
characteristics of enzymes by rational molecular design will create
new applications in the biocatalysis and biotransformation in-
dustries. Moreover, immobilization methods have proven effective
for overcoming the limitations of biocatalysis in many industrial
applications (Cui et al., 2016).
In recent years, traditional solid-state fermentation technology
has been applied for the improvement of many agricultural prod-
ucts. For instance, Seo and Cho (2016) reported improved nutritional
quality of soybean meal by solid-state fermentation using B. subtilis.
Such studies support the potential of solid-state fermentation
methods for shrimp/crustacean waste. However, sustaining
fermentation with high efciency is an outstanding challenge. Han
et al. (2016) performed a techno-economic analysis of dark
fermentative hydrogen production from molasses in a novel
continuous mixing immobilized sludge reactor and concluded that a
hydrogen-producing plant with higher scale (50 m3) was econom-
ically feasible. This analysis provides the theoretical foundation for a
bioreactor design with practical industrial applications that could
enhance the comprehensive utilization of crustacean waste.
8. Conclusion
The trend toward sustainable development of environment and
economy has resulted in large-scale discussions on the use of sea-
food waste. In particular, in recent years, biotechnological methods
have become the mainstream of future research instead of haz-
ardous chemical routes. A simple, fast, effective, clean, economic,
and controllable bioprocess for the comprehensive utilization of
crustacean waste needs to be further investigated. Moreover,
adapting laboratory-scale biotechnological methods for ecologi-
cally safe industrial-scale bio-extraction of nutrients from shrimp
waste is a major challenge of future research.
Acknowledgements
This work was supported by the Major Special Science and
Technology Projects in Shandong Province (2015ZDZX05003),
Applied Basic Research Program of Qingdao (16-5-1-18-jch), the
 X. Mao et al. / Journal of Cleaner Production 143 (2017) 814e823
Fundamental Research Funds for the Central Universities
(201564018) and Shandong Provincial Natural Science Foundation
(ZR2015CQ021).
Abbreviations
DH degree of hydrolysis
DP deproteinization
DM demineralization
DA deacetylation
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