Molecular epidemiology of cystic echinococcosis in human and production animals in Tunisia.
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\r\n\tThe authors are cordially invited to express their knowledge and awareness in this domain, to share their unpublished clinical trials pertaining to any type of STT, to analyse any new data emerged from their studies and to display their information in a methodical way, so that we may present an original book with novel and useful medical material.
Cystic echinococcosis (CE) or hydatidosis, caused by Echinococcus granulosus, is a widespread zoonosis in the world, especially in North African countries such as Libya, Algeria, Morocco, and Tunisia [1, 2]. Alveolar echinococcosis (AE), caused by the larval stage of E. multilocularis is extremely rare in Maghreb and only three autochthonous cases of human AE were reported in Tunisia and Morocco [3–5]. CE is of veterinary and medical importance because infection with metacestode may cause severe illness and significant socio-economic repercussions. With an annual surgical incidence (SI) averaging 12.6/100000 inhabitants [6] and approximately US$ 10–19 million losses annually in both humans and animals [7], Tunisia is one of the most endemic areas amongst the Mediterranean countries. Neighboring countries such as Italy (SI=1.6), Algeria (SI=3.6–4.6), Morocco (SI=4.6), Libya (SI=4.2), Spain (SI=0.3), and France (SI=0.1) presented lower surgical incidence [1, 8, 9]. In the E. granulosus vital cycle the adult tapeworm lives in the intestine of some carnivores (definitive hosts), and the larval stage develops in the herbivores (intermediate hosts) and humans, essentially in the liver and lungs.
E. granulosus is a complex in which four or five cryptic species are intermixed: E. granulosus sensu stricto (genotypes G1 to G3), E. equinus (genotype G4), E. ortleppi (genotype G5), E. canadensis (genotypes G6 to G10), and Echinococcus felidis (lion strain) [10–12]. The characterization of the species/genotypes responsible for human and animal hydatidosis is important in order to adapt the measures of control and prevention against this parasitic disease. Indeed, it is known that the genotype influences the life cycle patterns, the host specificity, and the pathology. Four genotypes (G1, G3, G4, and G6) have been described in Tunisia using molecular techniques such as PCR-RFLP analysis of the ribosomal DNA ITS1 fragment and mitochondrial cytochrome C oxidase (Cox1) or elongation factor 1-alpha (ef1a) gene sequencing (Table 1). The G1 genotype was identified in humans, sheep, cattle, and dromedaries [13–16], the G3 genotype in cattle and human isolates [17], the G6 genotype in one human case and in the Southern dromedaries [13, 16], and the G4 genotype in donkeys [16]. Recently, it has been demonstrated that donkeys can be infected by two different species, E. granulosus s.s (G1 genotype) and E equinus, occurring in sympatry [16].
\n\t\t\t\tOrigin\n\t\t\t | \n\t\t\t\n\t\t\t\tNo. of cysts\n\t\t\t | \n\t\t\t\n\t\t\t\tGene markers\n\t\t\t | \n\t\t\t\n\t\t\t\tGenotype frequency (%)\n\t\t\t | \n\t\t\t\n\t\t\t\tReference\n\t\t\t | \n\t\t
\n\t\t\t\tSheep\n\t\t\t | \n\t\t\t102 | \n\t\t\tITS1 and cox1 | \n\t\t\tG1 (100%) | \n\t\t\t[13] | \n\t\t
33 | \n\t\t\t12s rRNA | \n\t\t\tG1 (100%) | \n\t\t\t[18] | \n\t\t|
10 | \n\t\t\tCox1 | \n\t\t\tG1 (100%) | \n\t\t\t[17] | \n\t\t|
33 | \n\t\t\tCox1 and ef1a | \n\t\t\tG1 (100%) | \n\t\t\t[16] | \n\t\t|
\n\t\t\t\tCattle\n\t\t\t | \n\t\t\t79 | \n\t\t\tITS1 and cox1 | \n\t\t\tG1 (100%) | \n\t\t\t[13] | \n\t\t
4 | \n\t\t\t12sRNA | \n\t\t\tG1 (100%) | \n\t\t\t[18] | \n\t\t|
10 | \n\t\t\tCox1 | \n\t\t\tG1 (90%); G3 (10%) | \n\t\t\t[17] | \n\t\t|
19 | \n\t\t\tCox1 and ef1a | \n\t\t\tG1 (100%) | \n\t\t\t[16] | \n\t\t|
\n\t\t\t\tHuman\n\t\t\t | \n\t\t\t50 | \n\t\t\tITS1 and cox1 | \n\t\t\tG1 (100%) | \n\t\t\t[13] | \n\t\t
10 | \n\t\t\tCox1 | \n\t\t\tG1 (90%); G3 (10%) | \n\t\t\t[17] | \n\t\t|
241 | \n\t\t\tITS1 and cox1 | \n\t\t\tG1 (100%) | \n\t\t\t[15] | \n\t\t|
25 | \n\t\t\tCox1 and ef1a | \n\t\t\tG1 (100%) | \n\t\t\t[16] | \n\t\t|
\n\t\t\t\tDromedary\n\t\t\t | \n\t\t\t3 | \n\t\t\tITS1 and cox1 | \n\t\t\tG6 (100%) | \n\t\t\t[13] | \n\t\t
13 | \n\t\t\tCox1 | \n\t\t\tG1 (100%) | \n\t\t\t[14] | \n\t\t|
11 | \n\t\t\tCox1 and ef1a | \n\t\t\tG1 (72%); G6 (28%) | \n\t\t\t[16] | \n\t\t|
\n\t\t\t\tDonkey\n\t\t\t | \n\t\t\t37 | \n\t\t\tCox1 and ef1a | \n\t\t\tG1 (40%); G4 (60%) | \n\t\t\t[16] | \n\t\t
\n\t\t\t\tGoat\n\t\t\t | \n\t\t\t14 | \n\t\t\tCox1 and ef1a | \n\t\t\tG1 (100%) | \n\t\t\t[16] | \n\t\t
Molecular epidemiology of cystic echinococcosis in human and production animals in Tunisia.
The Tunisian situation is grossly the same as other Maghreb countries (Algeria and Libya) where the G1 and the G6 genotypes were reported in livestock and camels [19–22].
In Tunisia, the most frequent genotype associated with CE is the G1 genotype (E. granulosus sensu stricto) [13–16]. Currently, at least 43 different haplotypes were described for the Cox1 gene by molecular analysis in humans and different intermediate hosts (Genbank accession numbers: U50464, AY679144, AY679145, AY679146, KM014606-KM014644, Table 2) [13, 16]. The existence of genetic and phenotypic variants inside this genotype has been previously shown by using isoelectric-focusing techniques [23, 24]. Regarding the host origin, it has been demonstrated that there is a slight difference of the G1 genotype in parasite populations between sheep, human, and cattle (Fst values from 0.05 to 0.15) [16, 23]. The cysts originating from human (lung or liver) are intermediary between sheep and cattle origins as considering the genetic variability, whereas cattle and sheep isolates are slightly different [25].
\n\t\t\t\tGenBank accession numbers\n\t\t\t | \n\t\t\t\n\t\t\t\tMutation\n\t\t\t | \n\t\t\t\n\t\t\t\tCO1 protein mutation\n\t\t\t | \n\t\t\t\n\t\t\t\tHost\n\t\t\t | \n\t\t\t\n\t\t\t\tCyst localisation\n\t\t\t | \n\t\t\t\n\t\t\t\tReference\n\t\t\t | \n\t\t
U50464 | \n\t\t\tC56T | \n\t\t\tA27V | \n\t\t\tHuman, Sheep , Cattle | \n\t\t\tLung and Liver | \n\t\t\t[13] | \n\t\t
AY679144 | \n\t\t\tT123C | \n\t\t\tNone | \n\t\t\tSheep | \n\t\t\tLiver | \n\t\t\t[13] | \n\t\t
AY679145 | \n\t\t\tG312A | \n\t\t\tNone | \n\t\t\tHuman | \n\t\t\tLung | \n\t\t\t[13] | \n\t\t
AY679146 | \n\t\t\tT204G | \n\t\t\tNone | \n\t\t\tCattle | \n\t\t\tLung | \n\t\t\t[13] | \n\t\t
KM014606 to KM014644 | \n\t\t\t- | \n\t\t\t- | \n\t\t\tCamels, cattle, goat sheep, human, jackals, donkey, wild boar | \n\t\t\t- | \n\t\t\t[16] | \n\t\t
Molecular epidemiology and G1 genotype genetic diversity observed in Tunisia.
The E. granulosus adult stage infects the Canidae that releases the parasite eggs in the environment through their feces. Humans are a dead-end host that do not play a role in the natural cycle of the parasite. They are contaminated by an accidental consumption of the eggs, resulting from the contact with an infected dog or through the ingestion of contaminated vegetables. Eggs result in the development of one or several unilocular hydatid cysts (Figure 1) that could grow up to 20 cm in diameter [15, 26, 27]. Because of the slow rate of cyst growth, clinical symptoms do not usually arise until several years after infection. The liver and the lungs are the most commonly involved organs but the cyst can occur almost anywhere in the body.
Hydatid cysts from Tunisian children operated at F. Bourguiba Monastir teaching hospital. A: Pulmonary cyst from an eight-year-old child. B: Hepatic cyst from a six-year-old child. Photograph: LP3M: Laboratory of Medical and Molecular Parasitology-Mycology, Faculty of Pharmacy, University of Monastir, Tunisia.
CE remains an important public health problem in Tunisia and despite the deployed prevention program, a slight reduction in the mean annual surgical incidence rate (SI) from 15 to 12.6 cases/100,000 inhabitants was observed during the last 20 years [6, 28]. The endemic status differs from one region to another, based on the SI, and some areas have been defined as hyperendemic (SI>22.6), holoendemic (15 < SI <22.6), mesoendemic (7.5< SI <15), and hypoendemic regions (SI < 7.5) [6]. The geographical repartition of different endemic regions was shown in Figure 2. Hydatidosis is known to be more important in rural areas where the definitive hosts (domestic and wild Canid) and herbivore intermediate hosts are in close contact, but an extension in the urban zones was noted during the past decade.
Hydatidosis endemic status based on mean human annual surgical incidence (SI) published by [6]. (The maps of Africa and Tunisia come from http://d-maps.com/).
In Tunisia, several human studies were focusing on pediatric hydatidosis [15, 27, 29–31]. Cystic echinococcosis, which commonly starts during childhood or adolescence and described as a young adult disease, may be observed at any age. Two studies on children hydatid cysts (161 and 241 cysts) were carried out between 1999 and 2009 and assessed that the greatest number of cases was observed in the age groups of 4–9 years [15, 31]. Another retrospective study conducted between 1985 and 2009 and based exclusively on 757 pulmonary cysts of young children (3–7 years) and older children (8–15 years) demonstrated a mean age of 5.7 and 12 years old, respectively [27]. This early infestation has already been described in Turkey [32], Palestine [33], and Jordan [34].
In children, the lungs are the most common sites for hydatid cysts followed by the liver (Table 3) [15, 27, 31]. This could be explained by noisier and earlier symptoms in children where cough, chest pain, and hemoptysis are the most frequently encountered signs [27, 30]. Some exceptional cyst localizations (central nervous system, orbit, spleen, kidney, and heart) were also described [15, 35, 36]. Several organs may be contaminated simultaneously and about 20% of cases reported in literature were involved in multiple cysts with essentially liver associated with lung [15, 27]. The growth of the hydatid is independent of children’s age since cysts of high diameter (15 cm) were found in children of 4 years old [15, 31].
\n\t\t\t\tNo. of patients\n\t\t\t | \n\t\t\t\n\t\t\t\tNo. of cysts\n\t\t\t | \n\t\t\t\n\t\t\t\tCyst localization\n\t\t\t | \n\t\t\t\n\t\t\t\tFrequency (%)\n\t\t\t | \n\t\t\t\n\t\t\t\tFertility (%)\n\t\t\t | \n\t\t\t\n\t\t\t\tViability (%)\n\t\t\t | \n\t\t\t\n\t\t\t\tReference\n\t\t\t | \n\t\t
195 | \n\t\t\t241 | \n\t\t\tLiver | \n\t\t\t34.8 | \n\t\t\t68.5 | \n\t\t\t77 | \n\t\t\t[15] | \n\t\t
Lung | \n\t\t\t61.8 | \n\t\t\t83 | \n\t\t||||
Other | \n\t\t\t3.4 | \n\t\t\t71 | \n\t\t||||
121 | \n\t\t\t161 | \n\t\t\tLung | \n\t\t\t59.5 | \n\t\t\t70 | \n\t\t\t79 | \n\t\t\t[31] | \n\t\t
Liver | \n\t\t\t36 | \n\t\t\t81.5 | \n\t\t||||
Other | \n\t\t\t4.5 | \n\t\t\t71 | \n\t\t
CE prevalence, cyst fertility, and protoscoleces viability in Tunisian children.
Numerous pediatric studies have noted a slight male predominance compared to girls with a sex ratio of 1.2 to 1.8 [15, 27, 28, 31]. This observation was although reported in Algeria [37], Iran [38], Bulgaria [39], and Jordan [34]. The higher infestation of boys compared to girls in endemic countries is explained by the fact that school-age boys have more external activities than girls, with a greater promiscuity with dogs. In adults, in contrast to what is observed in children, women are more commonly affected than men [28, 40, 41]. This difference is due to their role in the home activities and that in rural areas, the women are more often at home and care very often for dogs and cattle, which increases the risk of contamination. In addition, the adult females have more regular medical follow-up (e.g., during pregnancy) that results in fortuitous cyst discoveries on ultrasound examinations.
The direct examination of the cyst allows studying its fertility (presence or absence of protoscoleces) and the protoscolex viability. The cyst fertility was analyzed by light microscopic observation. Protoscolex viability was determined using vital eosin 0.2% coloration (Figure 3). The fertility of the cyst is independent of its location and its size and no relation with the age of infected children was noticed (Table 3) [15, 27, 42]. The cyst fertility and protoscoleces viability in humans are not involved in the maintenance of the parasite life cycle because human is a dead-end host but they are parameters attesting of the perfect adaptation of the parasite to humans.
As mentioned before in the genotyping section, the most frequent species associated with human hydatidosis is the E. granulosus sensu stricto (G1 genotype). Nevertheless, for two children, the E. granulosus sensu stricto (G3 genotype) and the E. canadensis species were observed. The fact that humans could be infected by different species/genotypes is an epidemiological feature to be taken into account in CE control measures.
Determination of protoscolex viability by using 0.2% eosin coloration: Alive (A) and dead (B) protoscolex. Photograph: LP3M: Laboratory of Medical and Molecular Parasitology-Mycology, Faculty of Pharmacy, University of Monastir, Tunisia.
Livestock echinococcosis leads to economic repercussions because of animal liver and lung condemnations, decrease of the carcass weight, animal fertility, and milk production [43]. In Tunisia, breeding remains mainly traditional and the population practices extensive sheep farming. Livestock are ubiquitous all over Tunisia, especially in rural areas and about 4 million sheep female unit (FU), 700,000 goat FU, 420,000 cattle FU, and 17,000 camel FU are recorded [44]. The CE prevalence in food animals depends on the presence of the intermediate host of the parasite and their close contact with the final host (stray and semi-stray dogs). These intermediate hosts differ from one region to another in function of climatic factors and/or breeding or alimentary practices. Thus, dromedary breeding is essentially located in Southern Tunisia (desertic climate) where camels are most consumed, whereas sheep and cattle breedings are practiced throughout the country. Herbivores acquire the infection through ingestion of echinococcus eggs excreted by the dog feces. The breeding of small animals is practiced by farmers, but also commonly practiced by households in rural and urban areas (Figures 4 and 5). The importance of pastoral animal husbandry constitutes a significant risk factor for echinococcosis transmission because of the close contact of production animals with dogs. Thus, the infected livestock that died on the pastures are not buried and dogs or other carnivores are able to access these cadavers and lead to complete the transmission cycle (Figure 6).
Pastoral sheep breeding in rural areas where sheep are in close contact with dogs. Photograph: LP3M: Laboratory of Medical and Molecular Parasitology-Mycology, Faculty of Pharmacy, University of Monastir, Tunisia.
Sheep breeding in urban areas where sheep are in close contact with dogs. Photograph: LP3M: Laboratory of Medical and Molecular Parasitology-Mycology, Faculty of Pharmacy, University of Monastir, Tunisia.
Carcass of dead cattle abandoned on the pasture and accessible to stray dogs. Photograph: LP3M: Laboratory of Medical and Molecular Parasitology-Mycology, Faculty of Pharmacy, University of Monastir, Tunisia.
CE prevalence in production animals was estimated by post-mortem examination of slaughtered animals at abattoirs. A series of studies carried out in Tunisia assessed that the prevalence of E. granulosus infection ranged from 16.42% to 40.42% in sheep, 8.56% in cattle, 6% in Dromedaries, 2.9% in goats, and 8.48% in donkeys [14, 45–47] (Table 4). The highest prevalence was observed in sheep, as is the case in Libya (20%) and Italy (11.5%) [21, 48], while the lowest prevalence was noted in goats. In Morocco and Algeria, the cattle (23%) and the camels (25%), respectively, are the most infected animals whereas husbandry and slaughtering practices are grossly the same as in Tunisia [19, 49, 50]. It has been accepted that variations in animal CE prevalence rates can be related to the species/genotype involved in the infection [51]. Nevertheless, in Tunisia, the G1 genotype is predominant in all intermediate hosts with high fertility rates [13] and only some infection cases by others genotypes are reported (see genotyping section). Moreover, a study conducted in Tunisia has demonstrated that the sheep over 8 years old are more often contaminated than sheep of 1-2 years and sheep of 4 years (60% vs. 20% and 40%, respectively) [46]. Consequently, in Tunisia, the difference in prevalence rate is more related to the age of slaughtered herbivores than to the species/genotype implied in the infection. In camels, the prevalence varies largely according to the region because of the difference in camel slaughtering practices. Thus, camels are slaughtered at an older age in South of Tunisia, whereas they are slaughtered at an earlier age (before 3 years) in Center of Tunisia. It has been demonstrated, in opposition to other intermediate hosts, that the camel cysts do not or seldom develop before the age of three [14, 52]. Therefore, it can be assumed that only old camels are implied in the parasite life cycle dog-camel.
Liver and lung are the only organs observed to be infected in production animals. The prevalence of the liver localization of the cyst was higher than that of pulmonary cysts except for the dromadaries where the pulmonary cysts are predominant (Table 4) [14, 45, 46, 53]. The co-infection of both organs in the same host is frequently observed and, contrary to what is usually described in humans, several cysts (up to 50 cysts) may develop in the same organ (Figure 7).
\n\t\t\t\tHosts\n\t\t\t | \n\t\t\t\n\t\t\t\tNo. of\n\t\t\t\t \n\t\t\t\tanimals\n\t\t\t | \n\t\t\t\n\t\t\t\tPrevalence (%)\n\t\t\t | \n\t\t\t\n\t\t\t\tLocation\n\t\t\t\t \n\t\t\t\tof cysts\n\t\t\t | \n\t\t\t\n\t\t\t\tFrequency (%)\n\t\t\t | \n\t\t\t\n\t\t\t\tFertility\n\t\t\t | \n\t\t\t\n\t\t\t\tProtoscolex viability\n\t\t\t | \n\t\t\t\n\t\t\t\tPeriod\n\t\t\t | \n\t\t\t\n\t\t\t\tReference\n\t\t\t | \n\t\t
Sheep | \n\t\t\t2722 | \n\t\t\t16.42 | \n\t\t\tLiver | \n\t\t\t54.97 | \n\t\t\t19.24 | \n\t\t\t74.94 | \n\t\t\t2003-2010 | \n\t\t\t[45] | \n\t\t
Lung | \n\t\t\t45.02 | \n\t\t\t11.01 | \n\t\t\t66.49 | \n\t\t\t2003-2010 | \n\t\t\t[45] | \n\t\t|||
1039 | \n\t\t\t40.42 | \n\t\t\tliver | \n\t\t\t40.42 | \n\t\t\t- | \n\t\t\t- | \n\t\t\t2001-2004 | \n\t\t\t[45] | \n\t\t|
248 | \n\t\t\t- | \n\t\t\tLiver | \n\t\t\t67.33 | \n\t\t\t86 | \n\t\t\t- | \n\t\t\t2000-2005 | \n\t\t\t[53] | \n\t\t|
- | \n\t\t\tLung | \n\t\t\t32.66 | \n\t\t\t63 | \n\t\t\t- | \n\t\t\t2000-2005 | \n\t\t\t[53] | \n\t\t||
Cattle | \n\t\t\t3913 | \n\t\t\t8.56 | \n\t\t\tLiver | \n\t\t\t62.58 | \n\t\t\t0.55 | \n\t\t\t67.9 | \n\t\t\t2003-2010 | \n\t\t\t[45] | \n\t\t
Lung | \n\t\t\t37.41 | \n\t\t\t0.40 | \n\t\t\t89 | \n\t\t\t2003-2010 | \n\t\t\t[45] | \n\t\t|||
203 | \n\t\t\t- | \n\t\t\tLiver | \n\t\t\t60.6 | \n\t\t\t76.5 | \n\t\t\t- | \n\t\t\t2000-2005 | \n\t\t\t[53] | \n\t\t|
- | \n\t\t\tLung | \n\t\t\t39.4 | \n\t\t\t27.5 | \n\t\t\t- | \n\t\t\t2000-2005 | \n\t\t\t[53] | \n\t\t||
Goat | \n\t\t\t3779 | \n\t\t\t2.88 | \n\t\t\tLiver | \n\t\t\t67.82 | \n\t\t\t15.57 | \n\t\t\t25.63 | \n\t\t\t2003-2010 | \n\t\t\t[45] | \n\t\t
Lung | \n\t\t\t32.14 | \n\t\t\t14.75 | \n\t\t\t14.8 | \n\t\t\t2003-2010 | \n\t\t\t[45] | \n\t\t|||
Dromed-aries | \n\t\t\t404 | \n\t\t\t5.94 | \n\t\t\tLiver | \n\t\t\t86.27 | \n\t\t\t22.22 | \n\t\t\t65.86 | \n\t\t\t2003-2010 | \n\t\t\t[45] | \n\t\t
Lung | \n\t\t\t13.72 | \n\t\t\t22.22 | \n\t\t\t13.72 | \n\t\t\t2003-2010 | \n\t\t\t[45] | \n\t\t|||
291 | \n\t\t\t6.5 | \n\t\t\tLiver | \n\t\t\t7.69 | \n\t\t\t- | \n\t\t\t- | \n\t\t\t- | \n\t\t\t[14] | \n\t\t|
Lung | \n\t\t\t92.30 | \n\t\t\t- | \n\t\t\t- | \n\t\t\t- | \n\t\t\t[14] | \n\t\t|||
8 | \n\t\t\t- | \n\t\t\t- | \n\t\t\t- | \n\t\t\t100 | \n\t\t\t100 | \n\t\t\t2000-2005 | \n\t\t\t[53] | \n\t\t|
Donkey | \n\t\t\t2040 | \n\t\t\t8.48 | \n\t\t\tLiver | \n\t\t\t89.9 | \n\t\t\t3.58 | \n\t\t\t35.80 | \n\t\t\t2006-2007 and 2009 | \n\t\t\t[47] | \n\t\t
Lung | \n\t\t\t10.09 | \n\t\t\t15.38 | \n\t\t\t32.96 | \n\t\t\t2006-2007 and 2009 | \n\t\t\t[47] | \n\t\t
CE prevalence and epidemiological data in Tunisian production animals.
Multiple hydatid cysts in the liver (A) and lungs (B) of a bovine. Photograph: LP3M: Laboratory of Medical and Molecular Parasitology-Mycology, Faculty of Pharmacy, University of Monastir, Tunisia.
However, available CE prevalence in livestock species does not reflect the real endemic situation since it is not considered private or illegal slaughtering. Uncontrolled home slaughtering during religious or local festivities is very common in Tunisia and the infected viscera unsuitable for consumption are rejected and eaten by dogs. The knowledge of the parasite cycle and its transmission modalities are weak, in spite of a perception of the risk notably in rural areas. For example, a study conducted in 2007 (76 patients, 90 and 100 humans from urban and rural areas, respectively), has demonstrated that 40% of topics interrogated have the false notion that the humans’ contamination is consecutive to the consumption of viscera containing hydatic cysts, whereas only 25.8% among them incriminate the dog [54]. The livestock trade in Tunisia is mainly based on weekly markets and exchange of animals all over the country. Thus, the lack of information on the exact geographical origin of livestock makes very difficult the identification of grazing areas at risk and the targeting of prophylactic measures.
Molecular analyses have demonstrated that E. granulosus species circulating in Tunisia are E. granulosus sensu stricto (G1 and G3 genotypes), E. canadensis (G6 genotype), and E. equinus (G4 genotype) [13, 16, 17] (see Table 1 in genotyping section). The cyst fertility and the viability of protoscoleces are the main factors that allow the maintenance of the cycle between intermediate and definitive host and leads to the existence of particular life cycles. Cyst fertility varied amongst livestock host populations with average rates of 44.8% (range: 11–86%) in sheep, 15% in goats, 27.13% (range: 0.4–76.5%) in cattle, 48.5% (range: 22.22–100%) in camels, and 4.77% in donkeys [13, 14, 45, 47, 53] (Table 4). Contrary to what was observed in European countries [48], G1 genotype has a significant fertility rate (46%) in the cattle host in Tunisia [13] and Algeria (from 52% to 70%) [19]. In general, a significant fertility rate, as far as cattle was concerned, was due to the E. ortleppi species (G5 genotype) and rarely to E. granulosus sensu stricto (G1 genotype) [55, 56]. The findings of fertile hydatid cysts and viable protoscoleces in cattle, suggest that Tunisian and Algerian cattle can act as suitable hosts for the G1 genotype. The perfect adaptation of this genotype to bovine host is a significant parameter risk for human contamination via infected dogs and proved that cattle has more importance in the transmission cycle than previously believed.
The CE is highly endemic in many North African countries and very high infection rates have been reported in dogs (55–58% in Morocco [49], 20–25.8% in Libya [57] and 19–42% in Algeria [58]). In Tunisia, although a sylvatic life cycle involving wild carnivores (golden jackals and red foxes) as definitive hosts was described [59], the E. granulosus transmission is typically through a synathropic cycle between dogs and livestock (essentially sheep, cattle, and camels). In Tunisia, the dogs are used primarily to guard livestock and property. The canine population is estimated at 800,000 dogs and composed essentially of feral and semi-feral (free-roaming dogs that are fed by an owner) dogs that rarely receive deworming treatment (Figure 8) [60].
In rural, urban, and semi-urban areas, the canine density is one dog per 3.0 to 5.5 inhabitants, one dog per 16 inhabitants and one dog per 46 inhabitants, respectively. In urban regions, less than 20% of households own a dog, whereas in rural regions there are 7–30 dogs per km², and more than 80% of households own at least one dog [61].
The prevalence of E. granulosus in dogs is estimated by several techniques including the detection of worms at necropsy, worm antigen in feces (coproantigen), or direct examination of eggs in dog feces. The prevalence of E. granulosus infection in Tunisian dogs ranges from 3.75% to 27.1% depending on the regions [59, 62, 63] (Table 5).
\n\t\t\t\tNo. of dogs\n\t\t\t | \n\t\t\t\n\t\t\t\tPrevalence (%)\n\t\t\t | \n\t\t\t\n\t\t\t\tWorm burden (n)\n\t\t\t | \n\t\t\t\n\t\t\t\tDiagnostic procedure\n\t\t\t | \n\t\t\t\n\t\t\t\tPeriod\n\t\t\t | \n\t\t\t\n\t\t\t\tReference\n\t\t\t | \n\t\t|
60 | \n\t\t\t18.4 | \n\t\t\t848 | \n\t\t\tNecropsy | \n\t\t\t2007 | \n\t\t\t[59] | \n\t\t|
375 | \n\t\t\t3.75 | \n\t\t\t- | \n\t\t\tPurgation | \n\t\t\t2002-2003 | \n\t\t\t[62] | \n\t\t|
256 | \n\t\t\t6.9-27.1 | \n\t\t\t2534 | \n\t\t\tNecropsy | \n\t\t\t1998-1999 | \n\t\t\t[63] | \n\t\t
Prevalence and molecular epidemiology of E. granulosus dog infection in Tunisia.
n: mean number of parasites per infected dog
Stray and semi-stray dogs in rural area of Gafsa governorate (South of Tunisia). Photograph: LP3M: Laboratory of Medical and Molecular Parasitology-Mycology, Faculty of Pharmacy, University of Monastir, Tunisia.
The study of the vital areas of stray dogs, based on 52 to 285 locations in a semi-urban area of Tunisia, has identified an area ranging between 0.06 and 8.53 km² [61]. Thus, considering that the average number of worms per dog is estimated at several thousands [59] and more than 8,000 Echinococcus eggs were shed per day [64], dogs cause massive environmental contamination. Using direct examination of dog feces, the overall contamination index of dog feces by E. granulosus was estimated to 25.3% and was ranged between 8.3% to 41.3% depending on the regions [65]. Nevertheless, the canine echinococcosis is not necessarily correlated to human CE since transmission of echinococcus is influenced by human activities and behavior [65, 66]. As in many African countries, dog contamination is essentially due to the consumption of uninspected meat during familial or religious slaughtering, improper disposal of offal or carcasses unsuitable for consumption that are eaten by numerous stray and semi-stray dogs, and lack of knowledge about the transmission of the disease [50, 60, 67]. Thus, 38.4% and 44% of butchers and population, respectively, have an inappropriate behavior concerning the management of infected offal. They throw them directly into the trash or bury them superficially leaving them easily accessible to dogs [67].
Despite the control programs, essentially based on the systematic condemnation of infected offal in slaughterhouses, cystic echinococcosis remains a major public health in Tunisia. The endemic status differs from one region to another, and some areas have been defined as hyperendemic, holoendemic, mesoendemic, and hypoendemic regions. Hydatidosis is important in rural areas but an extension in the urban zones was noted during the past decade. The characterization of the species responsible for echinococcosis in Tunisia is a significant point that has to be taken into consideration in order to focus and to adapt the control measures. Three Echinococcus species (E. granulosus sensu stricto, E. canadensis and E. equinus) have been described in different intermediate hosts but E. granulosus sensu stricto G1 genotype remains predominant. The continuing presence of CE in Tunisia depends on a variety of factors and human behavior plays an important role in the perpetuation of the cystic echinococcosis. The importance of pastoral animal husbandry, the elevated number of unrestrained dogs, and their frequent contamination by E. granulosus infected viscera are the major causes of the CE spread. The inadequate deworming treatment, the close contact of untreated dogs with humans, and animals particularly in rural areas, the hygiene level, the poor public awareness about the disease, and the favorable ecological and climatic conditions for the survival of Echinococcus eggs in the environment constitute ideal conditions for the transmission of the infection to dogs, humans, and animals. Thus, the sanitary education concerning hydatidosis should be reinforced and efforts should be made to implement a targeted educational program. Awareness should be created for the animal attendants, farmers, customers, slaughterhouse workers, and butchers regarding the CE public health significance.
The authors thank Imen Hizem-Attig for her assistance with the linguistic part of this paper.
Due to the ever-growing demand for energy, environmental impact caused by pollution, and the depletion of fossil fuel reserves, the demand for materials from renewable resources has become an important matter [1, 2]. Lignocellulose biomass is considered one of replacing chemicals as well as fuels based on oil [3]. Agricultural residues is one of the most valuable and renewable lignocellulosic biomass as well as a promising alternative for cellulosic materials. Among different sources of agricultural residues, rice straw has been extensively investigated because it is one of the most consumed cereals in the world, about 650–975 million tons per year all over the world [4, 5]. Rice straw is composed of approximately 35% cellulose, 18% hemicellulose, and 15% lignin [6]. It can be used as raw material for conversion to high value-added products through chemical, biochemical and physical processes. However, cellulose is usually accompanied by other structural biopolymers, saying hemicellulose, and lignin. Therefore, determination of methods for the efficient separation of the constitutive biomass components has long been the major obstacles to its utilization.
Thus, novel environmental-friendly processes have been developed continuously, which include steam explosion [7], organosolv processing [8], the chlorine-free method [5], biological treatment [9], and ionic liquid isolation [10]. In this chapter, a brief review on the separation of rice straw cellulose in sustainable ways and the related utilization of the cellulose enhanced composites are systematically presented.
Steam explosion (SE) has been a well-known technology for the pretreatment of straws during the separation of cellulose [11, 12, 13]. The critical process is the high-temperature hydrothermal treatment followed by a sudden exposure to atmospheric pressure [14].
During processing, the major hemicelluloses are partially hydrolyzed (autohydrolysis) due to high temperature transforming the acetyl groups connected with hemicellulose into acetic acid. Part of lignin can be depolymerized and leaving on the cellulose [15]. It is reported that the hydrolysis of hemicellulose separation was related to the reaction time and the pressure in the reactor [16]. For example, with the optimum condition (215°C, 7.5 min), a high-yield of sugar (81%) and ethanol (12.4%) can be obtained [17]. Therefore, the critical issue is a suitable condition of parameters, such as cooking time and the pressure, for efficient removal of hemicelluloses during the steam explosion which has been optimized by Zhou et al. [18]with a regression method in statistics.
Naturally dried rice straw was cut into pieces of about 2–3 cm in length and then shredded in a high-speed pulverizing mill. The crushing process was necessary for the effective infiltration of steam into the cell. The rice straw was then put into the reactor with a solid to liquid ratio of 3:1 (w/w). The high-temperature steam was poured in by opening a valve and cooked for a specified time, followed by explosion procedure.
A series of experiments were designed to investigate the effects of pressure X1 (corresponding to temperature) and cooking time X2 on the final product of hemicelluloses (γ-Cellulose) and cellulose (α-Cellulose) in the process of steam explosion by using a regression method in statistics. The number of the experiments (N) meets the equation below:
where, m is the number of independent factors and “3” is the number of experiments in the central point, which could improve the accuracy of the regression.
The spatial distribution of these two factors (X1, X2) in this regression design is illustrated in Figure 1. According to the earlier works of exploratory, the interval factors have been determined, as shown in Table 1.
Spatial distribution of these two factors.
Normalized | Pressure X1 (MPa) | Cooking time X2 (min) |
---|---|---|
1.414 | 3.2 | 32 |
1.000 | 3.0 | 30 |
0.000 | 2.5 | 25 |
−1.000 | 2.0 | 20 |
−1.414 | 1.8 | 18 |
Parameter table.
The details of the experimental plan as well as the content of hemicelluloses and cellulose of the steam-exploded straw are shown in Table 2. The sample 11 of steam explosion in Table 2 is selected for the comparison of the main components with the original rice straw. It is clear that most of the hemicelluloses can be hydrolyzed and extracted at the proper set of conditions for the steam explosion.
No. | X1 | X2 | Treatment severity lgRoa | γ-C (%)b | α-C (%)b | DPc |
---|---|---|---|---|---|---|
Rice straw | — | — | — | 17.98 | 35.06 | 1123 |
1 | 1.000 | 1.000 | 6.19 | 3.01 | 70.43 | 229.5 |
2 | −1.000 | 1.000 | 5.60 | 8.66 | 69.85 | 303.3 |
3 | 1.000 | −1.000 | 6.01 | 3.84 | 65.03 | 198.4 |
4 | −1.000 | −1.000 | 5.42 | 3.25 | 67.58 | 272.7 |
5 | 0 | 1.414 | 5.92 | 6.29 | 72.96 | 360.2 |
6 | 0 | −1.414 | 5.67 | 6.68 | 70.51 | 276.2 |
7 | 1.414 | 0 | 6.25 | 2.72 | 61.77 | 246.0 |
8 | −1.414 | 0 | 5.37 | 8.46 | 70.49 | 418.6 |
9 | 0 | 0 | 5.81 | 1.45 | 64.80 | 268.3 |
10 | 0 | 0 | 5.81 | 1.38 | 64.72 | 266.0 |
11 | 0 | 0 | 5.81 | 1.10 | 64.65 | 270.9 |
Effect of reaction time and pressure on the final content of hemicelluloses and cellulose [18].
Refers to the strength of the steam explosion and could be calculated by Eq. (2).
The contents of α-Cellulose and γ-Cellulose, respectively, which are determined according to Tappi method T203 cm-99.
The intrinsic viscosity of α-Cellulose ([η]) that is measured in cupriethylenediamine (CED) solution, and from which the DP (degrees of polymerization) could be calculated by the equation below, according to SCAN-CM 15:88 standard.
The value of lgRo is proportional to the strength of steam explosion. Additional hydrolysis of α-Cellulose may happen along with higher lgRo, as indicated by sample 7 in Table 2.
Experimental data in Table 2 have been fitted to the following second-order polynomial:
where Yi refers to α-Cellulose, γ-Cellulose and SCAN viscosity, respectively. The symbols of a, b, c, d, e and f are the corresponding estimated parameters. Regression has been carried out using a nonlinear method with the SPSS software, and the result data are as shown in Table 3. R2 values of α-Cellulose and γ-Cellulose are close to 0.9, which indicate good models for these factors. However, the model of SCAN viscosity is not well fitted, as indicated by the low value of the R2.
Symbols | γ-Cellulose (%) | α-Cellulose (%) | SCAN viscosity |
---|---|---|---|
a | 1.31 | 64.72 | 268.42 |
b | −1.65 | −1.79 | −48.76 |
c | 0.50 | 1.34 | 26.80 |
d | 1.80 | 0.54 | 14.93 |
e | 2.25 | 3.28 | 4.85 |
f | −1.56 | 0.78 | 6.95 |
R2 | 0.896 | 0.861 | 0.332 |
Characteristic constants and R2 of the regression [18].
Since the regression has established good models (Eqs. 5 and 6) describing the relationships between independent factors (referring to X1 pressure and X2 cooking time of the steam explosion process) and the responses (referring to Yi, final content of γ-Cellulose and α-Cellulose). These relationships can be visualized, as shown in Figure 2. The values of coordinates of their nadirs are given in Table 5.
A 3D graphic of dependent factor (referring to final content of γ-cellulose Yi) vs. independent factors (referring to pressure X1 and reaction time X2 of the steam explosion process), (a) according to Eq. (5), and (b) according to Eq. (6).
As discussed earlier, the aims of the steam explosion maximizing the removal of the γ-Cellulose and the loss of α-Cellulose. From the observation, there is no serious contradiction between these two aims, since the nadir of γ-Cellulose and α-Cellulose locates at the center and the edge, respectively. Therefore, the optimal condition for the steam explosion is 2.74 MPa and 25.3 min, according to the data in Table 4. Under the optimal condition, the structure of steam-exploded rice straw showed the characteristics of soft, loose and porous with different sizes distributed though all of the fiber (as shown in Figure 3a), which supported further separation of cellulose and lignin. The most of hemicelluloses could be efficiently hydrolyzed, leaving only 1% residual hemicelluloses. However, the majority of cellulose, as well as the fragmentized lignin, were retained in the size, which could be identified by the SEM observations (Figure 3a) and the FTIR spectra (Figure 3b) [18].
γ-Cellulose | α-Cellulose | |||
---|---|---|---|---|
Scaled factor | Factor | Scaled factor | Factor | |
Pressure X1 (MPa) | 0.481 | 2.74 | 1.414 | 3.20 |
Reaction time X2 (min) | 0.056 | 25.3 | −0.374 | 23.1 |
Content of cellulose Yi (%) | 0.93 | 62.82 |
Factor values for the minimum of the content of γ-cellulose and α-cellulose.
(a) SEM images and (b) FT-IR spectra of rice straw (RS) and steam-exploded rice straw (SERS) [18].
Ionic liquids (ILs) are emerging as promising solvents for treatment of lignocelluloses [19, 20, 21, 22, 23], due to its low vapor pressures. These solvents are made up of large organic cations and small inorganic anions, which have the following key properties: (a) they are liquids below 100°C or even at room temperature; (b) high thermal stability; and (c) high polarity [24]. These properties allow to be easily adjusted to dissolve diversity of lignocellulosic biomass [25, 26, 27]. Since several kinds of ionic liquids have been found to be non-derivatized solvents for cellulose, they have been applied in such research fields as capturing the portrait of single cellulose molecule [25], and chemical modification of cellulose [19]. In this section, ionic liquids are used in separation of cellulose from steam-exploded rice straw. It is proved to be an efficient and environmentally friendly way to selectively dissolve and then recover cellulose [6].
Four kinds of ionic liquids: 1-butyl-3-methylimidazolium chloride (BMIMCl), 1-allyl-3-methylimidazolium chloride (AMIMCl), 1-benzyl-3-methyl-imidazolium chloride (BnMIMCl) and 1-benzyl-3-methyl-imidazolium trifluoroacetate (BnMIMTFA) have been synthesized according to literature [20]. Their solubility for cellulose and lignin are shown in Table 5. The results indicate that BnMIMCl and BnMIMTFA are efficient for dissolving lignin. BMIMCl and AMIMCl are efficient for dissolving cellulose.
Ionic liquid | AMIMCl (%) | BMIMCl (%) | BnMIMTFA (%) | BnMIMCl (%) |
---|---|---|---|---|
Cellulose | 5.2 | 4.9 | - | - |
Lignin | - | - | 4.9 | 3.9 |
Solubility of the four ILs for cellulose and lignin [6].
“-” represents nearly no sample can be dissolved.
AMIMCl is selected as a selective solvent to separate cellulose from the steam-exploded rice straw. The contents of acid-insoluble lignin and celluloses of the steam-exploded rice straw are 14.76 and 64.80% respectively. After dissolving in AMIMCl, there is only 0.90% acid insoluble lignin contained in the recovered cellulose. With a procedure of bleaching by immersing the separated cellulose into hydrogen peroxide aqueous solution together with ozone blowing (about 3.4 g/h produced by SZH5 ozone apparatus, Peking) was needed for bleaching. The bleached cellulose was finally obtained with a yield of 30.73%. The component analysis according to TAPPI standard methods (T 222 om-06 and T 203 cm-09) indicated no detectable acid-insoluble lignin and only 0.85% of hemicelluloses left in the final cellulose. The average degree of polymerization (DP) was 484.
The SEM image and XRD profile of the bleached cellulose are shown in Figure 4. As seen in Figure 4a, cellulose fibers could be observed clearly with average lengths more than 100 μm. The prominent peak at 22.13° denotes the (002) reflection (as shown in Figure 4b). However, the characteristic (101) and (101) peaks (2θ between 15 and 17°) are not as distinct as those in cotton [24], but combine into one broad peak at 15.728°.
(a) SEM image and (b) XRD profile of bleached cellulose [6].
The FTIR spectra of the original rice straw, steam-exploded rice straw as well as the bleached cellulose had been provided, Figure 5a. The peaks at 1510, 1465 and 1423 cm−1 in the sample RS, corresponded to the skeleton stretch of the benzene ring, mainly contributed by lignin. These peaks reduced in steam-exploded sample and disappeared in the bleached cellulose, indicating that the lignin could be removed by selective dissolving of IL. The 13C CP/MAS solid-state NMR spectrum of the bleached cellulose shown in Figure 5b suggests that highly purified cellulose is obtained. The chemical shifts at 62 and 64 ppm are assigned to C6 of the primary alcohol group of cellulose, 71 and 74 ppm attributed to C2, C3 and C5, the ring carbons of cellulose. The peaks at 83 and 88 ppm associated with C4, and 104 ppm associated with C1. The feeble signals at 173 and 20 ppm attributed to the carbonyl and the methyl resonances, respectively.
(a)FT-IR spectra and (b) 13C CP/MAS solid-state NMR spectrum of rice straw (RS); steam-exploded rice straw (SERS); bleached cellulose (BC) [6].
The organosolv process can effectively degrade lignin, which is mainly used osmosis to break and decompose the internal chemical bonds of cellulose and hemicellulose [28, 29, 30]. It is considered to be an environmentally friendly way because it can be recycled conveniently, which demonstrates its potential utilization in isolation of lignocellulosic biomass [8]. Moreover, the structure of the dissolved component will be protected from degrading under such moderate conditions, which is benefit for further utilization. The efficient solvent for delignification combined with steam explosion treatment has been realized as the separation of the three components that are cellulose, hemicellulose and lignin. However, few organosolv process have been proved attractive as regards efficiency and selectivity, even though intensive researches have been done [31, 32]. In this section, consideration has given on the effect of delignification with mixed solvent from steam-exploded rice straw under ambient pressure [18].
First, the rice straw is pretreated by steam explosion as the optimal conditions mentioned above. The steam-exploded rice straw was then washed with hot water (1:20 g/mL). After that, the residue was delignified by different mixed solvent to obtain crude cellulose. Finally, it was bleached with aqueous solution (1:30 g/mL, pH 11, 55°C) of 2% hydrogen peroxide and 0.2% TAED (tetraacetylethylenediamine) for 5 h.
The mixed solvent system and its results were summarized in Table 6. Comparing the delignification efficiency of H2O-Dimethyl Sulfoxide (DMSO) (sample 7, 63.27%), H2O-N-methylpyrrolidone (NMP) (sample 8, 75.14%) and H2O-N, N-dimethylformamide (DMF) (sample 9, 84.78%) solvent systems to H2O-methanol (sample 1, 57.13%), one could conclude that water-aprotic solvent system was better than water-protic solvent system for delignification. The delignification efficiency of H2O-methanol could be further improved to 79.99% with aniline additive as the catalyst (sample 6). Similarly, aniline additive as a catalyst in the H2O-DMF solvent system resulting in the efficiency improvement of delignification from 84.78 (sample 9) to 95.04% (sample 10).
No. | Solvent systema | Additives | Lignin residue (wt%) | Delignificationb (%) |
---|---|---|---|---|
1 | H2O-methanol | — | 7.52 | 57.13 |
2 | H2O-methanol | Formic acid | 5.09 | 70.98 |
3 | H2O-methanol | Terephthalic acid | 5.10 | 73.93 |
4 | H2O-methanol | Salicylic acid | 2.89 | 77.33 |
5 | H2O-methanol | Sodium hydroxide | 3.92 | 69.25 |
6 | H2O-methanol | Aniline | 3.51 | 79.99 |
7 | H2O-DMSO | — | 6.45 | 63.27 |
8 | H2O-NMP | — | 4.36 | 75.14 |
9 | H2O-DMF | — | 2.67 | 84.78 |
10 | H2O-DMF | Aniline | 0.87 | 95.04 |
Influence of the composition of mixed solvent on the delignification process [18].
Represents the residual cellulose-enriched fractions obtained with water, organic solvent and additives in ratios of 20:10:1 (ν: ν: ν).
The acid-insoluble lignin content in the steam-exploded rice straw is measured to be 12.75%.
Conclusively, the H2O-DMF-aniline solvent system (in a volume ratio of 20:10:1) demonstrated to be the most efficient solvent for removing lignin from the steam-exploded rice straw because the DMF and aniline included amino groups might improve the dissolution of lignin.
The FTIR spectrum of the original rice straw, the steam-exploded rice straw, the delignified sample with H2O-DMF-aniline and bleached cellulose shown Figure 6a indicated that the lignin in straw could be removed and obtained pure cellulose because the absorptions at 1511 and 1429 cm−1 assigned to the aromatic C=C stretch from aromatic ring in lignin became weak after the steam explosion (spectrum SERS) and delignification (spectrum DERS), and finally disappeared in the bleached cellulose (spectrum BC) [18].
(a) FTIR spectra and (b) 13C-NMR spectra of different samples (0, original rice straw); steam-exploded rice straw (1, SERS); H2O-DMF-aniline delignified rice straw (2, DERS); bleached cellulose (3, BC) [18].
Figure 6b indicated the 13C CP/MAS solid-state NMR spectra of the steam-exploded rice straw (SERS), delignified rice straw (DERS) and bleached cellulose (BC).All of these spectra were dominated by the resonances attributable to cellulose. Notably, two small peaks at 99.5 and 58.2 ppm in the spectrum of steam-exploded rice straw (pattern SERS) were assigned to lignin and they nearly disappear in spectrum DERS, BC, which were the delignified cellulose with the water-DMF-aniline solvent system and bleached cellulose, respectively.
Biological treatments employ microorganisms and their enzyme systems to break down the lignin present in lignocellulosic biomass. This approach has recently attracted increased attention because of its mild condition, low energy consumption, and the absence of pollution [33, 34]. Among the microorganisms those are capable of degrading lignocelluloses, white rot fungi has a higher selectivity toward lignin with lower energy input, as well as being environmentally friendly [35].
One white rot fungus, Phanerochaete chrysosporium (P. chrysosporium), harbors at least 10 lignin peroxidases (LiP), five manganese peroxidases (MnP), and several copper oxidases in its lignin-degrading system [36, 37]. Combinatorial treatment of steam explosion and biological treatment has been considered as an effective method of separating components for various kinds of biomass. Chen et al. [38] reported the effects of the solid-state fermentation (SSF) conditions on biodegradation of steam-exploded wheat straw with P. chrysosporium. Under the optimum conditions of SSF, the degradation amount of lignin reached 60% on the 5th day. Zhang et al. [39] indicated that steam explosion is an important pretreatment method for biodegradation of lignin in rice straw. After steaming under 2.5 MPa for 25 min, then completely decompressed within 3 min, the steam-exploded straw was collected and dried for biodegradation treatment. In their study, the degradation rate of lignin was 31.23% without steam explosion, and 55.40% lignin loss rate had been found on day 30 after steam explosion pretreatment. A two-stage process proposed by Zhou et al. [40] was based on the pretreatment of steam explosion and followed by a P. chrysosporium post-treatment for the isolation of cellulose.
As the above processes, the orthogonal experiments (using L16 (4)5 orthogonal table) were designed (Table 8) to investigate the relationship between the delignification in SERS and the five factors. Each factor had been set based on the pretest results and a literature review (Table 7). Curves of the factors vs. the delignification was shown in Figure 7.
No. | Factor | Level 1 | Level 2 | Level 3 | Level 4 |
---|---|---|---|---|---|
A | Spore suspensions concentration (%) | 0.5 | 1.0 | 1.5 | 2.0 |
B | T-80 concentration (%) | 0.1 | 0.2 | 0.3 | 0.4 |
C | Moisture content (%) | 65 | 70 | 75 | 80 |
D | Initial pH | 3 | 4 | 5 | 6 |
E | SSF time (day (d)) | 7 | 14 | 21 | 28 |
Factors levels for the orthogonal experiments [40].
Curves of the factors vs. delignification [40].
According to Table 8, the maximum lignin loss rate, 64.25%, was obtained with the conditions of 1% spore suspension, 70% moisture content, 0.1% T-80, initial pH of 5.0, and 28 days of fermentation. The experimental outcomes could not be used as the best SSF conditions, so the orthogonal analysis should be performed. Table 8 shows the results of orthogonal analysis for five factors used in the fermentation process and the order of importance of these factors on lignin removal was E > C > B > D > A. Based on the k value of the testing factors, the optimum process for fermentation is A3B3C2D3E4, corresponding to 1.5% spore suspension, 0.3% T-80, 70% moisture, initial pH of 5.0, and 28 days of SSF.
Test no. | Factors | Delignification (%) | ||||
---|---|---|---|---|---|---|
A | B | C | D | E | ||
1 | 0.5 | 0.1 | 65 | 3 | 7 | 16.63 |
2 | 0.5 | 0.2 | 70 | 4 | 14 | 49.68 |
3 | 0.5 | 0.3 | 75 | 5 | 21 | 56.37 |
4 | 0.5 | 0.4 | 80 | 6 | 28 | 37.92 |
5 | 1.0 | 0.1 | 70 | 5 | 28 | 64.25 |
6 | 1.0 | 0.2 | 65 | 6 | 21 | 52.78 |
7 | 1.0 | 0.3 | 80 | 3 | 14 | 28.75 |
8 | 1.0 | 0.4 | 75 | 4 | 7 | 17.02 |
9 | 1.5 | 0.1 | 75 | 6 | 14 | 48.43 |
10 | 1.5 | 0.2 | 80 | 5 | 7 | 8.65 |
11 | 1.5 | 0.3 | 65 | 4 | 28 | 62.65 |
12 | 1.5 | 0.4 | 70 | 3 | 21 | 53.46 |
13 | 2.0 | 0.1 | 80 | 4 | 21 | 39.15 |
14 | 2.0 | 0.2 | 75 | 3 | 28 | 57.97 |
15 | 2.0 | 0.3 | 70 | 6 | 7 | 24.06 |
16 | 2.0 | 0.4 | 65 | 5 | 14 | 43.06 |
K1 | 160.60 | 168.46 | 175.81 | 156.81 | 66.36 | |
K2 | 162.80 | 169.08 | 191.45 | 168.50 | 170.61 | |
K3 | 173.19 | 171.83 | 179.79 | 173.02 | 201.76 | |
K4 | 164.93 | 152.15 | 114.47 | 163.19 | 222.79 | |
k1a | 40.15 | 42.12 | 43.95 | 39.20 | 16.59 | |
k2a | 40.70 | 42.27 | 47.86 | 42.12 | 42.65 | |
k3a | 43.30 | 42.96 | 44.95 | 43.25 | 50.44 | |
k4a | 41.23 | 38.04 | 28.62 | 40.80 | 55.70 | |
Rb | 3.15 | 4.92 | 19.24 | 4.05 | 39.11 |
Experimental setup together with results of P. chrysosporium delignification [40].
k1, k2, k3, and k4 are the mean values of the sum of the evaluation indexes of all levels. By comparing k values, the optimal levels of the factors can be confirmed.
The range of factors (R = Max(kj)− Min(kj)) indicates the function of the corresponding factor. The larger value of R means the greater impact of the level of the factor on the experimental index.
According to the analysis presented above, the most important factor for delignification was SSF time. However, there was no top value of SSF time vs. delignification unlike the other four factors as shown in Figure 7. Hence, the effect of SSF time required further study.
Moisture was found to be significant to the delignification by P. chrysosporium. Water in SSF systems shows functions of transporting the nutrients and metabolites, which can contribute to the stability of the cellular and molecular structures. As displayed in Figure 7, until the moisture levels (70 and 75%), the delignification increased with increase of moisture. However, the delignification decreased after moisture exceeded 75% because the high moisture hampered the diffusion of oxygen into the liquid and solid phases thus limited aerobic SSF.
Figure 8 shows the effect of SSF time on the removal of lignin. The experimental results satisfactorily fitted the Boltzmann model with the decisive coefficient R2 = 0.9983. The lignin content of the steam-exploded rice straw was efficiently degraded after a prolonged period. Nearly half of the highest lignin removal rate was obtained on the 7th day of fermentation. When the treatment time of using P. chrysosporium was 10 days, the lignin removal increased to 50.13%. Thereafter, the lignin removal rate decreased and tends to stabilize after 12 days.
The effect of SSF time on the removal of lignin [40].
Although the lignin degradation of steam-exploded rice straw is very important, too much weight loss is unexpected. The relationship between the SSF time and weight loss is shown in Figure 9, which turned out to be a linear relationship with decisive coefficient R2 = 0.99843. The weight loss of SERS is proportional to the SSF time, differing from the results shown in Figure 8. Weight loss of SERS might be attributed to the removal of lignin and hemicellulose over 10 days of fermentation, after which the degradation of cellulose gradually became a major factor of SSF.
The effect of SSF time on the weight loss of SERS [40].
In comparison with the FTIR spectrum of untreated SERS (Figure 10a), prominent changes could be obtained in the samples degraded after different SSF timings. The increase in the intensity of peak at 1650 cm−1 showed higher abundance of C=O groups of lignin, demonstrating the aromatic lignin moieties altered by oxidation with lignin biodegradation. In addition, peaks at 1510 and 1431 cm−1 turned weakening with increasing treatment time, implying the aromatic skeletal carbon of lignin was destroyed by P. chrysosporium. The peaks at 896, 1060, and 1160 cm−1 disappeared with increasing SSF time, showing that the structure of cellulose has been degraded after longer treatment time. Peaks observed near 23.11° (Figure 10b) represent diffractions of the (002) crystal plane, indicating that the crystal type of cellulose in SERS is cellulose type-I in nature (as shown in Figure 10b). Fermentation appeared not to change the crystal type of cellulose. With increasing SSF time, the peak height of the (002) crystal plane decreased and the peak width at half height increased compared with untreated SERS. The morphologies of the samples before and after bio treatment had been examined by SEM (Figure 10c) to visually demonstrate the process of delignification and cellulose degradation by P. chrysosporium.
(a) FTIR spectra, (b) XRD patterns, and (c) morphologies of SERS before and after SSF with P. chrysosporium.
All-cellulose composites (AACs) have been proposed to meet the interfacial problem in the cellulose-based composites, where both the reinforcement phase and matrix are cellulose [41, 42, 43]. Zhou et al. [44] reported that ACCs, with microcrystalline cellulose (MCC) as the matrix and the straw cellulose fibers (SCFs) as the reinforcement agent exhibited an ultra-high tensile strength (650.2 MPa, Figure 11).
(a) Typical engineering stress–strain curves of the samples, (b) the corresponding tensile properties, where, a: Regenerated MCC, b: ACCs/activated SCF (A-SCF), c: ACCs/alkali-treated (N-SCF), and d: ACCs/activated SCF [44].
The mechanism for the high performance of alkali-treated SCF (N-SCF) and activated SCF (A-SCF) reinforced ACCs are shown in Figure 12. For the ACCs/N-SCF sample (Figure 12a), the reinforcement mechanism is possibly related to the removal of impurities and the increase of aspect ratio. For the ACCs/A-SCF sample (Figure 12b), the reinforcement mechanism can be possibly attributed to the fact that both A-SCF and MCC experience the same activation process. Due to successively pre-swell with solvents (water, ethanol, N, N-dimethylacetamide) gradually reducing polarity, the A-SCF and MCC molecular chains in SCF and MCC could be partially dissolved and penetrated into each other, resulting in improving entanglement density of molecular chains on the fiber surface.
Schematic representations showing the reinforcement mechanism of the N-SCF (a) and the A-SCF (b) in the ACCs [44].
Besides, the isolated cellulosic fibers of about 2–16 wt% can be introduced into the cement with a slurry vacuum de-watering technique [45]. It was found that the flexural strength and fracture toughness of the optimal sample were increased by 24.3% and 45 times, respectively.
Effective solvent system for cellulose dissolution is a long-standing goal due to the abundant hydroxyl groups on the cellulose chain form a strong, three-dimensional intermolecular and intramolecular hydrogen bonding network [46, 47]. Therefore, Zhou et al. [48] developed a new aqueous solvent for cellulose based on the quaternary ammonium hydroxide (TBAH). It was found that cellulose can be efficiently dissolved in a 40 wt% TBAH aq. solution under a cooling condition. The mechanism for the dissolution is believed to be the match of amphiphilicity between the solvent and cellulose crystal. Then, Zhou et al. [49] studied the effects of urea to the dissolution of cellulose in TBAH. It was found that a hybrid hydrate of TBAH and urea formed. Urea can serve as a hydrophobic contributor, by which the amphiphilic property of the solvent system can be adjusted. With a suitable amphiphilicity, interfacial resistance between the solvent and crystal surface can be reduced so that the crystal of natural cellulose can be effectively infiltrated and subsequently dissolved by the solvent. The schematic dissolution process of the cellulose is shown in Figure 13.
Schematic illustration for the mechanism of TBAH/urea aqueous solution dissolving cellulose. These two diagrams display various interfacial resistances between the crystal surface of natural cellulose and the solvent [49].
The steam explosion process is realized as a promising pre-treatment for the separation of cellulose from natural biomasses. It can make most of the hemicellulose hydrolyze and part of the lignin degrade, which results in loose and porous structures, which could ultimately support further separation of cellulose and lignin. One kind of ionic liquids, 1-allyl-3-methylimidazolium chloride (AMIMCl), is selected for the extraction of cellulose, due to its especially selective solubility for cellulose rather than lignin. This process with ionic liquids shows advantages of efficiency, environmentally friendly, and recyclability character. Isolation of cellulose with the organic solvent system that composes of H2O-DMF-aniline has proved that it also been considered as an environmental-friendly approach, and up to 95.04% lignin can be dissolved out from the steam-exploded rice straw, leaving quite a small amount of hemicellulose (<1%) and lignin (<0.85%). There is no obvious decrease in the degree of cellulose crystallinity during the delignification and bleaching processes. Approximately 58% lignin can be removed under selective delignification of the steam-exploded rice straw by P. chrysosporium. The isolated cellulose fiber from the rice straw can serve as a reinforcement material for the advanced mechanical property of composites. The prepared ACCs exhibit an ultra-high tensile strength. The cellulose/cement composites show a remarkable improvement in the flexural strength and fracture toughness. Cellulose can be efficiently dissolved in a 40 wt% TBAH aq. solution under a cooling process.
The authors acknowledge the financial support of the National Natural Science Foundation of China (No. 51303151), the National Key Technology R&D Program of the Ministry of Science and Technology of China (No. 2011BAE11B01), the Science and Technology Planning Project of Sichuan Province (No. 2016GZ0224,2016CZYZF0003), Sichuan Province Youth Science and Technology Innovation Team (No. 2016TD0026), Sichuan Province Science and Technology Innovation Talent Project (No. 2017072), and the Fundamental Research Funds for the Central Universities (No. 2682016CX069).
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',metaTitle:"Terms and Conditions",metaDescription:"These terms and conditions outline the rules and regulations for the use of IntechOpen Website at https://intechopen.com and all its subdomains owned by Intech Limited located at 7th floor, 10 Lower Thames Street, London, EC3R 6AF, UK.",metaKeywords:null,canonicalURL:"/page/terms-and-conditions",contentRaw:'[{"type":"htmlEditorComponent","content":"By accessing the website at www.intechopen.com you are agreeing to be bound by these Terms of Service, all applicable laws and regulations, and agree that you are responsible for compliance with any applicable local laws. Use and/or access to this site is based on full agreement and compliance of these Terms. All materials contained on this website are protected by applicable copyright and trademark laws.
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\n\nThe following terminology applies to these Terms and Conditions, Privacy Statement, Disclaimer Notice, and any or all Agreements:
\n\n“Client”, “Customer”, “You” and “Your” refers to you, the person accessing this website and accepting the Company’s Terms and Conditions;
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I am also a member of the team in charge for the supervision of Ph.D. students in the fields of development of silicon based planar waveguide sensor devices, study of inelastic electron tunnelling in planar tunnelling nanostructures for sensing applications and development of organotellurium(IV) compounds for semiconductor applications. I am a specialist in data analysis techniques and nanosurface structure. I have served as the editor for many books, been a member of the editorial board in science journals, have published many papers and hold many patents.",institutionString:null,institution:{name:"Sheffield Hallam University",country:{name:"United Kingdom"}}},{id:"54525",title:"Prof.",name:"Abdul Latif",middleName:null,surname:"Ahmad",slug:"abdul-latif-ahmad",fullName:"Abdul Latif Ahmad",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"20567",title:"Prof.",name:"Ado",middleName:null,surname:"Jorio",slug:"ado-jorio",fullName:"Ado Jorio",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Universidade Federal de Minas Gerais",country:{name:"Brazil"}}},{id:"47940",title:"Dr.",name:"Alberto",middleName:null,surname:"Mantovani",slug:"alberto-mantovani",fullName:"Alberto Mantovani",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"12392",title:"Mr.",name:"Alex",middleName:null,surname:"Lazinica",slug:"alex-lazinica",fullName:"Alex Lazinica",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/12392/images/7282_n.png",biography:"Alex Lazinica is the founder and CEO of IntechOpen. After obtaining a Master's degree in Mechanical Engineering, he continued his PhD studies in Robotics at the Vienna University of Technology. Here he worked as a robotic researcher with the university's Intelligent Manufacturing Systems Group as well as a guest researcher at various European universities, including the Swiss Federal Institute of Technology Lausanne (EPFL). During this time he published more than 20 scientific papers, gave presentations, served as a reviewer for major robotic journals and conferences and most importantly he co-founded and built the International Journal of Advanced Robotic Systems- world's first Open Access journal in the field of robotics. Starting this journal was a pivotal point in his career, since it was a pathway to founding IntechOpen - Open Access publisher focused on addressing academic researchers needs. Alex is a personification of IntechOpen key values being trusted, open and entrepreneurial. Today his focus is on defining the growth and development strategy for the company.",institutionString:null,institution:{name:"TU Wien",country:{name:"Austria"}}},{id:"19816",title:"Prof.",name:"Alexander",middleName:null,surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/19816/images/1607_n.jpg",biography:"Alexander I. Kokorin: born: 1947, Moscow; DSc., PhD; Principal Research Fellow (Research Professor) of Department of Kinetics and Catalysis, N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow.\r\nArea of research interests: physical chemistry of complex-organized molecular and nanosized systems, including polymer-metal complexes; the surface of doped oxide semiconductors. He is an expert in structural, absorptive, catalytic and photocatalytic properties, in structural organization and dynamic features of ionic liquids, in magnetic interactions between paramagnetic centers. The author or co-author of 3 books, over 200 articles and reviews in scientific journals and books. He is an actual member of the International EPR/ESR Society, European Society on Quantum Solar Energy Conversion, Moscow House of Scientists, of the Board of Moscow Physical Society.",institutionString:null,institution:{name:"Semenov Institute of Chemical Physics",country:{name:"Russia"}}},{id:"62389",title:"PhD.",name:"Ali Demir",middleName:null,surname:"Sezer",slug:"ali-demir-sezer",fullName:"Ali Demir Sezer",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/62389/images/3413_n.jpg",biography:"Dr. Ali Demir Sezer has a Ph.D. from Pharmaceutical Biotechnology at the Faculty of Pharmacy, University of Marmara (Turkey). He is the member of many Pharmaceutical Associations and acts as a reviewer of scientific journals and European projects under different research areas such as: drug delivery systems, nanotechnology and pharmaceutical biotechnology. Dr. Sezer is the author of many scientific publications in peer-reviewed journals and poster communications. Focus of his research activity is drug delivery, physico-chemical characterization and biological evaluation of biopolymers micro and nanoparticles as modified drug delivery system, and colloidal drug carriers (liposomes, nanoparticles etc.).",institutionString:null,institution:{name:"Marmara University",country:{name:"Turkey"}}},{id:"61051",title:"Prof.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"100762",title:"Prof.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"St David's Medical Center",country:{name:"United States of America"}}},{id:"107416",title:"Dr.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Texas Cardiac Arrhythmia",country:{name:"United States of America"}}},{id:"64434",title:"Dr.",name:"Angkoon",middleName:null,surname:"Phinyomark",slug:"angkoon-phinyomark",fullName:"Angkoon Phinyomark",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/64434/images/2619_n.jpg",biography:"My name is Angkoon Phinyomark. I received a B.Eng. degree in Computer Engineering with First Class Honors in 2008 from Prince of Songkla University, Songkhla, Thailand, where I received a Ph.D. degree in Electrical Engineering. My research interests are primarily in the area of biomedical signal processing and classification notably EMG (electromyography signal), EOG (electrooculography signal), and EEG (electroencephalography signal), image analysis notably breast cancer analysis and optical coherence tomography, and rehabilitation engineering. I became a student member of IEEE in 2008. During October 2011-March 2012, I had worked at School of Computer Science and Electronic Engineering, University of Essex, Colchester, Essex, United Kingdom. In addition, during a B.Eng. I had been a visiting research student at Faculty of Computer Science, University of Murcia, Murcia, Spain for three months.\n\nI have published over 40 papers during 5 years in refereed journals, books, and conference proceedings in the areas of electro-physiological signals processing and classification, notably EMG and EOG signals, fractal analysis, wavelet analysis, texture analysis, feature extraction and machine learning algorithms, and assistive and rehabilitative devices. I have several computer programming language certificates, i.e. Sun Certified Programmer for the Java 2 Platform 1.4 (SCJP), Microsoft Certified Professional Developer, Web Developer (MCPD), Microsoft Certified Technology Specialist, .NET Framework 2.0 Web (MCTS). 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