**3.2 Cloud computing storage**

Cloud storage provides companies with greener computing services. Cloud computing storage cuts companies' and organizations' energy consumption and computing expenses. By using cloud storage, purchasing additional storage capacity is minimized and storage maintenance tasks and expenses are reduced. Cloud storage is a service package in which data is stored, managed, backed up remotely, and made available to users over a network and internet services. Cloud storage is based on a virtualized infrastructure with accessible interfaces. Cloud-based data is stored in servers located in data centers managed by a cloud provider. A file and its associated metadata are stored in the server by using an object storage protocol. The server assigns an identification number, ID, to each stored file. When the file needs to be retrieved, the user presents the ID to the system, and the content is assembled with all its metadata, authentication, and security. The most common use of cloud services is cloud backup, disaster recovery, and archiving of infrequently accessed data. Cloud storage providers are responsible for keeping the data available and accessible and the physical environment protected and running. People and organizations buy or lease storage capacity from the providers to store and archive data files. Cloud storage services may be accessed via cloud computers and web services that use application programming interfaces, API, such as cloud desktop storage and cloud storage gateways. There are three main cloud-based storage architecture models public, private, and hybrid.


**Acknowledgements**

We thank hematologist Chiseko Okada for the assessment of AML.This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas "Fluctuation & Structure," Project Number: 2502, from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Grant Support: Ministry of Health, Labor, and Welfare of Japan.


**3. Starter bacteria**

In cheese processing lactic acid bacteria are used as starter cultures, contributing to organoleptic characteristics and preservation of the product. These strains are added in a different form, grown in milk or mediums. Also, in France or Italy the whey incubated the day before use is applied as starter culture. Starters comes from the fact that these bacteria start the lactic acid production. These belong to

mesophilic and thermophilic bacteria [2, 10]. Mesophilic bacteria include lactococci (*Lactococcus lactis* subsp. *lactis* and *Lactococcus lactis* subsp. *cremoris*,) and often leuconostocs (*Leuconostoc mesenteroides* subsp. *cremoris* and *Leuconostoc lactis*). Thermophilic cultures include *Streptococcus thermophilus* and different species of *Lactobacillus* as *Lb. delbrueckii* and *Lb. helveticus* [10].

Cheddar cheese, soft cheeses, and most Dutch cheeses are produced with mesophilic starters. Starter culture have to meet different expectations as lactic acid production, reduction of pH, to be resistant to bacteriophages, inhibition of pathogenic and spoilage bacteria, contribute to the flavour, texture formation and have health benefit. Depending on the type of cheese the starter culture contributes to the development of nature of the cheese due to lipolysis, proteolysis and autolysis of the cells [10, 11].

Beside the lactic acid bacteria *Debaryomyces hansenii* and *Yarrowia lipolytica* yeasts are promising cheese starter cultures affecting the cheese maturation [12]. An example of molds as secondary starter culture are *Penicillium roqueforti* and *Penicillium camemberti* used in the production of blue- veined cheese. They tolerate the high and medium salinity conditions and possess high proteolytic activity [13]. There are traditional processed cheeses without starter culture, for example Spanish varieties. In these case at the beginning the high proportion of microbiota came from the milks' microbiota as lactococci.

### **4. The characteristic cheese microbiota**

Dairy products are perishable foods providing a good medium for microbial growth. Foodborne pathogens as *Staphylococcus aureus* in raw milk can be originated from infected lactating animals, undesirable milking conditions that lead to milk contamination with animal faeces and other materials. Incorrectly handled equipment, cleaning, sanitizing equipment, water or air bleeds can provoke a multitude of contamination with various microorganisms [14].

Superior varieties of cheeses as Emmental, Gruyère, Comté, Parmigiano Reggiano, Reblochon and Roquefort are made from raw milk. The microbial safety of these products is ensured by the heat treatment at a higher temperature > 50°C, with duration of 1 h. The challenge of this type of cheeses represents the pathogen bacteria from raw milk that can grow during the process [15].

The different types of cheeses are characterized by diverse microbiota. The raw milk cheeses (soft and hard, short or long maturated, starter added or nonstarter added cheeses, and ripened in brine or unbrined cheeses) own their distinct microflora.

Gram-negative bacteria and salt-tolerant bacteria belonging to different genus, including *Halomonas, Vibrio*, and *Hafnia* spp., and salinity tolerant bacteria, including *Arthobacter, Micrococcus, Brevibacterium* and *Brachybacterium* spp., have been characteristic bacteria of raw milk cheese surface microbiota [16].

The environmental conditions of the dairy farm have an impact on the microflora of the raw milk and therefore influence manufacturing. The main spore formers in milk are species of *Clostridium*, *Bacillus* and *Geobacillus* genus. These bacteria are the follows: *C. sporogenes*, *C. butyricum*, *C. tyrobutyricum*, *B. cereus*, *B. sporothermodurans*, *G. stearothermophilus.* For the occurrence of defects in cheese as off-flavors, late blowing is responsible *C. tyrobutyricum.*

Bacteria species of *Enterococcus* are widespread in dairy products affecting negatively or positively the flavor development as the result of hydrolytic processes, proteolysis and peptidolysis. Their origin is related to raw material or to starter culture composition. In southern European artisanal cheeses, *Enterococci*'s number

*Microbial Diversity of Traditionally Processed Cheese from Northeastern Region of Transylvania… DOI: http://dx.doi.org/10.5772/intechopen.97591*

is higher than 107 CFU g−1. This bacteria is an opportunistic nosocomial pathogen. Also, *Enterococci* could play a role in antibiotic resistance transfer and hospitalacquired infections. Consequently, the occurrence of this in dairy products is suspicious [2, 17]. In Gouda cheese the *Ec. malodoratus*, as its name suggest lead to the development of bad flavours [15]. *Streptococcus* species also can be isolated from milk [17].

Fox et al. [15] reported that in cheese the non-lactic acid bacteria are bacteria belonging to genera *Agrococcus*, *Arthrobacter, Brevibacterium, Brachybacterium, Corynebacterium, Microbacterium, Propionibacterium, Pediococcus, Micrococcus, Kocuria, Kytococcus, Staphylococcus.*

Aged cheese microbiota includes non-starter lactic acid bacteria. The predominantly found bacteria are facultatively heterofermentative as *Lactobacillus casei* and *L. paracasei*. It occurs occasionally *Pediococcus* spp. and obligately heterofermentative *Lactobacillus* spp., such as, *L. brevis* and *L. fermentum*.

Non-starter lactic acid bacteria origin is the raw material and/or the processing environment. A little part of the lactobacilli are resistant to the thermal treatment, pasteurization, for example in Emmental cheese obtained from raw milk can survive the 52°C temperature.

Majority of non-starter lactic acid bacteria can be characterized as: facultative anaerobes, salt-and acid-tolerant and able to grow in cheese. The involvement of these bacteria in the production of cheese flavour is unclear [2].

The number of *Micrococcus* and *Staphylococcus* is higher than 106 CFU g−1 in different types of cheese. It was found on the surface of soft smear-ripened, semi hard and hard cheeses. *Staphylococcus saprophyticus*, *S. equorum*, *S. vitulus*, and *S. xylosus* were described as main staphylococci occurred in cheese. *Kocuria*, *Nesterenkonia*, *Kytococcus*, *Dermacoccus*, and *Micrococcus* were found in cheese. *Kocuria rosea*, *K. varians*, *Dermacoccus sedantarius*, *Micrococcus lylae*, and *M. luteus isolated from* the surface of cheese. The function of *Micrococcus* and *Staphylococcus* in cheese is unclear, but many of them are related to the production of proteinases and lipases [2].

Coryneform bacteria, especially *Brevibacterium linens* is involved in the red or orange colour formation of the surface in smear-ripened cheeses. Therefore, intentionally is applied on the surface of cheeses after brining. It was proved that *Arthrobacter*, *Agrococcus*, *Brachybacterium*, *Corynebacterium*, and *Microbacterium* spp. have an important role. Origin of these corynebacteria is the brine and shelving. It is believed that staphylococci, coryneforms and micrococci, can be originated from skin due to manual handling, comprising the microflora of the skin [2].

Swiss-type cheese as Emmental and Comte´ contain propionic acid bacteria, *Propionibacterium freudenreichii*. Their pivotal role is the metabolization of lactate produced by the starter bacteria to propionate, acetate, and CO2 [2].

Cheese can be a reservoir and carrier of probiotic bacteria for human. Due to the low redox potential, this product is a protective environment for probiotics. Their resistance to changes in pH represents an advantage in the acidic environment of gastrointestinal track. Probiotic bacteria applied in cheese production include *Lactobacillus acidophilus*, *L. casei*, *L. delbrueckii* subsp. *bulgaricus, L. johnsonii*, *L. reuteri, L. rhamnosus, Bifidobacterum bifidum, B. animalis, B. brevis, B. longum, B. infantis* [7].

*Escherichia coli* and coliforms are hygienic indicator bacteria. There are limit values for cheeses as well as for raw-milk cheeses.

The level of indicator microorganisms is less than 10 CFU/g and not more than 100 CFU/g, these representing the limit values in many countries.

In those instances, where cheeses had high levels of indicator bacteria, most studies attributed these to the use of poor-quality raw milk which contained high levels of indicator flora or to unsanitary conditions, or both.

The occurrence of coliforms in dairy products reveal low grade raw milk, poor hygienic conditions under production process. Coliforms could be avoided by respecting personal hygiene and sanitation process.

Human pathogens including *Salmonella* spp., *Streptococcus* spp., *L. monocytogenes*, *Helicobacter pylori*, *Campylobacter* spp., *Escherichia coli*, *Coxiella burnetti*, *Mycobacterium* spp., *Brucella* spp., tick-borne encephalitis virus (TBEV), *Staphylococcus aureus*, *Arcanobacter pyogenes*, *Toxoplasma gondii*, *Bacillus cereus*, *Leptospira*, *Clostridium* spp*.*, and *Yersinia enterocolitica* can be present in raw milk cheeses. The inadequate fermentation conditions contribute to the growth and development of these pathogens [16].

At the origin of unwanted yeasts and molds appearance in cheeses could stand environmental factors, improper processing conditions or infested starter culture. Several yeast could affect negatively (sometimes positively) the cheese. These microbes can be involved in taste and flavour development, affecting the quality attributes also, causing spoilage and release unpleasant aroma compounds. Clinically relevant yeast was detected in different type of cheese as *Candida albicans*, *C. glabrata, C. krusei* and *C. tropicalis* [3].

The advanced hygiene and quality control contributed to the decrease of gas production by yeasts during cheese manufacturing [2].

A diverse group of yeasts represent a high proportion of surface microflora of different type of smear- and mold-ripened cheeses, including Comte´, Tilsit, Limburger, Blue, and Camembert. These microorganisms level can reach 106 –107 CFU/g and are characterized by tolerance to low pH and high salt concentrations.

The most prevalent yeast species isolated from cheese were *Galactomyces candidus, C. parapsilosis, C. sake, C. batistae, Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces lactis, K. marxianus, Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia kudriavzevii* [2, 3, 7].

The most common molds in cheese include *Penicillium roqueforti* in Blue cheeses (e.g., Stilton, Roquefort, and Gorganzola) and *Penicillium camemberti* in surface mold-ripened cheeses (e.g., Camembert and Brie) [2, 3, 7]. Molds isolated from various retail cheese were: *Penicillium verrucosum*, *P. paneum*, *P. brevicompactum*, *P. gladioli*, *Aspergillus niger*, *A. flavus*, *A. phoenicis*, *Cladosporium sphaerospermum*, *C. cladosporioides*, *Eurotium rubrum* [3]. The presence of *P. commune*, which is closely related to *P. camemberti*, can cause discoloration of cheese surfaces and the development of off-flavours [15].

### **5. Microbiota and characteristics of traditionally processed cheeses**

Artisanal cheeses represent a particular category of cheeses that is appreciated worldwide. Particular properties of products as the individual sensory aspects are related to local environment, cultural and historical features [8].

One of the challenges of artisanal cheese producers is to keep the microbial diversity of the product and exploit their beneficial effects. The traditional process, including the wooden equipment, microorganisms are the main factor in food safety issues [18].

In raw milk, over 400 species of lactic acid bacteria, Gram-positive and negative, catalase-positive bacteria, yeast and mould have been found. In cheese cores the diversity is not very large, typical lactic acid bacteria are the predominant microorganisms, whereas the surface of cheeses is rich in several species of bacteria, moulds and yeast.

*Microbial Diversity of Traditionally Processed Cheese from Northeastern Region of Transylvania… DOI: http://dx.doi.org/10.5772/intechopen.97591*

Diversity within the cheeses results from considerable variation in distribution and growth of the same bacteria in the various cheese. Raw milk cheeses compared to the others are characterized by an intense and rich flavour. This results from the native microflora of the raw material and is not a character of cheeses made from treated milk [18].

The artisanal cheeses possess a heterogeneous microbiota characterized by a typical population dynamic. The unique microflora is associated with geographical indication for that region [19].

Perhaps natural milk cultures are used for italian traditional cheeses including Asiago, Pecorino Siciliano, Canestrato Pugliese, Castelmagno and Fossa. The origin of these cultures is the raw milk. The processing comprises a heat treatment of the raw milk for 15–30 min on 60–65°C, fast cooling and back slopping technique. Thermophilic species of that type of cultures are *Streptococcus thermophilus, Streptococcus macedonicus*, *Enterococcus*, and *Lactobacillus* spp. The whey based cultures contain aciduric and thermophilic strains. Among the cultivable bacteria *Lactobacillus helveticus, Lactobacillus delbrueckii* subsp. *lactis*, *Lactobacillus fermentum*, and *Str. thermophilus* were identified [20].

Central and Southern Italian cheese is "Pecorino" with PDO status, processed traditionally. The raw milk and natural microflora contribute to the development of typical organoleptic attributes. The main representants are *Enterococcus faecium*, *E. faecalis*, *Lactobacillus plantarum*, *L. brevis*, *Lactococcus lactis* subsp. *cremoris* [19].

In traditional cheese production crucial role have the autochthonous starter cultures, the main steps of manufacturing are more affected by it, but the final product are less. This refers only to that cheeses that are processed in the same geographical area and with settled circumstances. Numerous parameters have effect on the specific characteristics of cheese produced in different locations. This is case of PDO Pecorino Siciliano that is manufactured in different places of Sicily. There are some common parameters that must be applied during production as raw ewe's milk, lamb rennet paste, wooden equipment and no added starter.

The inconstancy in the raw milk quality and the variable autochthonous microbiota determine the alteration of the attributes as organoleptic characteristics of artisanal cheese manufactured in different locations. With the application of lactic acid bacteria, the characteristics of PDO Pecorino Siciliano cheese production could be stabilized [21].

Brazilian traditional processed cheeses differ from the commercial product in their unique characteristics. These are produced with tradition, the essence of production is transferred from one generation to the next. These type of cheese are characterized by unique organoleptic properties, typical aroma, flavour, and colour due to the typical endogenous microbiota. This microbiota includes lactic acid bacteria, yeasts, fungi that source is the raw material and the production environment. Among the lactic acid bacteria isolated from artisanal cheese are mainly *Lactobacillus plantarum* and other strains as *Lactobacillus brevis*, *Lactobacillus paracasei*, *Lactococcus lactis*, *Lactobacillus rhamnosus*, *Pediococcus pentosaceus*, *Lactobacillus curvatus*, *Lactobacillus paraplantarum*, *Lactococcus garvieae*, and *Pediococcus acidilactici* [22].

The traditionally processed cheeses from raw milk are very popular and preferred by the local population, therefor is a great need for the microbiological safety of this product. For this reason, there are characterized lactic acid bacteria with multi effect traits as anti-listerial effect isolated from artisanal cheeses. *Enterococcus faecalis* (1–37, 2–49, 2–388 and 1–400, *Lactobacillus brevi*s 2–392, *Lactobacillus plantarum* 1–399 a4) have antibacterial effect against *L. monocytogenes* growth during the low temperature storage soft cheese and decrease the ripening time of semi-hard cheeses aged at ambient temperature [23].

Based on Margalho et al. [24] research from the 220 lactic acid bacteria originated from artisanal Brasil cheese twenty-two isolates had probiotic traits and role in biopreservation. These strains are characterized by resistance to gastric acids as low pH values, bile salts, and also possess good adhesion properties, mostly to enterocyte-like cells (Caco-2). One of the lactic acid bacteria, namely *Lactobacillus plantarum* (1QB77) was able to produce bacteriocin and exhibited inhibition effect against enterotoxigenic *S aureus* and *L. monocytogenes.* It was able to survive the processing conditions also.

Valuable source of microorganisms with specific characteristics are the artisanal cheese due to their high biodiversity influenced by the local ecosystem.

The Russian artisanal Buryatia cottage cheese is characterized by typical taste and flavour determined by the numerous lactic acid bacteria. From the 62 isolated and assayed lactic acid bacteria of Russian cheese belonged to different 6 genera, 14 species/subspecies. The full-length 16S rRNA sequencing revealed a high diversity of bacteria including: *Lc*. *lactis*, *S. thermophilus*, *Lactococcus raffinolactis*, *Acetobacter cibinongensis*, *Lactobacillus helveticus*, *Klebsiella pneumonia*, *Acinetobacter johnsonii* and *Klebsiella oxytoca* [25].

Northern Moroccan artisanal cheese is characteristic of rural regions. Due to the raw material, the goat's milk, this cheese possesses unique organoleptic properties. Galiou et al. [26] revealed that the microbiota of 28 Northern Moroccan artisanal cheeses included *Enterococcus* spp. (249 isolates), *Lactococcus lactis* (36), *Lactobacillus plantarum* (7) and *L. paracasei* (2). Besides, there were still present coliforms, yeasts, microscopic fungi and in some sample *Listeria monocytogenes.*

The Spanish Manchego is traditionally processed product, obtained with enzymatic coagulation. It is a maturated, uncooked and pressed cheese, characterized by high fat content. The 248 strains of predominant lactic acid bacteria obtained from manufacture and ripening process of two dairies included species as *L. plantarum*, *L. brevis*, *L. paracasei* subsp*. paracasei, L. fermentum, L. pentosus, L. acidophilus* and *L. curvatus*. In samples from both dairies, the species *L. plantarum, L. brevis* and *L. paracasei* subsp*. paracasei* dominated during ripening [27].

Portuguese Serpa cheese is designated as Protected Designation of Origin (PDO) cheese that is characterized also, by particular appreciated aroma and flavour. The organoleptic attributes have resulted from the not pasteurized raw ovine milk components and the addition of extracts of *Cynara cardunculus* L.. Based on literature data, the most relevant bacteria species of Serpa cheese are *Lactobacillus* spp. (*L. paracasei, L. plantarum*, *L. brevis, L. pentosus, L. curvatus*), *Leuconostoc mesenteroides, Lactococcus* lactis, coliforms, *Enterococcus faecalis*, *E. faecium*, *E. hirae*, *Hafnia alvei*, *Galactomyces* spp., *Debaryomyces hansenii*, *Kluyveromyces lactis*, *Candida zeylanoides*, *Pichia fermentans*, *Cryptococcus oeirensis*, *Yarrowia lypolytica* [28].

Northen French PDO Maroilles cheese is a soft type cheese with washed rind. The duration of maturation are 4–6 weeks according to the size, while the rinds are washed weakly with salt water. It is characterized by a surface microbiota *Geotricum candidum*, *Kluyveryomyces lactis, Brevibacterium linens* and *B. aurantiacum.* The industrially and traditionally processed cheeses differ in microorganism's composition, total mesophilic flora, rate of lactic acid bacteria and *Enterobacteriaceae*. The occurrence of *Enterobacteriaceae* in traditionally processed Maroilles reveals unhygienic conditions during manufacturing process [1].

North-western Iranian Koopeh is obtained from raw sheep or cow milk in rural households, and the ripening is under the soil for four months. This cheese is semi-hard type, characterized by high fat content, by buttery and pungent flavour. It has beneficial effects as antioxidant and antimicrobial activities attributed to antioxidant peptides and aromatic herbs content (*Allium ampeloprasum, Cuminum*  *Microbial Diversity of Traditionally Processed Cheese from Northeastern Region of Transylvania… DOI: http://dx.doi.org/10.5772/intechopen.97591*

*cyminum, Nigella sativa*, *Thymes vulgaris*) [29]. The non-starter lactic acid bacteria are the dominant microorganisms of the ripening steps.

The artisanal Mexican cheese, named Chiapas cream cheese is semi-ripened type. Organoleptic characteristics include the creamy and acidic taste. The predominant acidifying bacteria are the endogenous lactic acid bacteria adapted to the local climatic, tropical conditions. The bacterial strains originated from this type of cheese are characterized by high antioxidant capacity and proteolytic activity. It was showed that these bacteria could to produce bioactive compounds with role in functional foods, resulted from hydrolysis of proteins and also contain angiotensinconverting enzyme [30].

The PDO Parmigiano Reggiano traditional cheese is obtained from raw cow's milk and added whey starters obtained with back sloping. It is a long ripened type of cheese, produced in defined geographical areas (Po river's valley). The biochemical characteristics of cheese matrix are the result of the adventitious bacterial dynamics.

In cheese post brining process, that can take a 24 month different lactic acid bacteria were detected applying cultural and non-cultural methods: *Lacticaseibacillus casei*, *Lacticaseibacillus rhamnosus, Limosilactobacillus fermentum, Pediococcus acidilactici, Levilactobacillus brevis, Lacticaseibacillus paracasei, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. lactis, Lentilactobacillus buchneri, Schleiferilactobacillus harbiniensis, Lentilactobacillus kefiri, Lactococcus lactis, Streptococcus suis, S. thermophilus* and *Lentilactobacillus parabuchneri* [31].

Merchán et al. [32] reported that the 149 yeast from Extremadura region traditional cheese, identified by ISSR-PCR technique included *D. hansenii*, *D. vindobonensis*, *K. marxianus*, *K. lactis*, *C. cabralensis*, *C. pararugosa*, *C. zeylanoides*, *Y. lipolytica*, *P. fermentans*, *P. cactophila*, *P. kudriazevii* and *P. jadinii. K. lactis* and *P. fermentans* were characterized by tolerance to the most restrictive stomach and bile salts stress conditions. *P. fermentans* was characterized by antimicrobial, antioxidant, hydrophobicity and auto-aggregation ability. *Kluyveromyces* spp. strains posed antimicrobial activity. The studied traditional cheese also contained strains with probiotic features as different strains of *P. fermentans*, *P. kudriavzevii,* K*. marxianus, D. hansenii, Y. lipolytica*.

The turkish artisanal cheese Tulum is a semi-hard cheese, ripened in goat skin and characterized by a unique flavour and microbial diversity. The texture is porous thus permeable to air and water. The microorganisms identified by 16 S rRNA sequencing from this cheese during ripening included: *Enterococcus faecium, E. hirae, E. durans, E. faecalis, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus helveticus, Lactobacillus brevis, Lactococcus lactis, Streptococcus gallolyticus, Weisella paramesenteroides, Eubacterium rangiferina*. During ripening the level of Lactococci and Streptococci decreased. At the end of the maturation the DGGE profile of the bacterial community formed from *Streptococcus gallolyticus*, *Lactobacillus delbrueckii* subsp. *bulgaricus*, *Streptococcus lutetiensis*, *Streptococcus* spp., and *Enterococcus hirae*. At the end of the maturation, the most prevalent species were *Enterococcus* spp. and *Streptococcus* spp. [33].

The MALDI-TOF MS method resulted that greek artisanal cheese non-starter lactic acid bacterial community consisted of *Lactobacillus brevis, Lactobacillus plantarum, Lactococcus lactis*, *Leuconostoc mesenteroides, Lactobacillus paracasei*, *Lactobacillus rhamnosus*, *Pediococcus pentosaceus* and *Enterococcus faecium.* The first two were the most predominant species. This type of cheese is obtained from raw sheep and goat milk without added starter cultures [34].

The PDO Robiola di Roccaverano soft Italian cheese is processed from raw goat milk with natural starters. From fresh and matured cheese samples the following microorganisms were identified *Lactococcus lactis* subsp*. cremoris, Lactococcus*

*lactis* subsp. *lactis, Leuconostoc mesenteroides, Lactobacillus plantarum, Leuconostoc pseudomesenteroides, Lactobacillus brevis, Leuconostoc citreum)* and microscopic fungi (*Geotrichum candidum, Kluyveromyces marxianus, Saturnispora silvae, Yarrowia lipolytica, Kluyveromyces lactis, Trichosporon coremiiforme* [35].

Kashkaval is an artisanal pasta filata cheese, obtained from sheep's raw milk without added starters, but now days appeared the industrialized form of this cheese using pasteurised milk with an added commercial starter. Microbiology studies of this raw milk cheese revealed that the dominant bacteria were the mesophilic non-starter lactobacilli and enterococci. In the pasteurized milk cheese *Lactococcus lactis* starter were dominant [36].

Nicastrese goat's cheese is a Southern Italian traditional product obtain from raw goat milk coagulated with lamb rennet, without any added starter cultures. The local aromatic Mediterranean plants impart unique sensorial characteristics to the milk. The typical flavour of this type of cheese is resulted from the macronutrient transformations during the maturation time. The autochthonous microbiota is responsible for the biochemistry of ripening leading to unique sensorial features. The following bacteria were detected from Nicastrese goat's cheese samples: *Lactococcus lactis* subsp*. lactis*, *Lactobacillus plantarum*, *Enterococcus faecalis*, *Lactococcus lactis* subsp. *cremoris*, *Lactobacillus casei*, *Leuconostoc mesenteroides*, *Lactobacillus brevis*, *Enterococcus faecium*, *Lactococcus garvieae*, *Enterococcus durans*, *Enterococcus hirae* and *Streptococcus thermophilus* [37].

The southern Mexican Poro de Tabasco cheese is an artisanal product obtained from raw whole cow's milk and with natural whey starters. The unique character is attributed to salting, pressing process and the use of a paraffin coating. It is a hard fresh type of cheese with full-fat content. Dominant bacteria strains of this type of cheese include *Lactobacillus* spp., *Lactococcus* spp., and *Streptococcus* spp.. The microbial level it was reduced during the processing. The final products were free of *E. coli*, *Salmonella* spp., and *L. monocytogenes*. Meanwhile, *S. aureus* and its toxins were detected in few samples. This could be eliminated with heat treatment and good manufacturing practices [38].

11 artisanal Irish cheeses (soft, hard, and semi hard) microbiome analysis by high throughput DNA sequencing revealed the presence of *Lactococcus*, *Leuconostoc*, and *Lactobacillus. Corynebacterium*, *Facklamia*, *Flavobacterium*, and *Cronobacter* were found in rind cheeses. The lactococci proportion were higher in naturally ripened rinds compared to smear and washed rinds [39].

A traditional fried cottage cheese contained strains of *Galactomyces geotrichum* molds responsible for the production of polyunsaturated fatty acids. This microscopic fungi is part of natural microflora and might have a role in omega-3 lipid production in foods and cheese [40].

The predominant bacteria of Fresh ovine cheese were represented by *Lactococcus lactis* strains added as starter culture. The characteristic of this type of cheese is given by the saffron (*Crocus sativus* L.) applied as additive. The total and lactococci bacteria level were high after storage of the product. Due to saffron content the cheese possesses antibacterial activity toward coliforms and enterococci [41].

The Calabrian (Italy) artisanal cheese lactic acid bacteria possess antibacterial effect. This group of bacteria contributed to the growth reduction of *L. monocytogenes* in vitro conditions [42].

The component of the microbiota of cheeses is influenced by the manufacture conditions as the used cheese making vessels. Generally, in artisanal cheese production, the wood is a representing material, that is a natural reservoir for microorganisms. It was showed that in these circumstances the lactic acid bacteria level is high. *Microbial Diversity of Traditionally Processed Cheese from Northeastern Region of Transylvania… DOI: http://dx.doi.org/10.5772/intechopen.97591*

In the maturation process the shelves made from wood also are responsible for appearance of coryneform bacteria, moulds, and yeasts [17].

Gram-negative bacteria strains belonging to different families were detected on the surface of maturated soft cheese. These were: *Enterobacteriaceae, Moraxellaceae, Pseudoalteromonadaceae, Pseudomonadaceae, Sphingobacteriaceae* and *Vibrionaceae* spp. The Gram-negative bacteria were attributed to undesirable and defective texture and flavour. This defects resulted from extracellular proteolytic and lipolytic enzymes activities of bacteria. An example is the occurrence of *Pseudomonas* spp. that influence negatively the organoleptic characteristics of cheese due to release of ethyl esters and alcohols. It is supposed that the Gram-negative bacteria belonging to natural microbiome affecting positively the product technological and sensorial features. A strain of *Proteus* spp. closely related to *P. hauseri* and strains of *Psychrobacter* spp. have been contributed to flavour development of smear type cheeses. In French traditional cheeses were detected Gram-negative bacterial strains with double effect. There were bacteria strains characterised by no tolerance to acidic conditions in simulated gastro-intestinal stress, to human serum and showed no adherence to Caco-2 cell and were no virulence. In contrast to *Hafnia paralvei* 920, *Proteus* spp. (close to *P. hauseri*) UCMA 3780, *Providencia heimbachae* GR4, and *Morganella morganii* 3A2A that were toxic in *in vitro* conditions possessing virulence factors [43].

## **6. Occurrence of allochthonous microorganisms in traditionally processed cheese**

The detection of allochthonous bacteria and microscopic fungi in raw milk cheese raise food safety issues because those products can harbour diverse spoilage and pathogenic microbes. We evaluated the microbial quality of 13 traditionally processed cheese from northeastern region of Transylvania. The processed cheese are made from unpasteurized cow and sheep milk. It is considered that the milk quality is affected by grazed grass, the special feeding strategy in that region.

The detection of the various pathogen bacteria was carried out by cultivation methods on different selective media: for the detection and enumeration of *Escherichia coli* TBX Chromo Agar was used, ChromoBio®Coliform agar was applied for the detection of coliforms, for the detection of *Salmonella* spp. Brilliance ™ Salmonella Agar Base was used, for the detection of *Shigella* spp. Salmonella Shigella (SS) Modified Agar was applied, for the detection of *Campylobacter jejuni* Campylobacter Blood-Free Selective Agar Base Modified CCDA was used, for *Yersinia enterocolitica* detection the Yersinia Selective Agar Base was used, for *Listeria monocytogenes* detection Listeria mono Differential Agar Base was used, for *Bacillus cereus* detection ChromoBio®Cereus Base was used, for *C. perfringens* Clostridial Differential Broth was used, for the detection of *Pseudomonas aeruginosa* Pseudomonas Isolation Agar Base was applied. The detection of microscopic fungi was carried out by spread plate method on ChromoBio®Candida and Czapek Dox Agar [44, 45].

Isolation of the most representative bacteria with high count and characteristic colony morphology was followed by identification of strains on species-level by 16 rDNA sequencing method. Genomic DNA isolation of isolated bacterial strains from the selective mediums was realized with AccuPrep® Genomic DNA Extraction Kit from Bioneer, according to the manufacturer's protocol. A part of the bacterial 16S rDNA gene was amplified with universal primers 27f and 1492r (5' AGAGTTTGATCMTGGCTCAG 3′, 5' TACGGYTACCTTGTTACGACTT 3′).

The PCR products were purified using *GeneJET PCR Purification Kit* (*Thermo Scientific*, K070) and sequencing was carried out [46].

Our results of the microbiological quality of the assayed artisanal cheese showed diverse values. The highest total count of coliforms, *Escherichia coli* and *Salmonella* spp. was detected in the cheese 7 sample (**Table 1**). The mean number of *Campylobacter* jejuni was 103 CFU/g and 102 –103 CFU/g in the case of *Yersinia enterocolitica.* The highest level was detected in the case of cheese 10 sample 8·104 CFU/g *C. jejuni* and 4.1·104 CFU/g *Y. enterocolitica.* The highest number of *Listeria monocytogenes* was detected in cheese 7 sample 7.5·105 CFU/g, in case of *Staphylococcus aureus* the highest count was 1.9·107 CFU/g in sample cheese 6. The highest level of *Bacillus cereus* was 2·105 CFU/g in sample cheese 7 (**Table 1**). The *Clostridium perfringens* was detected in two samples cheese 3, cheese 8 samples.

The count of microscopic filamentous fungi in the assayed samples obtained by us varied 102 –103 CFU/g. The highest load was found in cheese 4 sample 5·104 CFU/g. The maximum value detected in the case of yeasts was 4.5 · 105 CFU/g in cheese 9 sample (**Table 2**). The developed filamentous fungi belonged to *Aspergillus* and *Penicillium* genera (**Figure 1**).

The identified 27 bacterial strains isolated on various selective media according to 16S rDNA sequence analysis include: *Stenotrophomonas maltophilia*, *Escherichia coli*, *Staphylococcus equorum* subsp*. equorum*, *Staphylococcus equorum* subsp*. linens*, *Klebsiella michiganensis*, *Halomonas alkaliphila*, *Proteus vulgaris*, *Shigella flexneri*, *Enterococcus durans, Enterococcus faecalis, Bacillus licheniformis, Kocuria rhizophila, Hafnia paralvei* (**Figure 2**)*.*

The occurrence of *Stenotrophomonas maltophilia* in cheese samples originated from different regions of France was reported by Coton et al. 2012 [47]*.* This multidrug-resistant bacteria causing nosocomial infections, can be found in different environments as aquatic or humid, in natural soil or plant rhizosphere [48, 49]. In immunocompromised patients this bacteria was associated with pneumonia and bacteremia. Also can cause fatal hemorrhagic pneumonia. It has been found that the StmPr1 gene is involved in pathogenicity [48]. Biofilm formation (the presence of genes rpfF and smf-1 genes) was considered that play role in virulence [50].

In dairy products the presence of *Escherichia coli* indicates fecal contamination. It was detected in Brazilian raw milk that could be appear in Minas Frescal-type cheeses produced from raw milk. The risk could be eliminated with the heat treatment of milk [51]. Metz et al. 2020 [52] reported that *E. coli* and coliforms (detected in raw milk) number, rise during the early steps of processing. At the end of the cheese making, including fermentation and ripening, their level decline by numerous logs or the final product is free of this bacteria. Contaminated raw milk of domestic animals and dairy products could be the reservoir of verotoxin-producing *Escherichia coli* (VTEC). This bacteria can cause severe infections also. Outbreaks of *Escherichia coli* O157:H7 was related to contaminated raw milk. It was reported that *E. coli* O157 was growth in initial phase of preparation of Fontina PDO cheese. The heat treatment of curd cooking not affected the bacteria. Only during ripening decreased the level of *E. coli.* It was considered that the aw reduction and the lactic acid could contribute to the reduction of *E. coli* O157 level [53]. In Cacioricotta goat cheese *E. coli* O157:H7 could survive after 90 days of ripening. This fact gives reason for that the low level contaminated raw milk represents a threat to consumers [54].

The plasmid-mediated colistin resistance gene in *E. coli* from hard cheese represents a high risk factor because cheese is consumed without heat treatment [55]. During manufacturer process of raw milk cheese only the low temperature conditions of maturation affected the growth negatively of shigatoxigenic *E. coli* and *L. monocytogenes* [56].

*Microbial Diversity of Traditionally Processed Cheese from Northeastern Region of Transylvania… DOI: http://dx.doi.org/10.5772/intechopen.97591*

> *Microbial load of cheese samples (CFU/g).*

**Table 1.**

#### **Table 2.**

*Occurrence of microscopic fungi in the analyzed cheese samples (CFU/g).*

#### **Figure 1.**

*Colonies of aerobic spore-forming bacteria from cheese 6 sample (A) and microscopic fungi from cheese 10 sample (B) on selective agar plates.*

One of the predominant bacteria from staphylococci is *S. equorum* in cheeses owing antilisterial activity. *S. equorum* and *S. equorum* subsp. *linens* was mentioned as potential new starter culture [57]. *S. equorum* also characterized by high salinity tolerance (up to 25%) was the predominant species of the long maturation processes.

In traditional fermented foods staphylococci were the dominant species. These bacteria are involved in the development of sensorial features. *Staphylococcus carnosus*, *S. xylosus* and *S. equorum* with the production of low molecular-weight compounds, including aldehydes, amines, amino acids esters and free fatty acids take part in flavor development. This is possible due to various biochemical transformations as reduction of nitrates to nitrite and then to nitrous oxide, proteolysis and lipolysis [58]. With DNA–DNA hybridization method it was revealed that

*Microbial Diversity of Traditionally Processed Cheese from Northeastern Region of Transylvania… DOI: http://dx.doi.org/10.5772/intechopen.97591*

#### **Figure 2.**

*Phylogenetic tree based on similarities of 16S rDNA partial sequences of bacterial isolates using the neighborjoining method.*

*Staphylococcus equorum* subsp. *linens* subsp. nov. and *Staphylococcus equorum* subsp. *linens* was part of the dominant microflora of surface ripened Swiss mountain raw milk cheese. Characteristics of these two strains include the ability to grow between 6 and 40°C and in salinity conditions 13% NaCl [59].

Based on literature data *Klebsiella michiganensis* originated from traditionally processed cheese is considered an emerging pathogen, causing nosocomial and bloodstream infections. It was isolated from hospital samples. Different virulence factors were identified in this bacteria as capsule locus type, plasmid replicon types, cell adherence and iron permitting acquisition. *K. michiganensis* is a multidrug resistant bacteria harboring (KPC)-3 carbapenemase gene, carbapenemase gene *bla*OXA-181 and other 11 genes encoding different antibiotic resistance. The carbapenemase gene encoding plasmid is originated from *Klebsiella pneumoniae* [60, 61]. Mitra et al. [62] reported that K*. michiganensis* strain S8 possess cadmium accumulating capacity contributing to enhance the productivity of rice.

The other identified bacteria from cheese was *Halomonas alkaliphila*. According to Sun et al. [63] is a halophilic proteobacteria with role in nitrogen cycle of marine environments, converting inorganic nitrogen compounds.

It was reported that *Proteus vulgaris* potentially could be applied to enhance the flavor based on the production of widest varieties and the largest quantities of volatile compounds on the surface of a smear-ripened cheese [64]. In surface-ripened cheeses proteobacteria as *Proteus* are responsible for the development of purple pigmentation of cheese rinds [65].

*Shigella flexneri* is known as a human pathogen infecting the colonic epithelial cells. With reduction of intercellular tension is able to spread between cells [66].

Jamet et al. [67] reported that enterococci take part of the dominant microflora of raw milk cheeses ranging from 102 to 108 CFU/g. There are contradictory aspects regarding the *Enterococcus* lactic acid bacterial strains. It was considered that these strains of bacteria are capable of causing diseases. Studies are showing that *Enterococcus* bacterial strains are promising starter cultures owing relevant technological and probiotic characteristics. *E. durans* LM01C01 with beneficial characteristics in cream cheeses lowered the *S. aureus* contamination level [68]. *Enterococcus durans* LAB18s possess probiotic potential, antimicrobial, antioxidant characteristics and selenium bioaccumulation ability. The culture supernatant of aforementioned bacteria exhibited inhibitory effect against *Listeria monocytogenes, Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Aeromonas hydrophila* and *Corynebacterium fimi*. The intracellular extract of *E. durans* LAB18s showed antibacterial effect against *L. monocytogenes, E. coli, B. cereus, Staphylococcus aureus, S. enteritidis, Ps. aeruginosa, Ae. hydrophila* and *Corynebacterium fimi* [69].

*Enterococcus durans* SJRP29 originated from cheese is thought to be an advantageous safe strain with practical use due to the beneficial characteristics (tolerance to 3% NaCl, good adhesion and without bile salt hydrolase activity or mucin degradation) [70]. Enterococci are involved in antibiotic resistance gene transfer among non-pathogenic and pathogenic bacteria. It has been reported that the predominant antibiotic resistant enterococci in French cheeses were *E. faecalis, E. faecium* and *E. durans.* Antibiotic resistance was showed against tetracycline, minocycline, erythromycin, kanamycin and chloramphenicol [67]. *L. monocytogenes* and *E. faecalis* were true cases of horizontal gene transfer in Minas Frescal cheese [71]. In another study it was concluded that antibiotic resistance and virulence factors can be transferred with high rate during cheese and sausage fermentation to enterococci [72].

*B. licheniformis* due to high resistance of environmental factors and heat treatments is involved in the biofilm formation in milk processing plants, but this can be avoiding with the pH and milk residues control [73]. *B. licheniformis* is a food spoilage and poisoning bacteria causing serious diseases as enteric disease, septicaemia, peritonitis, ophthalmitis [74]. Also these spore forming bacteria are promising probiotic strains. It was reported that *Bacillus* strains could survive the harsh environment of Requeijão cremoso Brazilian cheese [75].

*Kocuria* as a non-pathogenic bacteria is part of skin microbiota. Several studies reported that it was associated with peritonitis and urinary tract infection. Recently it has been reported that *Kocuria rhizophila* is causing catheter-related bacteremia with endocarditis [76].

The genus *Hafnia* belonging to Enterobacteriaceae comprise bacterial strain predominant in nature, gastrointestinal tracts of mammals and also in food products. *Hafnia* sp. and *Enterococcus faecalis* was associated with cholecystitis [77].

### **7. Conclusions**

The microflora of traditionally processed cheese comprise of diverse and heterogeneous group of microbes. These microorganisms have specific role in product formation providing the unique characteristics of the cheese. Also, it could appear microorganisms that are emerging pathogens. For these reason in traditional manufacture process have to pay attention to personal hygiene and sanitation process. The support of autochthonous bacteria in fermented products have to be a priority to contribute to the preservation of the organoleptic characteristics typical of that geographical region.

*Microbial Diversity of Traditionally Processed Cheese from Northeastern Region of Transylvania… DOI: http://dx.doi.org/10.5772/intechopen.97591*


**Magnetic Multiwall Carbon Nanotube Decorated with Novel Functionalities: Synthesis and Application as Adsorbents for Lead Removal from Aqueous Medium**

**Ghadir Hanbali 1 , Shehdeh Jodeh 1,\* , Othman Hamed 1,\*, Roland Bol 2 , Bayan Khalaf 1 , Asma Qdemat 3 , Subhi Samhan 4 and Omar Dagdag 5**

Received: 19 July 2020; Accepted: 10 August 2020; Published: 14 August 2020

**Abstract:** Water pollution is one of the major challenges facing modern society because of industrial development and urban growth. This study is directed towards assessing the use of multiwall carbon nanotube, after derivatization and magnetization, as a new and renewable absorbent, to remove toxic metal ions from waste streams. The adsorbents were prepared by, first oxidation of multiwall carbon nanotube, then derivatizing the oxidized product with hydroxyl amine, hydrazine and amino acid. The adsorbents were characterized by various techniques. The adsorption efficiency of the multiwall carbon nanotube adsorbents toward Pb(II) was investigated. The effect of adsorbent's dose, temperature, pH, and time on the adsorption efficiency were studied and the adsorption parameters that gave the highest efficiency were determined. The derivatives have unique coordination sites that included amine, hydroxyl, and carboxyl groups, which are excellent chelating agents for metal ions. The thermodynamic and kinetic results analysis results indicated spontaneous adsorption of Pb(II) by the multiwall carbon nanotube adsorbents at room temperature. The adsorption process followed pseudo-second-order and Langmuir isotherm model. The adsorbents were regenerated using 0.1 N HCl.

**Keywords:** magnetic multiwall carbon nanotube; adsorption; kinetics; isotherm; thermodynamic; lead

#### **1. Introduction**

Metal water pollution is a major environmental concern as some are highly toxic elements, even at low concentrations [1]. The toxicity of heavy metals is an inevitable consequence as they accumulate in the soft tissues of the human body [2,3]. These metals might enter the human body through different ways, among these is drinking water, and unlike organic pollutants, heavy metal ions are nonbiodegradable.

Heavy metal ions exists in waste streams of various industries, including leather tanning, metal plating, radiator manufacturing, batteries manufacturing, mining operations, smelting, alloy industries, and fertilizer industries [4].

Lead is considered one of the most toxic metals, it has access to the human body via ingestion, inhalation or skin assimilation [5]. Once inside the body, lead is absorbed and stored in bones, blood, and tissues [6]. Lead can cause severe damage to the brain, kidneys and, ultimately, it could lead to death. By simulating calcium, lead can cross the blood-brain barrier and destroys myelin sheaths of neurons, decreases their numbers, inhibits with neurotransmission routes and reduces the growth of neurons [7].

In nature, lead can compete with other minerals in and on plants surfaces and may inhibit photosynthesis, and negatively effect on plant growth and survival at sufficiently high concentrations [8].

The bioaccumulation of lead in food sources and its toxicity to biological systems attributable to increased concentrations over time has called for a significant pressure for removal. Several technologies have successfully removed lead from polluted water; among these are adsorption, membrane filtration, ion exchange bioremediation, precipitation and coagulation, and heterogeneous photocatalyst [9–11]. Among the most effective technologies, adsorption has been proven the highest removal efficiency at a reasonable cost. In addition, adsorbents can be regenerated and reused [12,13]. An unlimited number of effective adsorbents were developed and reported in the literature. Among the most effective metal adsorbents is multiwall carbon nanotube (MWCNT), it has shown significant success and has been employed in many of commercial applications [14]. Several published studies reported that carbon nanotubes are among the most effective in water treatment and adsorption of heavy metals such as copper, lead, cadmium. It also showed high efficiency in polar and nonpolar organic molecules. This was related to the various physical and chemical interactions MWCNT can afford in the from π–π interactions, covalent bonding, hydrogen bonding [15], and electrostatic interactions [16].

Chemical modification or doping is believed to optimize MWCNT properties for a certain specific application. A common method is to enhance the dispersion, and optimize the use of multi-wall carbon nanotubes through chemical functionality. This enables the formation of chemical interconnection between MWCNT and targeted materials. Both non-covalent and covalent structures have been employed to improve solubility and physical properties of MWCNT [17].

MWCNT functionalization with P, O, and N containing groups on the surface of MWCNT allow the researchers to control the surface area, surface charge, hydrophobicity/hydrophilicity, dispersion. and improve adsorption capacity and selectivity towards the heavy metals [18,19]. For instance, Li et al. [20] showed that acid-refluxed carbon nanotubes (CNTs) have enhanced ability to adsorb Pb(II) ions from water. Wang et al. [21] showed that adding oxygen functional groups on acidic MWCNT improved the adsorption capacity of lead ion. Tofighy and Mohamadi [22] reported that the adsorption on oxidized CNTs sheets followed the order: Pb2+ > Cd2+ > Co2+ > Zn2+ > Cu2+. Wang et al. [23] showed that oxidized MWCNT caused a rise in the zeta potential value resulting in the formation of negative surface charge because of the creation and ionization of functional groups (–COOH &–OH). It has been observed that the adsorption of Pb(II) onto acidic MWCNT was not uniform and mainly collected on the cap and defective sites of the MWCNT as adsorbent.

During recent years, the focus has been on magnetic adsorbents to avoid problems associated with adsorption regeneration. The main advantages of magnetic composites include: high strength, strong adsorption rate and improved adsorption ability [24]. The composite adsorbent could effectively achieve the separation of solid–liquid under an active magnetic field without filtration or centrifugation. This results from the properties of the magnetism, which simplifies post-processing. In addition, magnetic recycling can help prevent nano-adsorbents from occurring in the natural environment, and mitigate future hazards [25].

This work focused on a MWCNT grafted with various functionalities as a potential new adsorbent for toxic metal ions. The MWCNT was first oxidized then converted to acid chloride. MWCNT functionalized with acid chloride was reacted separately with hydroxylamine, cystine, and hydrazine. The prepared grafted MWCNT were magnetized and then evaluated as an adsorbent for Pb(II) removal from water. The effect of various factors on the adsorption efficiencies of grafted MWCNT were evaluated.

The novelty of this work can be summarized as the grafted MWCNTs presented in this work represent the first example in the oxidized MWCNT literature modified with such functionalities and applied for removal of Pb(II). In addition, previous studies on lead removal using a single functional group provided lower removal than the one presented in this study.

#### **2. Materials and Methods**

Chemicals and MaterialsAll chemicals used in this work were purchased from Sigma-Aldrich (Jerusalem, Israel) and used as received.

#### *2.1. Instrument and Characterization*

Infrared spectra were recorded using (FTIR-SHIMADZU, Shimadzu Corporation, Kyoto, Japan, Model: FTIR-8700). The following parameters were used: resolution 4 cm−1 , spectral range 600–4000 cm−1 , number of scans 128. Flame atomic absorption spectroscopy (iCE 3300, Thermo Fisher Scientific, Cambridge, UK) was used to determine lead ion concentration. Raman spectra were recorded on Bruker RFS 100 FT-Raman spectrometer, scanning electron microscopy ((Hitachi SX-650 machine (Tokyo, Japan)) was used to study the surface morphology or the adsorbents, Brunauer−Emmett−Teller surface area analysis (Micromeritics, Norcross, GA, USA), Vibrating Sample Magnetometer (VSM-LAKESHORE 7404, Boston, MA, USA). All results of the characterization can be found in our previous study [26].

#### *2.2. Magnetization of Modified Multiwall Carbon Nanotube*

The procedure includes four steps, at the beginning, MWCNT was oxidized then the developed carboxylic acid functionality was converted to acid chloride by reacting it with oxalyl chloride. Then the acid chloride was converted to hydroxamate, hydrazine and amino acids by reacting it with hydroxyl amine, hydrazine and cystine, respectively (1).

In the first step, MWCNT (0.1 g) was treated with 100 mL of 69% HNO3 in a flask of 500 mL. The flask was vibrated in an ultrasonic bath for 30 min at 25 ◦C. Next, the mixture was diluted with deionized water to reach 400 mL and then filtered through a polycarbonate membrane (0.22 µm). The same procedure was repeated exactly with H2O2 (30% v/v) instead of HNO3. Hydrogen peroxide was used to complete the oxidation process, but mildly. The pH of the filtrate was roughly 7.0 by washing the solid with deionized water, then the product was dried under a 24 h vacuum to produce the carboxylic acid-functionalized MWCNT (MWCNT-COOH) [27].

Oxidized MWCNT (MWCNT-COOH) (0.1 g), were stirred in 2 mL of oxalyle chloride in the presence of 2–3 drops of dimethylformamide (DMF), and 2 mL of triethyl amine (TEA) at 70 ◦C for 24 h under N2. After cooling to room temperature, the excess TEA was washed repeatedly with anhydrous tetrahydrofuran (THF) and then dried at 70 ◦C under vacuum in order to remove any traces of adsorbed TEA on the surface of acylated MWCNT. This samples are labelled as MWCNT-COCl [28].

Three separates of solutions of hydroxylamine (0.2 g), cystine (0.5 g) and of hydrazine (200 uL) in 1 mL THF and 0.5 mL pyridine was prepared. To each solution, w 0.05 g of MWCNT-COCl was added. The mixtures were stirred for 30 min at room temperature, then refluxed at 100 ◦C for 96 h. The residual hydroxylamine, cystine, and hydrazine were removed by rinsing with ethanol and sonication for 15 min. This rinsing process was repeated three times, until a clear ethanol was produced. The remaining solid was suspended in dichloromethane, sonicated and centrifuged. The collected black solid was dried under vacuum and labelled as magnetized multiwall carbon nanotube functionalized by hydroxyl amine (MWCNT-HA), magnetized multiwall carbon nanotube functionalized by cysteine (MWCNT-CYS), and magnetized multiwall carbon nanotube functionalized by hydrazine (MWCNT-HYD) [29].

A mixed solution of 0.1 M ferric chloride hexahydrate (FeCl3·6H2O) and 0.05 M ferrous chloride tetrahydrate (FeCl2·4H2O) was prepared with one to two molar ratios and then mixed with functionalized MWCNT and suspended for 2 h.

To precipitate iron oxides, 5.0 M NH4OH solution was added dropwise until pH adjusted at 10 at 70 ◦C, and then kept under continuous stirring for 1 h. The solid was allowed to cool and magnet separate, and then rinsed with distilled water and ethanol. The composite obtained was dried for 2 h in a furnace at 100 ◦C. This samples are labelled as (m-MWCNT-HA, m-MWCNT-CYS, m-MWCNT-HYD) [30].

#### *2.3. Adsorption Study*

All adsorption runs were carried out in plastic vials (50-mL) that were placed in a water bath equipped with a thermostat and a shaker. The effect of various variables such as metal ion concertation, pH values, adsorbent dosage, adsorption time, and temperature on adsorption efficiency were studied. The adsorption experiments were performed on lead (II) ion. After every adsorption run, a clear supernatant sample from the adsorption mixture was collected via magnet and analyzed by a flame atomic adsorption analysis at 193.7 nm to determine the residual lead (II) concentration. All adsorption experiments were carried out in triplicate, and the mean was reported. The adsorbent efficiency and the amount of lead (II) ions adsorbed that was adsorbed (*qe* , mg/g) was calculated by the Equation (1):

$$q\_e = \frac{V(\mathbb{C}o - \mathbb{C}e)}{W} \tag{1}$$

where *V* is the volume of the solution (L), *Co* is the initial lead concentration (mg·L −1 ), *Ce* is lead concentration at equilibrium (mg·L −1 ), and *W* is the adsorbent's mass (g).

#### 2.3.1. Adsorption Isotherms Models

Adsorption isotherms models are important for the description of the interaction mechanism between adsorbate and adsorbent. Several models are available for this purpose, among these, the most common are Langmuir and Freundlich. These models take into account essential comparative guidelines with small distinction in their approaches [31].

The Langmuir model adopts the formation of a monolayer of adsorbate on a homogeneous surface of an adsorbent and expressed as:

$$\frac{\mathbf{C}\_{\varepsilon}}{q\_{\varepsilon}} = \frac{1}{q\_{m}} \mathbf{C}\_{\varepsilon} + \frac{1}{q\_{m}\mathbf{K}\_{L}}\tag{2}$$

where *Ce* is the equilibrium concentration of adsorbate (mmol·L −1 ), *qe* is the amount of adsorbate adsorbed per unit weight of adsorbent (mmol·g −1 ), *qm* is the adsorption capacity (mmol·g −1 ), or monolayer capacity, and *KL* is a constant (L·mmol−1 ).

Langmuir isotherm can be identified by constant dimensions separation factor (*RL*) as shown by the following equation [32]

$$R\_L = \frac{1}{(1 + K\_L \mathcal{C}\_o)}\tag{3}$$

where *Co* is the highest initial concentration of adsorbate (mg·L −1 ), *KL* (L·mg−1 ) is Langmuir constant.

The value of the *RL* refers to the form of isotherm to be either unfavorable (*RL* > 1), linear (*RL* = 1), favorable (0 < *RL* < 1) or irreversible if (*RL* = 0).

The Freundlich isotherm describes the adsorption between the adsorbates and the adsorbents with a heterogeneous surface. The rate of adsorption/absorption varies according to the degree of energy at the adsorptive sites. Freundlich's equation is expressed as shown in Equation (3):

$$\ln Q\_{\varepsilon} = i \frac{1}{n} \ln \text{ i} \mathbb{C}\_{\varepsilon} + \ln \mathbb{K}\_{F} \tag{4}$$

where *KF* (mmol/g) and 1/n are the constant characteristics of the system [32].

*KF* is an indicator of adsorption capacity of the adsorbent and 1/n is an indicator of favorability of adsorption process. If (10 > n > 0) this donates a favorable adsorption process.

Langmuir and Freundlich's isotherms could be used to define the relationship between the amounts of Pb(II) adsorbed on m-MWCNT-HA, m-MWCNT-CYS, m-MWCNT-HYD adsorbents and its equilibrium concentration in solutions.

#### 2.3.2. Adsorption Kinetics

Numerous adsorption kinetic models have been set to define kinetics and rate-determining steps. These models give evidence about the performance of the adsorption system and the rate at which a specific component is removed using a specific adsorbent. Besides, it determines whether the adsorption process is physical or chemical in nature and which step is the rate-determining step. Examples of the adsorption kinetic models include pseudo-first-order, intra-particle diffusion kinetic model, pseudo-second-order models, intra-particle diffusion kinetic model, first-order reversible reaction model, Elovich's model, etc. [33].

Pseudo-first-order kinetics are developed for describing adsorption kinetics, and are considered as the earliest model. The equation for this model can be written as follows:

$$\log(q\_\ell - q\_l) = \log q\_\ell - \left(\frac{K\_1}{2.303}\right)t\tag{5}$$

where *qe* and *qt* are the masses of the adsorbate in equilibrium or at time t per unit mass of adsorbent (mg g−1 ). k1 is the rate constant of the first-order pseudo-adsorption model (mg·g −1 ·min−1 ).

The graph of log (*qe* − *qt*) as a function of t gives a straight line for first-order pseudo adsorption with log qe as Y intercept [34].

The pseudo-second-order models are primarily based on the idea that the step to determine may be chemical adsorption, which involves exchanging or sharing electrons between adsorbate and adsorbent.

The net equation for pseudo-second-order can be addressed as an equation:

$$\frac{t}{q\_t} = \frac{1}{q\_\varepsilon} t + \frac{1}{\mathcal{K}\_2 q\_\varepsilon^2} i \tag{6}$$

where *K*2 is the equilibrium rate constant of the adsorption pseudo-second order (g·mg−1 min−1 ).

The graph of t/*qt* versus t should give a linear relationship that allows the calculation of a second order rate constants, *K*2 from the Y intersect and *qe* from the slope [30,35,36].

The metal ions adsorption is mainly achieved through three steps, including migration of metal ion from liquid to surface of the adsorbents (film diffusion process), diffusion of metal ions within the porous structure (intraparticle diffusion process, and metal ion adsorption on the surface to adsorbent). In general, the third step isn't time-consuming and is not considered as a rate-controlling step.

Intra-particle diffusion kinetic model suggested by Weber and Morris. The net equation of this kinetic model is:

$$q\_t = K\_{id} t^{0.5} + \mathbb{C} \tag{7}$$

where *Kid* is the constant diffusion rate (mg·g −1min−1/2 ). C is a constant representing the thickness of the boundary layer (mg g−1 ). A graph of *qt* with respect to t0.5 will show a linear relationship with the constant C as Y intersect. When the curve moves through the origin, adsorption is controlled by the process of inter-particle diffusion. Otherwise, it is dominated by film diffusion [37].

Activation energy can also be measured by an Arrhenius equation:

$$\text{Ln } K\_2 = \text{Ln A} - \frac{\text{Ea}}{\text{RT}} \tag{8}$$

In general, if Ea between 5 kj/mol to 40 kj/mol the adsorption is physisorption whereas value (40 kj·mol−1 to 800 kj·mol−1 ) refers to chemisorption.

#### 2.3.3. Adsorption Thermodynamics

The thermodynamic study is done by ascertaining enthalpy, free energy, entropy.

Thermodynamic parameters are required to determine whether the process is spontaneous or not. Gibbs free energy change, ∆*G o* , is an indication of the spontaneity of a chemical reaction and therefore is an important criterion for spontaneity. Both enthalpy (∆Ho ) and entropy (∆S o ) factors must be considered to determine Gibb's free energy of the process. Reactions occur automatically at a certain temperature if ∆*G o* is a negative quantity.

The following equation is the general equation that Connect between thermodynamics parameters

$$
\Delta \mathbf{G}^o = \Delta \mathbf{H}^o - \mathbf{T} \Delta \mathbf{S}^o \tag{9}
$$

where T is the absolute temperature (K).

The change in Gibbs energy can be expressed by the following equation:

$$
\Delta G^\circ = -\text{RT } \ln \text{K}\_\text{D} \tag{10}
$$

where, KD is the constant of thermodynamic equilibrium equal to (*qe* /*ce*) with a unit of (L·g −1 ). R is the gas constant, 8.314 J·mol−1K −1 .

The net equation of the last two equations can be expressed as the follows:

$$
\ln K\_d = -\frac{\Delta \mathcal{H}^o}{RT} + \frac{\Delta \mathcal{S}^o}{R} \tag{11}
$$

The plot of In*Kd* against (1/T) gives a line with (∆S o /R) as the Y-intersect and (−∆H o /R) as the slope. The resulting graph is identified as a Van't Hoff diagram [38].

#### 2.3.4. Regeneration of Adsorbents

The adsorbent was separated by magnet, washed with 0.1 N HCl (10 mL) solution to release adsorbed metal ions, and then washed with distilled water. It was then left to dry at room temperature for 24 h. The three adsorbents were treated in the same manner.

#### *2.4. Real Water Sample*

The actual sample was used to study adsorbents efficacy for removal heavy metals. A real water sample was collected from Jericho city. The initial and final concentrations of the metals were measured using ICP/MS (Elan 9000 model, PerkinElmer, Inc., Waltham, MA, USA). A 0.02 g of adsorbent was mixed in 50 mL plastic vials with 10 mL of water sample under maximum condition; the amount adsorbed was calculated by comparing the concentrations of the metals before and after the adsorption.

#### **3. Results and Discussion**

#### *3.1. Adsorbents Analysis*

MWCNT was oxidized following a published procedure [26]. The developed carboxylic acid functionality was converted to acid chloride by reacting it with oxalyl chloride as shown in Scheme 1. Then the acid chloride was converted to hydroxamate, hydrazine and amino acids by reacting it with hydroxyl amine (HA), hydrazine (HYD) and cystine (CYS), respectively. The presence of these functionalities was confirmed by FTIR, which shows that when MWCNT-COOH was modified by HA, HYD, and CYS, several new peaks appeared in the spectra. Based on the literature: the peak at 1640 cm−1 is due to C=O stretching vibration. C–N stretching vibration and N–H bending vibration

appear at 1550 cm−1 (Figure S1). Raman spectroscopy shows the spectra of MWCNT, oxidized MWCNT, and devised as MWCNT. It could be clearly noticed that the D-band of MWCNT-COOH shifted from 1360 to 1349 cm−1 and G-band from 1605 to 1600 cm−1 and this shows that a large defection was created after oxidation. Additionally, the ratio of the intensity of the D-band peak to that of the G-band peak (ID/IG) of the pure MWCNT was 1.33, while that for the MWCNT–functionalized with HA, CYS, HYD was 1.71 (Figure S2). The images of scanning electron microscopy of all samples show disordered and highly porous morphologies. The unmodified MWCNT showed less agglomeration than others (Figure S3). In addition, chemical modification leads to removal of the ends of MWCNT, which increases surface area as confirmed by Brunauer−Emmett−Teller (BET) (Table 1). The magnetization of prepared MWCNT confirmed by VSM, which shows that the magnetization curve for MWCNT sample has a semi-linear representation, which indicates a paramagnetic state while magnetization curve for m-MWCNT-HA, m-MWCNT-CYS, and m-MWCNT-HYD, showed a sigmoidal response with no hysteresis, which is an indication of the presence of a saturated superparamagnetic component in the samples (Figure S4). All characteristic techniques confirmed that HA, CYS and HUD were successfully drafted on MWCNT, and also created functionals, which are expected to have excellent activities toward metal ions [39]. *Processes* **2020**, *8*, x FOR PEER REVIEW 7 of 18 functionalized with HA, CYS, HYD was 1.71 (Figure S2). The images of scanning electron microscopy of all samples show disordered and highly porous morphologies. The unmodified MWCNT showed less agglomeration than others (Figure S3). In addition, chemical modification leads to removal of the ends of MWCNT, which increases surface area as confirmed by Brunauer−Emmett−Teller (BET) (Table 1). The magnetization of prepared MWCNT confirmed by VSM, which shows that the magnetization curve for MWCNT sample has a semi-linear representation, which indicates a paramagnetic state while magnetization curve for m-MWCNT-HA, m-MWCNT-CYS, and m-MWCNT-HYD, showed a sigmoidal response with no hysteresis, which is an indication of the presence of a saturated superparamagnetic component in the samples (Figure S4). All characteristic techniques confirmed that HA, CYS and HUD were successfully drafted on MWCNT, and also created functionals, which are expected to have excellent activities toward metal ions [39].

**Scheme 1.** Preparation of magnetic functionalized multiwall carbon nanotube. **Scheme 1.** Preparation of magnetic functionalized multiwall carbon nanotube.

With reference to previous studies**,** there are several studies related to oxidation and surface modification of MWCNT. The novelty of this work can be summarized as the grafted MWCNTs

With reference to previous studies, there are several studies related to oxidation and surface modification of MWCNT. The novelty of this work can be summarized as the grafted MWCNTs presented in this work comprise the first example in the literature of oxidized MWCNT modified with such functionalities and applied for lead removal. Additionally, previous studies on the removal of lead using a single functional group provided lower removal than that presented in this study (Table 1). Other studies with higher removals than that presented in this study were based on using photodegradation or membranes, which are extremely expensive methods compared with the method presented in our study.

#### **Table 1.** Previous studies on lead removal from water.

#### *3.2. Lead Ion Adsorption*

The adsorption was carried out using a batch process. During the prior process, a known weight of adsorbent (0.02 g) was suspended in an aqueous solution of lead ion, separated by a magnet, and analyzed. The analysis was performed on the filtrate to determine the amount of the residual lead ions. The effects of several variables such as contact time, adsorbent dosage, temperature, and pH were evaluated to determine the best conditions for the highest adsorption efficiency. The adsorption study was performed on Pb(II) ions using (m-MWCNT-HA), (m-MWCNT-CTS), (m-MWCNT-HYD) adsorbents. Each experiment was repeated three times, and the average was used when we analyzed the data.

### 3.2.1. Effect of Contact Time

This experiment was performed to determine the optimal adsorption time. A 10 mL solution of Pb(II) with 10 ppm concentration was placed in a vial and shaken with 0.02 g of an adsorbent for a varied period of time; ranging from 1 to 120 min.

Adsorption curves are presented in Figure 1a. The figure shows that in the beginning, the development of monolayer was very rapid and the maximum removal was attained after approximately 30 min for all three adsorbents. After that, the adsorption leveled off and became nearly constant.

The high percentage of removal of Pb(II) in the beginning was due to the presence of the vacant sites which decrease steadily with time passing. In general, excellent adsorption efficiency for Pb(II) was exhibited by the three adsorbents.

The same type of interaction appears when using m-MWCNT-HA and m-MWCNT-CYS.

The high percentage of removal of Pb(II) in the beginning was due to the presence of te vacant sites which decrease steadily with time passing. In general, excellent

**Figure 1.** Effect of (**a**) contact time, (**b**) pH, (**c**) experimental mass titration curves, (**d**) temperature, (**e**) Pb(II) initial concentration on magnetized multiwall carbon nanotube functionalized by hydroxyl amine (m-MWCNT-HA), magnetized multiwall carbon nanotube functionalized by cysteine (m-MWCNT-CYS), and magnetized multiwall carbon nanotube functionalized by hydrazine (m-MWCNT-HYD).

MWCNT-HYD).

#### 3.2.2. Effect of pH 3.2.2. Effect of pH

The effect of pH on the adsorption efficiency is shown in Figure 1b. The pH effect on adsorption is an important factor as it affects the surface charge of the adsorbents. At pH values of 3.0 or less, the amine functionality becomes ammonium whereas, at high pH value over 5.5, the amine is not protonated and the carboxylic acid functionality becomes carboxylate. The highest adsorption efficiency was observed at a pH 8.0 then it started to decline. At pH value of about 8.0, the functional groups present in the adsorbents (amine and carboxyl) carry a lone of pair of electrons causing them to behave as a strong chelating agent. The effect of pH on the adsorption efficiency is shown in Figure 1b. The pH effect on adsorption is an important factor as it affects the surface charge of the adsorbents. At pH values of 3.0 or less, the amine functionality becomes ammonium whereas, at high pH value over 5.5, the amine is not protonated and the carboxylic acid functionality becomes carboxylate. The highest adsorption efficiency was observed at a pH 8.0 then it started to decline. At pH value of about 8.0, the functional groups present in the adsorbents (amine and carboxyl) carry a lone of pair of electrons causing them to behave as a strong chelating agent.

The same type of interaction appears when using m-MWCNT-HA and m-MWCNT-CYS.

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**Figure 1.** Effect of (**a**) contact time, (**b**) pH, (**c**) experimental mass titration curves, (**d**) temperature, (**e**) Pb(II) initial concentration on magnetized multiwall carbon nanotube functionalized by hydroxyl amine (m-MWCNT-HA), magnetized multiwall carbon nanotube functionalized by cysteine (m-MWCNT-CYS), and magnetized multiwall carbon nanotube functionalized by hydrazine (m-

The pH effect can also be explained in terms of pHpzc of adsorbents. In pHpzc the positive and negative groups are equal and thus sum surface charges become zero. The pH effect can also be explained in terms of pHpzc of adsorbents. In pHpzc the positive and negative groups are equal and thus sum surface charges become zero.

Mass titration technique (MT) was used to determine zero-point charge (pzc) [45]. When observing Figure 1c, it can be investigated that pzc equals 3.5, 3.7, 3.2 for m-MWCNT-HA, m-MWCNT-CYS, m-MWCNT-HYD, respectively, with the highest adsorption value appeared at pH > pHpzc. Consequently, electrostatic attractions among negatively charged surface of adsorbent and metal-ions could occur and influence to sorption sorbent mass (Figure 2). Mass titration technique (MT) was used to determine zero-point charge (pzc) [45]. When observing Figure 1c, it can be investigated that pzc equals 3.5, 3.7, 3.2 for m-MWCNT-HA, m-MWCNT-CYS, m-MWCNT-HYD, respectively, with the highest adsorption value appeared at pH > pHpzc. Consequently, electrostatic attractions among negatively charged surface of adsorbent and metal-ions could occur and influence to sorption sorbent mass (Figure 2).

**Figure 2.** Mechanism of lead adsorption on m-MWCNT-HYD. **Figure 2.** Mechanism of lead adsorption on m-MWCNT-HYD.

#### 3.2.3. Effect of Temperature on Lead Adsorption 3.2.3. Effect of Temperature on Lead Adsorption

Adsorption equilibrium is reached when the chemical potential of the adsorbed solute and the chemical potential the solute in aqueous solution are equal. This equilibrium is temperature-Adsorption equilibrium is reached when the chemical potential of the adsorbed solute and the chemical potential the solute in aqueous solution are equal. This equilibrium is temperature-dependence since the chemical potential is a function of temperature.

dependence since the chemical potential is a function of temperature. In this study, the dependence of adsorption equilibrium of Pb(II) on m-MWCNT-HA, m-MWCNT-CYS, and m-MWCNT-HYD was evaluated using different temperatures (5–50 °C) at a pH value of 8. The effect of temperature on the percentage of lead adsorption is shown in Figure 1d, as shown in the figure the maximum adsorption was at room temperature for the three adsorbents. In this study, the dependence of adsorption equilibrium of Pb(II) on m-MWCNT-HA, m-MWCNT-CYS, and m-MWCNT-HYD was evaluated using different temperatures (5–50 ◦C) at a pH value of 8. The effect of temperature on the percentage of lead adsorption is shown in Figure 1d, as shown in the figure the maximum adsorption was at room temperature for the three adsorbents.

#### 3.2.4. Effect of Lead Initial Concentration

3.2.4. Effect of Lead Initial Concentration The results of Pb(II) initial concertation on adsorption efficiency is shown Figure 1e. The highest removal percentage of Pb(II) for the three adsorbents at 10.0 ppm concentration.

The results demonstrate that at lower concentrations, the ratio of the number of metal ions to the existing sorption sites is low and thus the adsorption under these conditions is independent of the initial concentration. However, as the concentration of Pb(II) increases, the sites available for adsorption become less, and therefore, the removal of metals becomes highly dependent on the initial concentrations. The results show that the rate of Pb(II) ion removal decreases as the initial concentration of metal ions increases. concentrations. The results show that the rate of Pb(II) ion removal decreases as the initial concentration of metal ions increases.

adsorption become less, and therefore, the removal of metals becomes highly dependent on the initial

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removal percentage of Pb(II) for the three adsorbents at 10.0 ppm concentration.

The results of Pb(II)initial concertation on adsorption efficiency is shown Figure 1e. The highest

The results demonstrate that at lower concentrations, the ratio of the number of metal ions to the existing sorption sites is low and thus the adsorption under these conditions is independent of the

#### *3.3. Equilibrium Isotherm Models for Lead Adsorption 3.3. Equilibrium Isotherm Models for Lead Adsorption*

Langmuir and Freundlich's isotherms were applied to describe the relationship between amounts Pb(II) adsorbed on m-MWCNT-HA, m-MWCNT-CYS, and m-MWCNT-HYD adsorbents and its equilibrium concentration in solutions, results are presented in Figure 3a,b. Langmuir and Freundlich's isotherms were applied to describe the relationship between amounts Pb(II) adsorbed on m-MWCNT-HA, m-MWCNT-CYS, and m-MWCNT-HYD adsorbents and its equilibrium concentration in solutions, results are presented in Figure 3a, b. Freundlich and Langmuir adsorption isotherms parameters were calculated. The values

Freundlich and Langmuir adsorption isotherms parameters were calculated. The values of R2 using Langmuir adsorption isotherm are approximately one. The value of RL was found to be. 0.002, 0.012, 0.007 for m-MWCNT-HA, m-MWCNT-CYS, m-MWCNT-HYD, respectively, and this indicates that Langmuir adsorption isotherm is favorable. of R2 using Langmuir adsorption isotherm are approximately one. The value of RL was found to be. 0.002, 0.012, 0.007 for m-MWCNT-HA, m-MWCNT-CYS, m-MWCNT-HYD, respectively, and this indicates that Langmuir adsorption isotherm is favorable. From Freundlich isotherm data shown in Table 2, the value of R2 is much smaller than

From Freundlich isotherm data shown in Table 2, the value of R2 is much smaller than 1 (0.15, 0.02, 0.26) for m-MWCNT-HA, m-MWCNT-CYS, m-MWCNT-HYD, respectively), this indicates that the adsorption is not suited with Freundlich isotherm. 1 (0.15, 0.02, 0.26) for m-MWCNT-HA, m-MWCNT-CYS, m-MWCNT-HYD, respectively), this indicates that the adsorption is not suited with Freundlich isotherm.

**Figure 3.** (**a**) Langmuir isotherm plot, and (**b**) Freundlich isotherm plot for Pb(II) adsorption. **Figure 3.** (**a**) Langmuir isotherm plot, and (**b**) Freundlich isotherm plot for Pb(II) adsorption.

#### m-MWCNT-HYD 9.09 0.20 0.007 0.72 4.30 28.50 0.26 *3.4. Adsorption Kinetic Model*

*3.4. Adsorption Kinetic Model*  To study the mechanism of the adsorption process, pseudo first-order, pseudo second-order and intra-particle diffusion kinetic models were evaluated.

Adsorption kinetic models are represented in Figure 4. The kinetic study revealed that, the adsorption of Pb(II) on m-MWCNT-HA, m-MWCNT-CYS, m-MWCNT-HYD follows the pseudosecond-order kinetic mechanism. The correlation coefficient R2 and qe experimental and theoretical which in this kinetic model are approximately one (Table 3).

*Processes* **2020**, *8*, 986 *Processes* **2020**, *8*, x FOR PEER REVIEW 12 of 18 The obtained value of Ea indicates that the adsorption processes of Pb(II) ions by the three adsorbents

intra-particle diffusion kinetic models were evaluated.

which in this kinetic model are approximately one (Table 3).

m-MWCNT-HA, m-MWCNT-HYD and m-MWCNT-CYS are physical.

To study the mechanism of the adsorption process, pseudo first-order, pseudo second-order and

Adsorption kinetic models are represented in Figure 4. The kinetic study revealed that, the adsorption of Pb(II) on m-MWCNT-HA, m-MWCNT-CYS, m-MWCNT-HYD follows the pseudosecond-order kinetic mechanism. The correlation coefficient R2 and qe experimental and theoretical

In the intraparticle diffusion plot shown in Figure 3, the curve is not passing through the origin, so the adsorption is controlled by the external diffusion, and the interparticle diffusion mechanism.. The Ea value was calculated form Arrhenius equation by plotting ln *K2* versus 1/T (Figure 4d).

**Figure 4.** (**a**) Pseudo first-order plot. (**b**) Pseudo second-order plot. (**c**) Intra-particle diffusion plot. (**d**) Arrhenius plot for the adsorption of Pb(II) on m-MWCNT-HA, m-MWCNT-CYS, and m-MWCNT-HYD. **Figure 4.** (**a**) Pseudo first-order plot. (**b**) Pseudo second-order plot. (**c**) Intra-particle diffusion plot. (**d**) Arrhenius plot for the adsorption of Pb(II) on m-MWCNT-HA, m-MWCNT-CYS, and m-MWCNT-HYD.

**Table 3.** Parameters for Pb(II) adsorption of kinetic models of pseudo-first order, pseudo-second and intraparticle diffusion on m-MWCNT-HA, m-MWCNT-CYS and m-MWCNT-HYD. **Table 3.** Parameters for Pb(II) adsorption of kinetic models of pseudo-first order, pseudo-second and intraparticle diffusion on m-MWCNT-HA, m-MWCNT-CYS and m-MWCNT-HYD.

In the intraparticle diffusion plot shown in Figure 3, the curve is not passing through the origin, so the adsorption is controlled by the external diffusion, and the interparticle diffusion mechanism. **Intra-Particle Diffusion C (mg g−1) Kid (mg g−1min−0.5) R2** 4.29 0.07 0.51

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m-MWCNT -HA 4.96 4.03 –0.0023 0.33 4.99 0.32 10.50 0.99

m-MWCNT-CYS 4.93 3.35 –0.0034 0.51 5.15 0.03 5.65 0.99

**(mg g−1 min−1)**

**(mg g−1**

**min−1) (kj)**

**(mg g−1 min-1)**

The Ea value was calculated form Arrhenius equation by plotting ln *K2* versus 1/T (Figure 4d). The obtained value of Ea indicates that the adsorption processes of Pb(II) ions by the three adsorbents m-MWCNT-HA, m-MWCNT-HYD and m-MWCNT-CYS are physical. 1.172 0.41 0.60 1.90 0.34 0.40

#### *3.5. Adsorption Thermodynamic 3.5. Adsorption Thermodynamic*

**exp (mg g−1) Calc**

The Van't Hoff plot was used to calculate the common thermodynamics parameters: ∆S o , ∆H o , and ∆*G o* for Pb(II) adsorption on m-MWCNT-HA, m-MWCNT-CYS, m-MWCNT-HYD as shown in Figure 5. The Van't Hoff plot was used to calculate the common thermodynamics parameters: ∆S୭, ∆H୭, and for Pb(II) adsorption on m-MWCNT-HA, m-MWCNT-CYS, m-MWCNT-HYD as shown in Figure 5.

As shown in Table 4, the values of free energy were negative. This sign is a clear indication that the adsorption is spontaneous and favorable while the values of enthalpy were positive. This is a clear indication of the endothermic process. The positive ∆S o values reveal an increase in randomness in the solid and liquid surface, indicating the accumulation of Pb(II). As shown in Table 4, the values of free energy were negative. This sign is a clear indication that the adsorption is spontaneous and favorable while the values of enthalpy were positive. This is a clear indication of the endothermic process. The positive ∆S୭ values reveal an increase in randomness in the solid and liquid surface, indicating the accumulation of Pb(II).

**Figure 5.** Van't Hoff plot for Pb(II) adsorption on m-MWCNT-HA, m-MWCNT-CYS, and m-MWCNT-HYD. **Figure 5.** Van't Hoff plot for Pb(II) adsorption on m-MWCNT-HA, m-MWCNT-CYS, and m-MWCNT-HYD.

**Table 4.** Thermodynamic parameters for the adsorption of Pb(II) on m-MWCNT-HA, m-MWCNT-CYS, and m-MWCNT-HYD. **Table 4.** Thermodynamic parameters for the adsorption of Pb(II) on m-MWCNT-HA, m-MWCNT-CYS, and m-MWCNT-HYD.

m-MWCNT-HYD 50.60 0.19 −6.90 As shown in Table 4, the adsorption of Pb(II) on m-MWCNT-HA, m-MWCNT-CYS, m-As shown in Table 4, the adsorption of Pb(II) on m-MWCNT-HA, m-MWCNT-CYS, m-MWCNT-HYD endothermic and spontaneous process (∆H o > 0) and spontaneous (∆*G o* < 0).

#### MWCNT-HYD endothermic and spontaneous process (∆H୭ > 0) and spontaneous ( < 0). *3.6. Adsorbent Regeneration*

*3.6. Adsorbent Regeneration*  The adsorbents regeneration and reuse as absorbents for Pb(II) was evaluated. The three adsorbents were tested in three cycles, m-MWCNT-HA maintained its efficiency toward Pb(II), while the other two adsorbent m-MWCNT-CYS and m-MWCNT-HYD showed some reduction in the efficiency in the third cycles (Figure 6). To remediate the MWCNTs after regeneration, future studies will be needed.

Biodegradation of CNTs by enzymes was studied by Allen et al. (2008). They reported that oxidized single-wall CNTs (SWCNTs) could be degraded by enzymatic oxidation with horseradish peroxidase (HRP), a plant enzyme. They also reported that degradation took place within 10 days using hydrogen peroxides. This degradation will be leading to production of oxidized polyaromatic will be needed.

hydrocarbons and ultimately CO2 [46]. Serra et al., studied the circular zero-residue process using microalgae for efficient water decontamination, biofuel production, and carbon dioxide fixation [47]. using hydrogen peroxides. This degradation will be leading to production of oxidized polyaromatic hydrocarbons and ultimately CO2 [46]. Serra et al., studied the circular zero-residue process using microalgae for efficient water decontamination, biofuel production, and carbon dioxide fixation [47].

peroxidase (HRP), a plant enzyme. They also reported that degradation took place within 10 days

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The adsorbents regeneration and reuse as absorbents for Pb(II) was evaluated. The three adsorbents were tested in three cycles, m-MWCNT-HA maintained its efficiency toward Pb(II), while the other two adsorbent m-MWCNT-CYS and m-MWCNT-HYD showed some reduction in the efficiency in the third cycles (Figure 6). To remediate the MWCNTs after regeneration, future studies

Biodegradation of CNTs by enzymes was studied by Allen et al. (2008). They reported that

**Figure 6.** Three trials of adsorption—desorption of MWCNT using different functional groups towards removal efficiency of Pb(II). **Figure 6.** Three trials of adsorption—desorption of MWCNT using different functional groups towards removal efficiency of Pb(II).

#### *3.7. Real Water Samples 3.7. Real Water Samples*

The removal of metal by prepared adsorbents from real environmental water sample was studied to investigate the applicability of the proposed solid adsorbent. The results were presented in Table 5. It is clear from the table that m-MWCNT-HA, m-MWCNT-CYS, m-MWCNT-HYD has excellent efficiency towards most of the metal present in real water sample. The results obtained showed that the efficiency for the removal of lead were decreased when using real sample due to presence of dissolved organic matter and other metals. The removal efficiency decreased to about 64%, 73.1% and 67.1% when using m-MWCNT-HA, m-MWCNT-HYD and m-MWCNT-CYS respectively. This confirm the applicability of adsorbents for environmental. The removal of metal by prepared adsorbents from real environmental water sample was studied to investigate the applicability of the proposed solid adsorbent. The results were presented in Table 5. It is clear from the table that m-MWCNT-HA, m-MWCNT-CYS, m-MWCNT-HYD has excellent efficiency towards most of the metal present in real water sample. The results obtained showed that the efficiency for the removal of lead were decreased when using real sample due to presence of dissolved organic matter and other metals. The removal efficiency decreased to about 64%, 73.1% and 67.1% when using m-MWCNT-HA, m-MWCNT-HYD and m-MWCNT-CYS respectively. This confirm the applicability of adsorbents for environmental.

**Table 5.** Effect of adsorption of metals form real water sample. **Table 5.** Effect of adsorption of metals form real water sample.

### **4. Conclusions**

In this study, a magnetic multi-wall carbon nanotube with hydroxylamine, cystine and hydrazine was synthesis and tested for the removal of Pb(II) from water as the first example in the literature of oxidized MWCNT modified with such functionalities. The efficiency of the prepared derivatives toward Pb(II) was studied as a function of adsorbent dose, pH, metal ion initial concertation, temperature and time. The three adsorbents showed excellent efficiency toward Pb(II) and % of removal was quantitative. The highest efficiency was determined to be at room temperature and a pH of 8.0. The kinetic study revealed that the Pb(II) adsorption by the three adsorbents followed pseudo-second-order and Langmuir isotherm model. The thermodynamic analysis showed a negative free energy, indicating a spontaneous adsorption process at room temperatures. The adsorbents were regenerated by treatment with 0.1 N HCl. After third regeneration and reuse cycles, the efficiency of the regenerated adsorbents has shown a small reduction in efficiency. The presence of dissolved organic matter and other metals in real water sample has a significant effect on lead removal efficiency.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2227-9717/8/8/986/s1, Figure S1: FT-IR spectrum for MWCNTS, m-MWCNT-COOH, m-MWCNT-HA, m-MWCNT-Cys, m-MW CNT-HYD, Figure S2: Raman spectrum for MWCNTS, m-MWCNT-COOH, m-MWCNT-HA, m-MWCNT-Cys, m-MWCNT-HYD, Figure S3: SEM images of the original and the modified carbon nano tube (right) together with diameter distribution (left). (a) MWCNTs, (b) MWCNT-COOH, (c): m-MWCNT-HA, (d) m-MWCNT-CYS and (e) m-MWCNT-HYD. Scale bars: 5 µm, Figure S4: Magnetic field dependence of the magnetization measured at (a) 300K (M–H loops) of m-MWCNT-HA, m-MWCNT-CYS, m-MWCNT-HYD and MWCNT; (b) the magnification of the central area of the hysteresis loops.

**Author Contributions:** G.H.: Investigation, formal analysis, writing—original draft. S.J. and O.H.: investigation, formal analysis, writing—original draft, funding acquisition. R.B., O.D., and A.Q. assisted in sample analysis; B.K. and S.S. assisted in sample preparation. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by *Middle East Desalination Research* Center (MEDRC) and Palestinian Water Authority (PWA). Part of research was funded by the German Federal Ministry of Education and Research (BMBF).

**Acknowledgments:** The authors express their thanks to the MEDRC and PWA for their financial support during this study. Other thanks go to PADUCO 2 for some financial support. Many thanks go to the department of chemistry at An-Najah National University (ANNU) for facilitating the use of their instrumentations. This work was in part supported by the research project "Palestinian German Scientific Bridge (PGSB)" carried out by the Forschungszentrum Jülich and Palestinian Academy for Science and Technology- PALAST and funded by the German Federal Ministry of Education and Research (BMBF).

**Conflicts of Interest:** The authors report no relationships that could be construed as a conflict of interest.

### **Abbreviations**

#### **References**

2C%20Fertility%2C%20and%20Reproductive%20Toxicity%3B%20CRC%20Press%3A%202005&f=false (accessed on 14 May 2020).

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


4. Importance of product purity in chemical engineering

Synthesis of various chemicals usually is carried out in a reactor which may or may not be followed by separator. Either the case may be choice of design variable is very important. The market value of overhead product or the bottom product relies on its purity. Also the need of any further treatment for enhancing the purity relies on the initial product composition. In view of this, the degree of freedom for the column should be zero; that means the number of variables should be the same or equal to the number of equations involved in modeling. For example for a distillation column, if a designer specifies reflux ratio or boil up ratio and a distillate rate, then there will be corresponding unique set of distillate and bottom composition with respect to a fixed feed flow rate.