Hı̇droksı̇apatı̇t bazlı bı̇yomateryallerı̇n modı̇fı̇kasyonu ve denı̇z ortamında ağır metal gı̇derı̇mı̇ ve bı̇yofı̇lm oluşumuna yönelı̇k davranışları
Modification of hydroxyapatite from cattle bones with starch and l- cysteine to remove of heavy metal from environmental samples and limit the biofilm formation
Yazar: GÜLŞEN MELTEM
Danışman: PROF. DR. ASLI BAYSAL ; DOÇ. DR. SALİHA ESİN ÇELİK ; DOÇ. DR. NİL ÖZBEK
İstanbul Teknik Üniversitesi / Lisansüstü Eğitim Enstitüsü / Kimya Ana Bilim Dalı
Derece: Yüksek Lisans, 2024
Konu: Kimya = Chemistry

Özet:
"Ağır metaller" terimi yüksek yoğunluğa sahip olan herhangi bir metalik elementi ifade eder. Bazı ağır metaller eser miktarda canlı hayatında önemli bir konuma sahip iken düşük konsantrasyonda bile toksik veya zehirli olan birçok ağır metal bulunmaktadır. Ağır metal kirliliği günümüzün en önemli çevre sorunlarından biridir. Çevre hava, su, toprak gibi en somut ve yiyecek vb. gibi daha az somut olan fakat önemsiz olmayan birçok unsurları barındıran içerisinde yaşadığımız topluluklardır. Günümüzde gelişen teknoloji ile birlikte birçok endüstriyel işlemler sonucu ağır metal derişimi yüksek olan atıklar ve atıksular oluşmaktadır. Bu atıkların kontrolsüz bir şekilde çevreye salınması, ağır metallerin biyolojik olarak bozunabilme özelliklerinin sınırlı olması nedeniyle çevrede ve canlı vücudunda birikime sebep olarak hem çevre hem de canlı sağlığı açısından ciddi problemleri beraberinde getirmektedir. 
 
Günümüzde atıksulardan ağır metal giderimine yönelik birçok teknoloji (kimyasal çöktürme, iyon flotasyonu, iyon değişimi, koagulasyon ve topaklanma, elektroliz, membran ile ayırma, elektrokimyasal işlemler, nano teknoloji) bulunmaktadır. Fakat bu yöntemlerin uygulanabilirliği hem ekonomik hem de zaman açısından sınırlıdır. Son zamanlarda kolay, hızlı, ekonomik ve etkili ağır metal giderim potansiyeline sahip olan adsorpsiyon yötemi büyük ilgi görmektedir. Atıksulardan adsorpsiyon yöntemi ile ağır metal giderimi, gözenekliliği nedeniyle yüksek yüzey alanına sahip olan nano gözenekli adsorbanların yüzeyine ağır metallerin yapışması temeline dayanmaktadır. Adsorpsiyon prosesi bir veya daha fazla metal için diğerlerinden daha seçici olabilir.

Bu çalışmanın amacı ise adsorpsiyon yöntemi ile atıksulardan ağır metal giderimine yönelik etkili bir adsorban geliştirmektir. Bu amaç doğrultusunda, biyouyumlu bir materyal olan hidroksiapatit (Hap), farklı biyomoleküllerle (karbohidrat ve aminoasit) modifiye edilmiştir ve çevresel sulu çözeltilerden ağır metal giderim kapasitesi incelenmiştir. Ayrıca, modifiye edilmiş Hap sorbentin biyofilm oluşumunu farklı bakteriler kullanarak incelenmiştir.
Koyun kemiğinden elde edilen Hap, nişasta ve L-sistein biyomateryalleri ile modifiye edilerek kullanıma hazır hale getirilmiştir. Adsorpsiyon deneyleri için laboratuvar ortamında hazırlanan model bakır (Cu), krom (Cr) ve kurşun (Pb) çözeltilerindeki ağır metallerin bu sorbentler üzerindeki adsorpsiyonu Alevli Atomik Absorpsiyon Spektrofotometresi (FAAS) ile incelenmiştir. Sorpsiyona etki eden önemli paramatreler içerisinde bulunan pH, madde miktarı ve süre çalışılmıştır. 
Elde edilen sonuçlar doğrultusunda, her iki sorbentte de incelenen ağır metaller için 100 %'e yakın alıkonma kapasitesi pH 6'da gözlemlenmiştir. Madde miktarının adsorpsiyon kapasitesi üzerindeki etkisi ise her sorbent ve her ağır metal için değişkenlik göstermiştir. 0.20 g L-sistein-Hap sorbenti 92 % adsorpsiyon kapasitesi ile Cu giderimi sağlamıştır. Sürenin adsorpsiyon üzerinde ki etkisi incelendiğinde ise, Nişasta-Hap sorbenti her üç ağır metal için 90 dk çalkalama süresinde maksimum adsorpsiyon kapasitesi gösterirken, L-sistein-Hap sorbentinde bu durum 4 saat çalkalama süresinde gözlemlenmiştir. 
L-sistein-Hap ve Nişasta-Hap sorbentlerinin bakteriyel etkileşimi iki ayrı bakteri (Staphyloccus aureus (S.aureus) ve Pseudomonas aeruginosa (P. aeruginosa)) kullanılarak incelenmiştir. Her iki bakterinin iki ayrı sorbent ortamı içerisinde canlılık, biyofilm oluşumu, protein sentezi, antioksidan, süperoksit dismutaz (SOD) ve lipit peroksidaz (LPO) aktivite ölçümleri gerçekleştirilmiştir. Sonuçlar doğrultusunda sorbentler, iki farklı bakteriye karşı bakteriyel aktivitesi ve biyofilm oluşum özellikleri sınırlandırılmış özellikler göstermiştir.
Çalışma kapsamında çevre dostu, düşük maliyetli, Cr, Pb ve Cu ağır metallerine seçiciliği yüksek ve etkili olmakla birlikte bakteriyel etkileşimi sınırlandırılmış özelliklere sahip olan özgün adsorbanlar geliştirilmiştir.

Abstract:
"Heavy metals" refer to a subgroup of chemical elements characterized by their high density, toxicity, and tendency to accumulate in biological systems. Some common heavy metals include mercury, lead, arsenic, and cadmium. They can be released into the environment from various sources such as industrial activities, agricultural pesticides, and waste. These metals can have serious health effects on humans, including poisoning, cancer, neurological disorders, and environmental pollution. Therefore, prevention and control of heavy metal pollution are essential.


Heavy metals which are mentioned as metallic elements characterized by high densities, atomic weights, or atomic numbers. While certain heavy metals, such as iron, copper, and zinc, play crucial roles in biological processes at trace levels, many others are highly toxic or poisonous, even at low concentrations. Heavy metal pollution has emerged as a significant environmental issue, impacting both natural ecosystems and human health. The environment, which encompasses air, water, and food, as well as the less tangible but equally important communities we live in, is under threat from heavy metal contamination. Rapid industrialization and technological advancements have led to the generation of waste and wastewater containing high concentrations of heavy metals. The uncontrolled release of these wastes results in the accumulation of heavy metals in the environment and living organisms, posing severe ecological and health risks due to their limited biodegradability.


Various technologies have been developed to address heavy metal pollution, including chemical precipitation, ion flotation, ion exchange, coagulation and flocculation, electrolysis, membrane separation, electrochemical processes, and nanotechnology. Despite their effectiveness, these methods often face limitations regarding economic viability and practical implementation. In recent years, adsorption has gained attention as a promising method for heavy metal removal from wastewater. This technique is favored for its simplicity, cost-effectiveness, and efficiency. Adsorption relies on the adhesion of heavy metal ions onto the surface of nanoporous adsorbents, which offer high surface areas due to their porosity. The process can be tailored to selectively target specific metals, enhancing its applicability in various environmental contexts.


The primary aim of this study is to develop and characterize an effective adsorbent for the removal of heavy metals from wastewater using the adsorption method. Hydroxyapatite (Hap), can be used as an adsorbent. Hap is a biocompatible material, was chosen as the base adsorbent and modified with different biomolecules, including carbohydrates and amino acids, to enhance its heavy metal removal capacity. Additionally, the study investigates the biofilm formation properties of the modified Hap sorbents using different bacteria.


Hap is a naturally occurring mineral form of calcium apatite with the chemical formula Ca5(PO4)3(OH). It is the main mineral component of tooth enamel and bone. Hap crystals provide strength and rigidity to bones and teeth, making them crucial for structural support and protection. In addition to its natural occurrence, Hap is widely used in various applications. Hap was sourced from sheep bone and modified with starch and L-cysteine biomaterials to prepare the adsorbents. Starch is a carbohydrate polymer composed of glucose units linked together in a linear chain with occasional branching. It is the most abundant carbohydrate reserve in plants and serves as an essential source of energy for humans and animals.

The main types of starch are amylose and amylopectin, with amylopectin being highly branched and amylose having a more linear structure. The ratio of amylose to amylopectin varies among different starch sources and affects their properties, such as gelatinization temperature and texture. In addition to its culinary uses, starch has industrial applications in papermaking, heavy metal removal from aqueous systems and biodegradable plastics production. Moreover, starch is increasingly being explored as a renewable and sustainable source of biofuel and biochemicals, contributing to efforts to reduce reliance on fossil fuels and mitigate environmental impact.

L-cysteine is a non-essential amino acid, meaning it can be synthesized by the human body and is not solely reliant on dietary intake. It plays crucial roles in protein synthesis, detoxification, and the formation of important biological molecules such as glutathione, which acts as an antioxidant. One of the notable features of L-cysteine is its sulfhydryl group (-SH), which allows it to participate in various biochemical reactions, including the formation of disulfide bonds in proteins. These disulfide bonds contribute to the stability and structure of many proteins, including enzymes and antibodies. L-cysteine is found in various protein-rich foods such as meat, poultry, eggs, dairy products, and legumes. Additionally, it can be obtained through dietary supplements or synthesized industrially for use in food additives, cosmetics, and pharmaceuticals.

In the food industry, L-cysteine is commonly used as a dough conditioner and flavor enhancer, particularly in the production of baked goods such as bread and pastries. Its ability to break down gluten bonds helps improve dough elasticity and texture. Additionally, it can be used as a adsorbent for heavy metal removal from aqueous solutions. Beyond its culinary applications, L-cysteine is utilized in pharmaceutical formulations for its antioxidant properties and in cosmetic products for its skin-conditioning effects. However, its industrial production process, which often involves the use of human or animal hair, has raised ethical concerns, leading to the development of alternative production methods using microbial fermentation or plant-based sources. Overall, L-cysteine is a versatile amino acid with diverse applications in various industries, highlighting its importance in both biological and commercial contexts.

The adsorption experiments were conducted using model solutions of copper (Cu), chromium (Cr), and lead (Pb) prepared in the laboratory. The adsorption capacities of the modified Hap sorbents were assessed using Flame Atomic Absorption Spectrophotometry (FAAS). Key parameters affecting sorption, such as the amount of sorbent, pH, and contact time, were systematically studied to optimize the adsorption process. The experiments revealed that both modified sorbents exhibited nearly 100% retention capacity for the heavy metals at pH 6. The adsorption capacity varied depending on the type of sorbent and the specific heavy metal. For instance, the L-cysteine-Hap sorbent achieved a 92% removal rate for Cu with an optimal sorbent amount of 0.20 g. In terms of contact time, the Starch-Hap sorbent reached maximum adsorption capacity for all three heavy metals within 90 minutes of shaking, whereas the L-cysteine-Hap sorbent required 4 hours to achieve similar results.


The study also examined the interaction of the L-cysteine-Hap and Starch-Hap sorbents with two different bacteria: Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). Various biological assays, including viability, biofilm formation, protein synthesis, antioxidant activity, superoxide dismutase (SOD) activity, and lipid peroxidase (LPO) activity, were performed to evaluate the bacterial response to the sorbents. The results indicated that the sorbents exhibited distinct activities against the two bacteria, with limited biofilm formation observed in both cases. 


The modification of Hap with starch and L-cysteine significantly enhanced its heavy metal adsorption capabilities. The optimal conditions for heavy metal removal varied between the two modified sorbents, reflecting the influence of the specific biomolecules on the adsorption process. The high retention capacities observed at pH 6 suggest that the modified Hap sorbents can effectively remove heavy metals from aqueous solutions under mildly acidic conditions, which are common in industrial effluents.


The bacterial interaction studies provided valuable insights into the potential biocompatibility and antibacterial properties of the modified sorbents. The differential responses of S. aureus and P. aeruginosa to the sorbents highlight the importance of considering microbial interactions in the development of adsorbents for environmental applications. The limited biofilm formation observed suggests that the modified Hap sorbents may have potential applications in scenarios where bacterial contamination is a concern.


This study successfully developed a novel adsorbent based on hydroxyapatite modified with starch and L-cysteine, demonstrating high effectiveness in removing Cu, Cr, and Pb from wastewater. The modified sorbents are environmentally friendly, low-cost, and exhibit high selectivity and efficiency for heavy metal removal. Additionally, the sorbents showed limited bacterial interaction, making them suitable for applications where microbial contamination is a potential issue. Future research should focus on scaling up the production of these adsorbents and exploring their long-term stability and performance in real-world wastewater treatment scenarios. The integration of these adsorbents into existing treatment systems could provide a robust solution to the persistent problem of heavy metal pollution, contributing to the protection of both environmental and public health.