Süt endüstrisi atıksuyu ve organik evsel katı atığın hammadde olarak kullanılmasıyla karışık kültürden polihidroksialkanoat (PHA) eldesi
Polyhydroxyalkanoate (PHA) production from mixed microbial culture by using dairy wastewater and food waste as raw materials
Yazar: SELİN KARACA
Danışman: DOÇ. DR. NEVİN YAĞCI
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Çevre Mühendisliği Ana Bilim Dalı / Çevre Bilimleri, Mühendisliği ve Yönetimi Bilim Dalı
Derece: Yüksek Lisans, 2018
Konu: Biyoteknoloji = Biotechnology ; Çevre Mühendisliği = Environmental Engineering

Özet:
Petrol türevli plastikler yaygın kullanımlarına rağmen doğada çok uzun süreler bozunmadan kalması ve çevreye olan zararlarıyla bilinirler. Günümüzde bu plastiklerin yerini almaya aday olan biyoplastikler yenilenebilir biyokütle kaynaklarından elde edilen bir plastik türüdür fakat biyoplastikler yüksek maliyetlerinden dolayı dünyada halen yeterli endüstriyel üretim seviyesine ulaşamamışlardır. 
Bu sebeple bu yüksek lisans tez projesinin amacı biyoplastiklerin hammaddesi olan polihidroksialkanoat (PHA) üretiminde, PHA depolayan mikroorganizmaların karışık kültür içeren atık aktif çamurdan sağlanması, yüksek maliyetten sorumlu faktörlerden biri olan karbon kaynağı olarak atıksu ve/veya katı atıklar kullanılması ile hem maliyetin düşürülmesi hem de atıkların yenilenebilir kaynak olarak değerlendirilip atığın faydalı ürüne dönüştürülmesidir. 
Bu amaca yönelik olarak PHA depolanması, depolama miktarının arttırılması ve sistem konfigürasyonunun belirlenmesini hedefleyen deneysel bir çalışma yürütülmüştür. Bu çalışmada hammadde olarak süt endüstrisi atıksuyu ve evsel organik katı atık (gıda atığı) kullanılmıştır. Çalışma üç aşamada yürütülmüştür.
Birinci aşamada PHA depolayan mikroorganizma grubunun üretilebilmesi için sentetik evsel atıksu ile beslenen ve açlık-tokluk rejimi ve anaerobik-aerobik-anoksik rejimde işletilen iki ayrı ardışık kesikli reaktör (AKR) işletilmiştir. İşletme esnasında açlık-tokluk rejiminde 3 ve 6 gün, anaerobik-aerobik-anoksik rejimde 8 ve 12 gün olmak üzere ikişer farklı çamur yaşı denenmiştir. Her bir reaktör 96 gün çalıştırılmış ve 12 gün sentetik atıksuya UYA beslemesi yapılmıştır. Reaktörlerden atılan çamurda kesikli PHA depolama deneyleri gerçekleştirilmiştir.
İkinci aşamada hammadde kaynağı olarak seçilen süt endüstrisi atıksuyu ve organik katı atıkların asidifikasyonu amacıyla fermantasyon deneyleri yapılmıştır. Deneyler her iki karbon kaynağı için de farklı F/M oranlarında (1, 2,5, 5, 10) 6 gün yürütülmüş ve optimum değeri veren koşullar 4. gün ve F/M=10 olarak belirlenmiştir. Asidifikasyon deneyi sonuçlarına göre, seçilen koşullarda süt endüstrisi atıksuyunun üst suyundan 7,2 gKOİ/L, organik katı atık üstsuyundan ise 5,8 gKOİ/L uçucu yağ asidi (UYA) elde edilmiştir.
Üçüncü aşamada ise kültür seçilimi yapılan biyokütle ve ön deneylerin verilerine göre tekrarlanan fermentasyon deneylerinden elde edilen UYA, PHA üretimi için uygun işletme koşullarını araştırmak üzere kesikli deneyler yürütülmüştür. Kesikli deneyler toplamda 6 set için farklı F/M oranlarında denenmiş ilk iki sette elde edilen verilere göre değişkenler indirgenerek diğer 4 sete uygulanmıştır.
PHA üretiminde en yüksek verim organik katı atıktan sağlanmış olup kuru biyokütle ağırlığı başına %67,3 PHA içeriği açlık-tokluk rejimiyle zenginleştirilmiş çamur yaşı 3 gün olan biyokütle ile elde edilmiştir. Hammadde olarak süt endüstrisi atıksuyu kullanılması durumunda ise PHA içeriği %4,9 değerinde kalmış olup çamur yaşı 8 gün olan anaerobik-aerobik-anoksik biyokütleden elde edilmiştir.
Bu çalışmada, atıkların (özellikle gıda atıklarının) hammadde kaynağı olarak kullanılmasıyla, PHA depolama kabiliyeti olan alışmış atık aktif çamurun bulunduğu atıksu arıtma tesislerinde kurulabilecek fermentasyon sistemleri ile entegre çalışarak biyopolimer üretimi yapılabileceği gösterilmiştir.

Abstract:
Petroleum-derived plastics are known for their long-term deterioration in nature and their environmental hazards, despite their widespread use. Today, bioplastics, which are candidates for replacing these plastics, are a kind of plastic derived from renewable biomass sources, but bioplastics have not yet reached the sufficient level of industrial production in the world due to their high costs.
The purpose of this master thesis: in the production of polyhydroxyalkanoate (PHA) which is the raw material of bioplastics, PHA-storing microorganisms are produced from waste-activated sludge with mixed culture and the carbon source, which is one of the factors responsible for high cost, is obtained from wastewater and/or solid wastes.
For this purpose, an experimental study was conducted aiming to increase PHA storage, storage amount and system configuration. In this study, milk wastewater and domestic organic solid waste were used as raw materials. The study was carried out in three stages.
Firstly, in order to enrich PHA-storing microbial culture, two lab-scale activated sludge systems consisted of sequencing batch reactors (SBRs) with working volume of 4 L were operated parallel to offer feast/famine (FF) (SBR-1) and anaerobic/aerobic/anoxic (AnAA) (SBR-2) growth conditions. Each reactor was initially seeded with excess activated sludge obtained from a municipal wastewater treatment plant for enrichment of PHA storing community, and fed with synthetic wastewater reflecting similar characteristics and COD fractions with domestic wastewater.
In order to offer feast/famine (FF) environment, SBR-1 operated with 5 h reaction phase followed by 1 h of settling, decanting and idle periods to provide substrate gradient. Simultaneous feeding and aeration were initiated at the beginning of each cycle. The influent wastewater flow-rate of 12 L/d was fed into the reactor with tCOD concentration of around 500 mg COD/L during the operation period of SBR-1. The lab-scale reactor was operated for more than 90 days and 12 days VFA feeding duration in a constant temperature room maintained at 20±2°C under organic loading rate of 1.5 gCOD/L.d and sludge age of 3 and 6 days.
The experimental data required for the evaluation of PHA storage capability of anaerobic/aerobic growth conditions, SBR-2 was continuously operated in an anaerobic/aerobic/anoxic (AnA) sequence with a short post-aerobic phase to improve sludge settleability. This system was designed for organic carbon and nutrient removal. The reactor was operated in sequential cycles of 4 h: 60 min of anaerobic reaction including fast feeding of 5 min with a flow-rate of 15 L/d; 60 min of aeration; 45 min of anoxic; 15 min of aeration to improve setting properties of the sludge; 60 min of settling, decanting and idle. The COD concentration of the wastewater was around 400 mg COD/L during the operation period of SBR-2. The lab-scale reactor was operated for more than 90 days and 12 days VFA feeding duration in a constant temperature room maintained at 20±2°C under organic loading rate of 1.5 g COD/L.d, which is identical to the SBR-1, and sludge age of 8 and 12 days.
In the second stage, fermentation tests were carried out for acidification of milk wastewater and organic solid wastes, which were selected as two separate raw material sources. Fermentation experiments were performed in 1L glass bottles. The TS and VSS values of anaerobic inoculum sludge were 357 and 73 g/L, respectively. For dairy wastewater and organic solid waste, 7300 mg COD was calculated in each bottle. The experiments were carried out for both carbon sources at different F/M ratios (1, 2.5, 5, 10) for 6 days and the optimum values were determined as 4 days and F/M= 10. The VFA production was monitored during the tests by measuring acetic acid, propionic acid, valeric acid, butyric acid, isobutyric, valeric acid, isovaleric acid, caproic acid and heptanoic acid. Total VFA was calculated by conversion of each acid concentration to equivalent COD concentration. According to the results of the acidification test, 7.2 gCOD/L of dairy wastewater and 5.8 gCOD/L UYA for organic solid waste were obtained in the optimum condition.
In the third stage, the PHA production potential of the fermented dairy wastewater and organic food wastes was examined using acclimated activated sludge in SBRs as initial biomass under aerobic and anaerobic batch conditions. The food-to-microorganism (F/M) ratios used for these batch experiments were selected between 1-5 and identical for both VFA sources. The tests were initiated with the supernatant of acidified sources and biomass collected from sludge wastage line of each SBR systems. The acidification efficiencies were varied from batch to batch, thus the actual characterization was used for estimation of production of PHA derived from VFA produced in the experiments. VFA and PHA samples were collected every 30-60 minutes intervals for a period of 5h.
High concentrations of PHA were achieved with acidified food waste, while lower PHA concentration observed with acidified dairy wastewater during the entire accumulation tests. The highest yield in PHA production was obtained from organic solid waste and 67.3% PHA content per dry biomass weight was obtained by biomass enriched with FF regime with sludge age 3 day. In the case of the use of dairy wastewater as raw material, PHA content was 4.9%, and it was obtained from anaerobic-aerobic biomass with sludge age 8 day. The results show that PHA content of the activated sludge may be significantly different compered to F/M ratio and the source of fermented VFA. In this study, C / N = 21 for milk wastewater and C / N = 44 for organic solid waste. In terms of PHA production capacities, it was observed that organic solid waste with a high C / N ratio produced a higher yield of PHA. Due to high concentration of TKN in milk wastewater, nitrogen-restricted condition could not be achieved and PHA production was not seen as seen in organic solid waste.
According to data gathered in this study, production of 173 kg polymer from 1000 m3 of dairy wastewater, and 20.3 kg polymer from 1 tone of food waste is possible. The composition of resulting copolymer (PHBV) is suitable for the production of disposable items and food packaging materials.
This study shows that biopolymer production can be done by integrating the systems that can be installed in the wastewater treatment plants where the waste activated sludge will be obtained by using the wastes as the raw material source.