Control of biomass in anaerobic reactors using ultrafiltration membranes

PhD ThesisAnaerobic processing has become recognized as a simple and energy-efficient means of treating and stabilizing many high strength organic industrial wastes and is also not subjected to the operating limitations of aerobic processes. The literature review presented in this thesis outlines the advances made in the understanding of the microbiology and biochemistry of the process and the considerable advances in reactor configurations in achieving high SRTIHRT ratios. The efficacy of the anaerobic process is dependent on maintaining a high biomass concentration in the reactor, which in tum is dependent on the performance of the solids separator. The anaerobic contact process employs gravity settling for solid-liquid separation but the poor settleability of the anaerobic sludge may result in a poor performance of the contact process. By employing a much more efficient separation process such as ultrafiltration (UF), the performance of the anaerobic system can be significantly improved. In this study, therefore, a new method of operating a completely mixed anaerobic digester using a crossflow UF membrane technique was investigated in order to control the concentration of active biomass in the reactor and to determine the extent of any other advantages that can be gained over other reactor configurations. The study was carried out in four stages. In the first stage the performance of an anaerobic contact digester using a crossflow UF membrane technique was investigated for brewery wastewater treatment. The results obtained from this stage showed that under steady-state conditions, at an influent substrate concentration of approximately 120 g COD/l (100 g BOD/I) with a hydraulic retention time of 4.2 days giving an organic loading rate of 28.5 kg COO/m3.d , overall COO and BOD removal efficiencies of 99% and almost 100% respectively were achieved and the system had not reached its maximum loading capacity. Throughout the operation, HRT was maintained in the range of 2.5-4.2 days, largely determined by the flux rate. Microbiological analyses including Microscopic Count, Plate Count, Most Probable Number and Specific Methanogenic Activity test confirmed that there was almost no biomass loss through the membrane which, in tum, resulted in the maintenance of a high stability of the system under load changes. The UF membrane showed a remarkable consistency throughout the study. retaining a high concentration of active biomass in the digester and demonstrating that fouling by anaerobic biomass will reach a limiting level. In the second stage of the study the effect of Mixed Liquor Suspended Solids (MLSS) on the kinetics of the membrane reactor was investigated. The results showed that the kinetic coefficients estimated from the four steady-state runs had slight variations from each other but which could be mainly due to the changes in the numbers and the dominant species throughout the operation of the system. The increase in the MLSS concentrations did not significantly affect the kinetics of the system, In the third stage of the study the Specific Methanogenic Activity (SMA) technique was used to determine the methane production capacity of the membrane reactor, thus allowing suitable OLRs to be applied and to assess the effects of MLSS concentration on the activity of acetoclastic methanogenic bacteria in the digester. The results showed that any deterioration in acetoclastic methanogenic capacity of the system can be improved by increasing the sludge wastage rate. Ratios of the actual methane production rate to the potential methane production rate of less than 0.7 were found to be satisfactory in order to run the system efficiently in terms of COD removal and methane yield. In the final stage of the study the possible effects that membrane systems may have on the microbial population in the reactor was investigated. Therefore, microscopic examinations have frequently been carried out in order to determine the effects of the new configuration on any variation in the morphology or on the properties of methanogens as well as any change in the number of non-methanogens throughout the operation of the membrane reactor. This investigation showed that the membrane system configuration did have an apparent effect on the dominant methanogenic species throughout operation of the membrane reactor. For example Methanococcus species were the most dominant group at the beginning of the start-up period, becoming the third most dominant group at the end of the study. As a result, studying the changes in the number of viable methanogens and the dominant species may help to determine a reason for the deterioration in performance of a digester

Determination of Anaerobic and Anoxic Biodegradation Capacity of Sulfamethoxasole and the Effects on Mixed Microbial Culture

status: publishe

Effect of sludge age on the diversity of nitrification bacteria and reactor stability

Bir arıtma tesisinin başarısı büyük ölçüde arıtma işini yapacak mikrobiyal komünitenin oluşumuna bağlıdır. Fonksiyonel öneme sahip populasyonların kaybolması veya aktivitesini yitirmesi arıtma sisteminde verim kaybına yol açar. Kısaca, bir sistemin kararlı halde çalışması sahip olduğu mikroorganizma türlerinin sistemde kararlı halde tutulabilmesine bağlıdır.Yeterli miktarda ve ceşitlilikte mikroorganizmanın sistemde tutulabilmesi, optimum çamur yaşının belirlenebilmesi ile mümkündür. Ancak, son yıllarda yapılan çalışmalarla, çevre veya işletme koşullarında bir farklılık olmadığı durumlarda dahi arıtma sistemlerinin bir süre sonra stabilitesini kaybettiği gösterilmiştir; ancak stabil (kararlı) arıtma verimi elde etmek için, kararlı bir komünite yapısına sahip olmak gerektiği hala kesinlik kazanmamıştır. Bu durum, düşük büyüme hızına sahip nitrifikasyon bakterileri için daha fazla önem arzetmektedir. Bu çalışmada nitrifikasyon prosesi model olarak seçilmiş ve bu sistemlerin kararlı yapısı üzerine araştırma yapılmıştır. Büyüme hızı ile doğrudan ilişkilendirilmesi nedeniyle çamur yaşı bifürkasyon parametresi olarak seçilmiş ve laboratuar ölçekli atıksu arıtma reaktörleri farklı iki çamur yaşında işletilmiştir. Kimyasal analizler yanında moleküler analizler kullanılarak reaktörlerdeki nitrifikasyon bakterilerinin sayısı ve yapısındaki değişimler incelenmiştir. Elde edilen sonuçlar, replika reaktörlerde toplam amonyağı oksitleyici bakteri (AOB) sayısı açısından anlamlı bir ilişki olmadığını göstermiştir. Bunun yanında; 3 gün çamur yaşına sahip tüm reaktörlerde, 10 gün çamur yaşına sahip reaktörlere göre AOB çeşitliliğinin daha fazla olduğu, ancak zamana bağlı olarak çeşitliliğin azaldığı görülmüştür. Çeşitlilikteki bu azalma, yüksek çamur yaşında işletilen reaktörlerde daha fazla gözlenmiştir. Dizi analizi sonucu baskın olan türlerin Nitrosomonas türleri ve henüz kültüre alınmamış beta-proteobakteri olduğu bulunmuştur.  Anahtar Kelimeler: Atıksu arıtma, nitrifikasyon, stabilite,  FISH, DGGE. Variations in the relative abundance of microbial species are proved responsible of problems in biological treatment reactors. Quantitative ecology of wastewater treatment processes is the way of understanding these variations in community composition. In this point of view, many studies have been performed not only to understand the microbial structure of a wastewater treatment plants, but also to link microbial community dynamics to process stability. There are studies which investigated the instability caused by environmental conditions using lab- and full-scale treatment reactors. However, it is still unclear that functional stability implies a persistent community. Purkhold et al. (2000) found, for example, that few of the nitrifying bacteria classically studied in the laboratory were present in full-scale nitrification systems and functionally stable ecosystem. Saikaly et al. (2005) studied the stability of the treatment performance of laboratory scale sequencing batch reactors operated under different sludge ages. Results of the work by Fernandez et al. (1999) revealed that an extremely dynamic community sustains a more reliable treatment performance in terms of stable efficiency. However, similar studies revealed differences in the community structure of any changes in AOB community structure. However, it is not clear whether persistent community composition secures to get stable treatment performance. This is especially important for the microorganisms which have lower growth rate (e.g. nitrifiers) as they would be less abundant compared to others (e.g. heterotrophs). Loss or alteration of key functional groups might cause unstable treatment performance. Therefore, nitrification process is selected as a main investigation subject in the present study. Samples were collected for chemical analysis and also for molecular analysis. FISH and DGGE were applied to reveal total AOB numbers and any changes in community composition. To achieve this, two sets of replicate reactors were operated under identical conditions under two different sludge ages (3 and 10 days) with the same external conditions. During the whole investigation period, reactor 1 and 2 (3 days sludge age) achieved more than 88% COD removal whilse reactor 3 and 4 (10 days sludge age) achieved more than 96% COD removal. Results also showed that replicate reactors with high sludge age performed more stable nitrification performance than the reactors with low sludge age. Nitrification performance was assessed with amommium, nitrite and nitrate concentrations. In reactor 1 and 2, nitrate concentrations were in a range of 0 and 28.48 mg/l; whilst it was up to 40 mg/l in reactor 3 and 4. Nitrite concentration was maximum 10 mg/l in reactor 1 and 2; whereas no higher than 2.5 mg/l in reactor 3 nd 4 during the first 30 days of experiment. FISH quantification revealed no correlation in total AOB numbers within  replicate reactors. DGGE analysis also revealed decreasing similarities between replicate reactors over time. This was more obvious in reactors with higher sludge. On the day 32, the similarity values were 70.6% and 26.7% between reactor 1-2 and reactor 3-4, respectively. Beside statistical analysis, predominant bands were excised from DGGE gels and sequenced. Results showed that they match to the following sequences:  Nitrosomonas oligotropha (97.6 % similarity; accession number: AF272422); Nitrosomonas sp. IS79A3 (97.4% similarity, accession number: AJ621026) and uncultered beta-proteobacteria (higher than 97.7% similarity; accession numbers: AY062126, DQ413103, DQ376558, AY064177). Sequencing results were in aggrement with the literature which states that Nitrosomonas species are  dominant in wastewater treatment reactors. This study is of particular importance as there is still a gap in knowledge on performance stability and community composition in wastewater treatment reactors. Since, replicate reactors were run under identical conditions, our findings could be helpful to solve the link between performance and microbial diversity. Furthermore, we hope that our findings would  be in use for designing treatment reactors with new strategy. Keywords: Wastewater treatment, nitrification, stability, FISH, DGGE

Methanogenic population dynamics in full-scale UASB reactors

Bu çalışmada, alkol distilasyon atıksularını arıtan, kısaca IUASB, TUASB ve CUASB olarak adlandırılan üç farklı gerçek ölçekli yukarı akışlı anaerobik çamur yatağı (UASB) reaktörünün 2002-2004 yılları arasındaki işletme performansları, Metan Arke komunite yapıları ve Potansiyel Metan Üretim (PMÜ) hızları tartışılmıştır. Aynı süreçte UASB reaktörlerinden 2-12 kg KOİ/m3.gün aralığındaki organik yükleme hızlarında, %60-95 aralığında KOİ giderim verimleri elde edilmiştir. Spesifik Metan Aktivite (SMA) test sonuçları IUASB, TUASB ve CUASB reaktörlerinin PMÜ hızlarının 2002 yılında sırasıyla 321, 344 ve 256 mL CH4/gUAKM.gün iken, 2004 yılında  sırasıyla 133, 109 ve 108 mL CH4/gUAKM.gün değerlerine düştüğünü göstermiştir. Reaktörlerden elde edilen gerçek metan üretim (GMÜ) hızları, reaktör çamurlarının PMÜ hızları ile oranlandığında elde edilen değerler, 0.1-0.4, reaktörlerin maksimum kapasitelerinin çok altında yüklendiklerini göstermektedir. Floresanlı yerinde hibritleşme (FISH) sonuçları her üç reaktörde de baskın metanojenlerin, asetoklastik bir cins olan Methanasaeta’ya ait olduğunu göstermiştir. Hidrojen kullanan metan arkelerinden Methanobacteriales TUASB ve CUASB reaktörlerinde, Methanococcales ise IUASB reaktöründe baskın halde bulunmaktadır. IUASB ve TUASB reaktörlerinin asetoklastik metan üretim kapasitelerinde meydana gelen, sırasıyla %59 ve %68’lik kayıplara pararel olarak, reaktörlerde rastlanan tek asetoklastik cins olan Methanosaeta’nın rölatif miktarında sırasıyla %25 ve %11’lik kayıplar, hidrojen kullanan metanojenlerin rölatif miktarlarında ise sırasıyla %20 ve %24’lük bir artış meydana gelmiştir. CUASB reaktöründeki metanojenik komünite yapısı zaman içerisinde stabil kalmıştır. Anahtar Kelimeler: Floresanlı yerinde hibritleşme, spesifik metan aktivite testi, metanojenler, UASB reaktör, alkollü içki endüstrisi atıksuları.A prerequisite for stable performance in an anaerobic treatment system is maintenance of active methanogenic populations in the system. However, there are few published studies available that assess performance of a full-scale anaerobic reactor in relation to the make up of reactor biomass in terms of qualitative and quantitative measures of methanogenic species and their activities. This study was undertaken to assess changes in performance of three full-scale upflow anaerobic sludge blanket (UASB) reactors, namely IUASB, TUASB and CUASB, at wastewater treatment plants of Istanbul Alcohol (Raki), Tekirdag Alcohol (Raki) and Canakkale Alcohol (Cognac) distilleries in relation to qualitative and quantitative measures of the relevant archaeal methanogenic populations. Composition and quantity of archaeal methanogens were determined using fluorescent in situ hybridization (FISH) combined with epifluorescence microscopy. Specific methanogenic activity (SMA) test was used to determine the potential methane production (PMP) rates of the anaerobic sludges. The IUASB reactor performed well achieving COD removal efficiencies of no lower than 80% at a range of OLRs from 6-11 kg COD m-3 day-1 in years between between 2001 and 2004. The TUASB reactor also performed well achieving COD removal efficiencies between 70% and 85% at OLRs in a range of 2-12 kg COD m-3 day-1 between 2001 and 2004. COD removal efficiency of the TUASB reactor varied between 60% and 80% at OLRs in a range of 2.5-12 kg COD m-3 day-1 between 2002 and 2004. According to the SMA tests results PMP rates of the IUASB, TUASB and CUASB sludges were 321, 344 and 256 mL CH4 gVSS-1 day-1 respectively in the year 2002 and decreased to 133, 109 and 108 CH4 gVSS-1 day-1 respectively after two years of operation. When the PMP rates were compared with actual methane production (AMP) rates obtained from the three UASB reactors, AMP/PMP ratios were evaluated to be between 0.1 and 0.4. These results can be interpreted that the three UASB reactors were under loaded compared to their potential acetoclastic methanogenic capacities. This could have been due to retaining high amount of granular sludges within the UASB reactors resulting in F/M (food to microorganisms) ratios in a range of 0.02-0.07 gCOD gTVS-1 day-1 which is much lower than the typical values reported for similar reactors in literature. Since all other operational parameters such as pH, temperature, alkalinity, nutrients etc. have been maintained within their typical ranges, it was concluded that the prolonged operation of  the three UASB reactors under very low F/M ratios might have lead to the significant decreases in the PMP rates of the reactor sludges. FISH results revealed that the relative abundance of archaeal cells within the IUASB, TUASB and CUASB sludges were in range of 14-18%, 15-17% and 14-15% respectively. Methanosaeta spp. were the predominant methanogen in all of the anaerobic sludges. However, over time the relative abundance of acetoclastic Methanosaeta in the IUASB and TUASB reactors reduced from 83% ± 1.6 and 90% ± 1.2 to 58% ± 2.1 and 79% ± 1.4 of the archaeal population respectively. These decreases coincided with 59% and 68% decreases in the acetoclastic methanogenic capacities and 20% and 24% increases in the relative abundance of hydrogenotrophic methanogens in the archaeal population of the IUASB and TUASB sludges respectively. Although a 58% decrease in the acetoclastic methanogenic capacity of the CUASB sludge was detected, the reactor sludge had stable archaeal community structure. Among the hydrogenotrophic methanogens, Methanococcales followed by Methanobacteriales were dominant methanogens within the IUASB reactor. This study is the first that reports dominance of Methanococcales among the hydrogenotrophic methanogens within UASB reactors. Methanobacteriales was the predominant hydrogenotrophic methanogen within the both TUASB and CUASB reactors. In this study the significant decreases in the activity of methanogens was detected at an early stage by using the SMA test so that process conditions can be changed, and collapse of the reactors can be avoided. The SMA test and FISH results showed that the reduction of acetoclastic methanogenic activity in IUASB and TUASB reactors is related to loss of Methanosaeta spp. However, acetoclastic methanogenic activity losses in the CUASB reactor were not reflected in the archaeal community structure of  the reactor sludge. Keywords: Fluorescence in situ hybridization, specific methanogenic activity, methanogens, UASB reactor, alcohol distillery effluents

Effect of sludge age on the diversity of nitrification bacteria and reactor stability

Bir arıtma tesisinin başarısı büyük ölçüde arıtma işini yapacak mikrobiyal komünitenin oluşumuna bağlıdır. Fonksiyonel öneme sahip populasyonların kaybolması veya aktivitesini yitirmesi arıtma sisteminde verim kaybına yol açar. Kısaca, bir sistemin kararlı halde çalışması sahip olduğu mikroorganizma türlerinin sistemde kararlı halde tutulabilmesine bağlıdır.Yeterli miktarda ve ceşitlilikte mikroorganizmanın sistemde tutulabilmesi, optimum çamur yaşının belirlenebilmesi ile mümkündür. Ancak, son yıllarda yapılan çalışmalarla, çevre veya işletme koşullarında bir farklılık olmadığı durumlarda dahi arıtma sistemlerinin bir süre sonra stabilitesini kaybettiği gösterilmiştir; ancak stabil (kararlı) arıtma verimi elde etmek için, kararlı bir komünite yapısına sahip olmak gerektiği hala kesinlik kazanmamıştır. Bu durum, düşük büyüme hızına sahip nitrifikasyon bakterileri için daha fazla önem arz etmektedir. Bu çalışmada nitrifikasyon prosesi model olarak seçilmiş ve bu sistemlerin kararlı yapısı üzerine araştırma yapılmıştır. Büyüme hızı ile doğrudan ilişkilendirilmesi nedeniyle çamur yaşı bifürkasyon parametresi olarak seçilmiş ve laboratuar ölçekli atıksu arıtma reaktörleri farklı iki çamur yaşında işletilmiştir. Kimyasal analizler yanında moleküler analizler kullanılarak reaktörlerdeki nitrifikas-yon bakterilerinin sayısı ve yapısındaki değişimler incelenmiştir. Elde edilen sonuçlar, replika reaktörlerde toplam Amonyağı Oksitleyici Bakteri (AOB) sayısı açısından anlamlı bir ilişki olmadığını göstermiştir. Bunun yanında; bütün 3 gün çamur yaşına sahip reaktörlerde, 10 gün çamur yaşına sahip reaktörlere göre AOB çeşitliliğinin daha fazla olduğu, ancak zamana bağlı olarak çeşitliliğin azaldığı görülmüştür. Çeşitlilikteki bu azalma, yüksek çamur yaşında işletilen reaktörlerde daha fazla gözlenmiştir. Dizi analizi sonucu baskın olan türlerin Nitrosomonas türleri ve henüz kültüre alınmamış beta-proteobakteri olduğu bulunmuştur. Anahtar Kelimeler: Atıksu arıtma, nitrifikasyon, stabilite, FISH, DGGE.Variations in the relative abundance of microbial species are proved responsible of problems in biological treatment reactors. Quantitative ecology of wastewater treatment processes is the way of understanding these variations in community composition. In this point of view, many studies have been performed not only to understand the microbial structure of a wastewater treatment plants, but also to link microbial community dynamics to process stability. There are studies which investigated the instability caused by environmental conditions using lab- and full-scale treatment reactors. However, it is still unclear that functional stability implies a persistent community. Purkhold et al. (2000) found, for example, that few of the nitrifying bacteria classically studied in the laboratory were present in full-scale nitrification systems. and functionally stable ecosystem. Saikaly et al. (2005) studied the stability of the treatment performance of laboratory scale sequencing batch reactors operated under different sludge ages. Results of the work by Fernandez et al. (1999) revealed that an extremely dynamic community sustains a more reliable treatment performance in terms of stable efficiency. However, similar studies revealed differences in the community structure of any changes in AOB community structure. However, it is not clear whether persistent community composition secures to get stable treatment performance. This is especially important for the microorganisms which have lower growth rate (e.g. nitrifiers) as they would be less abundant compared to others (e.g. heterotrophs). Loss or alteration of key functional groups might cause unstable treatment performance. Therefore, nitrification process is selected as a main investigation subject in the present study. Samples were collected for chemical analysis and also for molecular analysis. FISH and DGGE were applied to reveal total AOB numbers and any changes in community composition. To achieve this, two sets of replicate reactors were operated under identical conditions under two different sludge ages (3 and 10 days) with the same external conditions. During the whole investigation period, reactor 1 and 2 (3 days sludge age) achieved more than 88% COD removal whilst reactor 3 and 4 (10 days sludge age) achieved more than 96% COD removal. Results also showed that replicate reactors with high sludge age performed more stable nitrification performance than the reactors with low sludge age. Nitrification performance was assessed with ammonium, nitrite and nitrate concentrations. In reactor 1 and 2, nitrate concentrations were in a range of 0 and 28.48 mg/L; whilst it was up to 40 mg/L in reactor 3 and 4. Nitrite concentration was maximum 10 mg/L in reactor 1 and 2; whereas no higher than 2.5 mg/L in reactor 3 and 4 during the first 30 days of experiment. FISH quantification revealed no correlation in total AOB numbers within replicate reactors. DGGE analysis also revealed decreasing similarities between replicate reactors over time. This was more obvious in reactors with higher sludge. On the day 32, the similarity values were 70.6% and 26.7% between reactor 1-2 and reactor 3-4, respectively. Beside statistical analysis, predominant bands were excised from DGGE gels and sequenced. Results showed that they match to the following sequences: Nitrosomonas oligotropha (97.6 % similarity; accession number: AF272422); Nitrosomonas sp. IS79A3 (97.4% similarity,accession number: AJ621026) and uncultered beta-proteobacteria (higher than 97.7% similarity; accession numbers: AY062126, DQ413103, DQ376558, AY064177). Sequencing results were in aggrement with the literature which states that Nitrosomonas species are dominant in wastewater treatment reactors. This study is of particular importance as there is still a gap in knowledge on performance stability and community composition in wastewater treatment reactors. Since, replicate reactors were run under identical conditions, our findings could be helpful to solve the link between performance and microbial diversity. Furthermore, we hope that our findings would be in use for designing treatment reactors with new strategy. Keywords: Wastewater treatment, nitrification, stability, FISH, DGGE

Nutrient enhanced bioremediation of petroleum hydrocarbons in anoxic marine sediments

Marmara Denizi petrol hidrokarbonlarıyla yoğun bir şeklide kirletilmektedir. Bu kronik kirliliğin giderilmesi için sürdürülebilir, az insan müdahelesi gerektiren ve ekonomik bir ıslah stratejisi geliştirilmesi şarttır. Eğer Marmara Denizi sedimentlerinde anaerobik hidrokarbon ayrıştırıcı mikroorganizmalar aktif bir şekilde bulunuyorsa ve aktivitelerini arttırmanın bir yolu bulunabilirse, kirliliğin giderilmesi için en iyi yöntem anaerobik koşullarda biyoıslah uygulanmasıdır. Anoksik koşulların hâkim olduğu, petrol hidrokarbonlarıyla aşırı şekilde kirletilmiş olan Haliç Körfezi sediment süzüntü sularında mevcut N ve P seviyeleri mikrobiyal çoğalmayı destekleyecek seviyelerin çok altındadır. Nutrient takviyesi ile sedimentlerin mikrobiyal aktivitelerinin arttırılabilirliğini sınamak için anaerobik koşullar altında hidrokarbon ayrışım mikrokozmosları kurulmuştur. Mikrokosmaslardaki nutrient seviyelerinin doğal seviyelerden başlanarak giderek arttırılması, hidrokarbon ayrıştırma aktivitesinde ~9× artışla sonuçlanmıştır. Sedimentlerin doğal hidrokarbon içeriklerinin tümü bu şekilde giderilebilmiştir. Sedimentler bir çok farklı aromatik (18 farklı 1-5 halka aromatikler) ve alifatik (n-C9-31 alkanlar ve asiklik isoprenoidler) hidrokarbonları ayrıştırabilmiştir. Mikrokozmoslarda metanojenesis ve dissimilatif sülfat indirgenmesi prosesleri hidrokarbonların ayrıştırımasında rol almıştır. Sonuç olarak, bu çalışma Haliç Körfezi’ndeki yoğun ve kronik kirliliğin, sediment organizmalarının aktivitelerinin nutrient takviyesi ile arttırılması yoluyla giderilebilmesinin mümkün olduğunu göstermiştir. Bu çalışmanın çıktıları, daha az maliyet ve insan müdahalesi gerektiren biyoıslah uygulamalarının dünya çapında uygulanmaya başlanmasına öncülük edecek niteliktedir. Anahtar Kelimeler: Petrol hidrokarbonları, deniz kirliliği, biyoıslah, Haliç Körfezi.Anoxic Halic Bay sediments have been extremely polluted with petroleum hydrocarbons, and N and P are limited in the sediment porewaters for biological activity. These raised the question that hydrocarbon degradation activity of Halic Bay sediments can be increased by N-P amendment under anaerobic conditions to overcome the pollution. In this study, anaerobic hydrocarbon degradation microcosms were set up to increase activity of Halic Bay sediments by gradually decreasing the natural TOC/N/P ratio of the sediment porewaters to the unlimited nutrient conditions. Microcosms were set up in an anaerobic cabinet with a regulated atmosphere of nitrogen. Each microcosm was fed with a defined substrate mix consisting of 31 different hydrocarbon types. Two control microcosms were included: (1) hydrocarbon mix was not added to determine the extent of anaerobic degradation on natural hydrocarbon content of the sediments; (2) NaN3 treatment was applied to suppress microbial activity. The anaerobic microcosms were incubated for 224 days. Decreasing the natural TOC/N/P ratio of the sediment porewater (1000/5/1) to 1000/40/6 resulted in ~9× increase in gas production (CH4+CO2) and hydrocarbon removal. Addition of external hydrocarbons to the microcosms was also resulted in ~2× higher gas production and hydrocarbon removal. A high proportion (92 %) of aromatic hydrocarbons and all n-alkanes were removed from the microcosms under unlimited nutrient supply conditions without external hydrocarbon addition. The sediment microorganisms degraded wide range of aliphatic (n-C9-31 alkanes and acyclic isoprenoids) and aromatic (18 different 1-5 ring aromatics) hydrocarbons. The anaerobic degradation hierarchy of hydrocarbons was as follows: the most easily degradable n-alkanes, followed by more resistant branched acyclic and monocyclic hydrocarbons, the most resistant polycyclic steroidal and triterpenoidal hydrocarbons, and aromatic hydrocarbons. Monitoring functional gene and transcript abundances revealed that methanogenesis and dissimilatory sulfate reduction took place simultaneously during the first 126 days, afterwards, only the syntrophic methanogenic consortium was active. Microbial activity and abundance were very high and related to the C removal all through the incubation period. Archaea abundance increases were ~1.5× higher than those of Bacteria. 74 and 67 % of the total C removal occurred between the days 126 and 168 during which period microbial activity levels increased 6-8×. Overall microbiological results implied that C removal in this period can be attributable to activities of syntrophic consortium of fermentative bacteria and methanogens. In this study, we obtained anaerobic hydrocarbon degradation rates (700 ug/gSediment.L.day) as fast as enrichment cultures' rates. Although the obtained rates were comparable to the aerobic ones, they are still much lower than the aerobic hydrocarbon degradation rates. On the other hand, an aerobic bioremediation strategy is unfeasible for Halic Bay since oxygen penetration into the anoxic sediments is poor and oxygen mass transfer enhancement by mechanical means is inappropriate for the inaccessible sediments. Under these conditions anaerobic hydrocarbon degradation is the only alternative. In summary, we have obtained three lines of evidence for demonstrating anaerobic bioremediation feasibility of petroleum HC pollution in Halic Bay sediments: (1) the anaerobic hydrocarbon degrading microbiota was highly abundant in the sediments; (2) anaerobic hydrocarbon degradation was taking place in the sediments; (3) the sediments were able to degrade wide range of hydrocarbons under anaerobic conditions; (4) high anaerobic hydrocarbon degradation rates were achieved via biostimulation of the sediments through nutrient amendment. We are now making the preliminary preparations to carry out a field-scale bioremediation trial to remove the accumulated hydrocarbons from the subsurface of Halic Bay through biostimulation of the sediments. Success of this trial will certainly lead to less human intervened and more economical field-scale bioremediation applications for over polluted anoxic marine environments worldwide. Keywords: Petroleum hydrocarbons, marine pollution, bioremediation, Haliç Bay.

A novel activated sludge process for production of bioplastic from nitrogen deficient wastewaters

Bu çalışmada, biyolojik olarak tümüyle ayrışabilir nitelikte poliesterler oldukları için biyoplastik olarak bilinen malzemenin hammaddesi olarak kullanılan polihidroksialkanoatların (PHA), karışık mikrobiyal kültürler tarafından, farklı işleteme koşullarındaki üretimleri karşılaştırılmıştır. Biyokütle zenginleştirmek amacıyla aerobik dinamik besleme (ADB) koşullarında üç ardışık kesikli reaktör (AKR) işletilmiş ve AKR çevrimi süresince azot varlığının popülasyon dinamiklerine ve seçilen biyokütlenin depolama yeteneğine etkisi araştırılmıştır. Reaktörlerden biri ilk defa bu çalışmada önerilen ve gecikmiş azot besleme (GAB) olarak adlandırılan bir yöntemle beslenmiştir. Azot ve karbonun bir arada bulunmalarının engellendiği bu prosesten alınan biyokütle ile gerçekleştirilen kesikli deneylerde, elde edilen spesifik polimer depolama hızı, substratın polimere dönüşüm oranı, depolanan polimer miktarı ve biyokütlenin polimer içeriğinin genellikle daha yüksek olduğu gözlenmiştir. Her bir AKR’deki bakteriyel çeşitlilikte gözlenen değişim farklılık gösterse de β-proteobacteria’nın Rhodocyclaceae ailesine bağlı türler, özellikle de Zoogloae her üç reaktörde de sürekli baskın olmuştur. AKR’lerden alınan biyokütle tarafından depolanan polimer konsantrasyonu, artan substrat yüklemesine bağlı olarak artmıştır. Azotsuz olarak gerçekleştirilen deneylerde artan substrat yüklemeleri spesifik polymer depolama hızında artışa yol açarken, azotla gerçekleştirilen deneylerde bu durum depolama hızında düşüşe yol açmıştır. Substratın polimere dönüşüm oranı her üç biyokütle için de artan substrat yüklemesine bağlı olarak düşmüştür. AKR işletimi sırasında ve bu reaktörden alınan çamur ile gerçekleştirilen deneyler sırasında uygulanan koşullar arasındaki uyumun polimer depolamasına olumlu yönde etki ettiği belirlenmiştir. Substrat türü ile depolanan polimerin yapısı arasında sıkı bir ilişki olduğu tespit edilmiştir. Anahtar Kelimeler: Aktif çamur, polihidroksialkanoatlar (PHA), biyoplastik, aerobik dinamik besleme (ADB), gecikmiş azot besleme (GAB).Polyhydroxyalkanoates (PHAs), which are biologically-derived and completely biodegradable polyesters, represents a potentially sustainable substitution to synthetic polymers known as plastics. Currently, high production and recovery costs are the main limitations for the bulk production of bioplastics. PHA production processes based on mixed microbial cultures, such as activated sludge systems, are being investigated as a possible technology to decrease production costs. In activated sludge systems no sterilization is required and bacteria can adapt quite well to the complex and cheap substrates, such as wastewaters. To understand the impact of different enrichment strategies on PHA production, and population dynamics is an obligation because selection of organisms with high storage ability is one of the most critical factors effecting on development of a competitive process for PHA production based on mixed cultures. In this study, two sequencing batch reactors (SBR) were operated under aerobic dynamic feeding (ADF) conditions for biomass enrichment in order to investigate the effect of nitrogen (N) availability during a SBR cycle, on PHA accumulation ability of selected biomass. In one of the reactors nitrogen was depleted completely together with carbon source at the end of feast phase. The second SBR was operated with delayed nitrogen feeding (DNF) strategy which was proposed in this study. In this feeding regime synthetic wastewater without nitrogen was fed to the SBR and nitrogen source was fed to the reactor following substrate depletion to hinder being substrate and ammonia simultaneously in the reactor. Changes in polymer storage ability of two biomasses were determined in terms of specific polymer storage rate, yield of polymer on substrate consumed, amount of polymer accumulated, and biomass polymer content. Polymer storage ability of biomasses enriched under ADF conditions were considerably higher when compared to those obtained for inoculum sludge. Substrate was accumulated mainly in the form of Polyhydroxybutyrate (PHB) because acetate was supplied as sole carbon source. Experimental data showed that, throughout biomass enrichment, nitrogen restraint during substrate uptake stimulated polymer accumulation. Accordingly, polymer content of biomass enriched under dynamic conditions with DNF and also polymer yield and polymer uptake rate obtained for this biomass was higher. Amounts of PHAs accumulated were 199.5 and 234.9 mg/L on COD basis during cycles of SBRs operated without and with DNF respectively. Accordingly, yields of polymers on substrate consumed were 0.61 and 0.71 Cmmol PHA/Cmmol HAc for SBRs operated without and with DNF respectively. Specific polymer storage rate of biomass enriched under DNF conditions increased to 0.283 Cmmol PHA/Cmmol X.h, whereas that of biomass enriched under ADF conditions increased to 0.383 Cmmol PHA/Cmmol X.h. Two batch experiments were conducted by using two different biomasses and applying a substrate loading of 0.4 g COD S/g COD X, which was four times of applied during SBR operation. Results obtained from batch experiments showed that concentrations of polymer accumulated by both sludges increased directly with substrate loading and higher polymer accumulation obtained for the biomass enriched under delayed nitrogen feeding conditions. Sludge polymer content (41.2% on COD basis) obtained for the biomass enriched under these conditions was also higher than that obtained for biomass enriched under ADF conditions. Yield of polymer on substrate consumed decreased with substrate loading for both sludges. Substrate was accumulated mainly in the form of hydroxybutyrate (HB) because acetate was supplied as sole carbon source. Restriction of nitrogen availability during substrate uptake improved polymer storage ability of biomass. Among two different enrichment strategies, DNF process, which was proposed first time in this study, was found to be more effective. If this process optimized and combined with other strategies, such as pulsewise feeding control, it can be a stronger alternative to industrial production of PHAs achieved by pure cultures. Ammonia deficient organic wastes can be used as a cheap carbon source in this process for PHA production after a fermentation process. Keywords: Activated sludge, polyhydroxyalkanoates (PHA), bioplastic, Aerobic Dynamic Feeding (ADF), Delayed Nitrogen Feeding (DNF)