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.

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

Determination of the microbial community in pulp and paper mills effluents

Bu çalışmada, kâğıt endüstrisi atıksularını arıtan gerçek ölçekli bir anaerobik kontak reaktörün 3 farklı yüksekliğinden 2 farklı zamanda alınan çamur numunelerinin mikrobiyal komünite yapıları Denatüran Gradyan Jel Elektroforez (DGGE) yöntemi kullanılarak karşılaştırılmıştır. 2 aylık izleme dönemi içinde sistem 2 hafta süreyle bakıma alınmıştır. Kontak reaktörün 1.6-1.8 kg KOİ/m3.gün organik yükleme hızında, KOİ giderim verimi % 47-55, metan üretim verimi 0.18-0.20 m3CH4/kgKOİgiderilen aralığında değişmiştir. DGGE analizleri sonucu, arkeyal popülasyona ait 31, bakteriyel popülasyona ait 57 farklı tür tespit edilmiştir. Arkeyal popülasyona ait 3 farklı tür Ağustos 2005'te tespit edilememiş, buna karşın 6 yeni tür gözlenmiştir. Bakteriyel popülasyonda ise Temmuz 2005 numunesine ait 10 farklı tür Ağustos 2005 numunesinde tespit edilemezken Ağustos 2005'te 10 yeni türün varlığı gözlenmiştir. Bu çalışmada incelenen reaktöre ait asetoklastik metan üretim kapasitesi önceki bir çalışmada Spesifik Metan Aktivite (SMA) test düzeneği kullanılarak ölçülmüş ve potansiyel metan üretiminin yaklaşık % 45 azaldığı tespit edilmiştir. Sistemde bulunan metanojenlerin ve Sülfat İndirgeyici Bakterilerin (SRB) tür ve sayıları Floresanlı Yerinde Hibritleme (FISH) yöntemi ile belirlenmiştir. SMA testi ve FISH tekniği ile tespit edilen mikrobiyal komünite değişimi DGGE yöntemi ile de doğrulanmıştır. DGGE yöntemi, iki farklı zamanda alınan numunelere ait komünite değişimini açıkça yansıtmakla birlikte sayısal değerlendirmede yetersiz kalmaktadır. Bu nedenle, anaerobik reaktörlerin mikrobiyal komünite yapılarının gerek DGGE gibi detaylı kalitatif sonuç veren gerekse FISH gibi mikroskobik sayıma dayalı, kültürden bağımsız yöntemlerle çalışılması gerektiği sonucuna varılmıştır. Anahtar Kelimeler: Denatüran gradyan jel elektroforezi, arkeyal popülasyon, bakteriyel popülasyon, anaerobik kontak reaktör, kâğıt endüstrisi atıksuları.The use of anaerobic technologies in the fields of wastewater treatment, sludge stabilization, bioremediation and management of hazardous and solid wastes has grown in importance during the last few decades. Although the general processes occurring in anaerobic biological wastewater treatment plants, such as hydrolysis, fermentation, acetogenesis, methanogenesis and sulfidogenesis are well understood, the microbial community responsible for these conversions is often considered as a black box. Physical and chemical parameters only give rough estimations about the operational conditions of the system. Therefore, understanding the biodiversity and the dominant species of the microbial community is of great importance in studying contaminant degradation pathways, optimizing treatment processes, and improving removal efficiencies of engineer-designed systems. The culture dependent methods used for the investigation of the biomass are not sufficient for the identification of the complex microbial diversity in wastewater treatment systems. The use of the culture-independent methods in microbial ecology allowed the determination of the complex microbial populations and community contents more representatively. Denaturing Gradient Gel Electrophoresis (DGGE) is a Polymerase Chain Reaction (PCR) dependent method used for the electrophoretic separation of the 16S rDNA genes due to the difference in the nucleotide sequences. The separation is observed as an individual band on the DGGE gel. Every DGGE band represents a single species and the DGGE pattern gives the fingerprint of that community. Since DGGE technique allows the analysis of many samples simultaneously and gives rapid results, the use of DGGE is getting extensive in the investigation of bioreactors and natural ecosystems which inhabit rich microbial diversity. In the context of this study, a full-scale anaerobic contact reactor treating pulp and paper mills effluents was investigated. Samples were taken from 3 different levels at 2 different times. There was a 2-week off-period of the reactor between sampling times. Denaturing Gradient Gel Electrophoresis (DGGE) was used for the fingerprinting of the microbial community in the sludge samples. Performance of the reactor in terms of COD removal efficiency and methane yield varied between 47% and 55% and 0.18 and 0.20 m3CH4/kgCODremoved at Organic Loading Rates (OLRs) in a range of 1.6-1.8 kg COD/m3day, respectively. DGGE analysis revealed that 31 species from archaeal population and 57 species from bacterial population were present in the anaerobic reactor. 3 species from the archaeal population were not detected in August 2005 whereas 6 species were newly observed. In bacterial population, 10 species belonging to July 2005 samples were not detected where 10 other species were found in August 2005. Acetoclastic methanogenic activity of the reactor had previously been investigated by specific Methanogenic Activity (SMA) test. A decrease of 45% in the potential methane production was observed during the monitoring period of 2 months. The quantities and species of methanogens and Sulfate Reducing Bacteria (SRB) in the reactor were determined by Fluorescent In Situ Hybridization (FISH). Parallel to SMA results, the quantities of SRB and methanogens were decreased in August 2005. The shift in the microbial community observed by SMA test and FISH quantifications were supported by DGGE analysis. During the monitoring of 2 months, 2 weeks shut-down of the anaerobic reactor might have caused activity loss and microbial community change. DGGE allows the comparison of microbial communities taken from the anaerobic reactor at two different sampling times and FISH informs quantities of present microbial species in the reactor. However, DGGE does not give numerical information but clearly depicts the community shift. Therefore, it is concluded that, the microbial community structures of anaerobic reactors should be determined by culture independent methods of both qualitative and quantitative techniques such as DGGE and FISH respectively. Keywords: Denaturing gradient gel electrophoresis, archaeal population, bacterial population, anaerobic contact reactor, pulp and paper mills effluents

Effects of high-concentration influent suspended solids on aerobic granulation in pilot-scale sequencing batch reactors treating real domestic wastewater

The aim of this study was to investigate the effect of high-influent-concentration suspended solids (SS) on the cultivation, structure and long-term stability of aerobic granular sludge (AGS). Cultivation and long-term stability of AGS were monitored in two pilot-scale sequencing batch reactors fed with raw (R1) and settled (R2) domestic wastewater, representing high and medium SS content, respectively. The real domestic wastewater had high chemical oxygen demand (COD) content (1100 +/- 270 mg COD L-1). Aerobic granular sludge was cultivated in 44 days (R1) and 25 days (R2) under the conditions of high settling velocity (18 m h(-1)) and high organic loading rate (OLR) (2.1-2.4 kg COD m(3) day). The AGS in both reactors had similar structural properties during long-term operation and remained structurally and functionally stable during the last five months of operation. Comparative evaluation of the results indicated that the high influent SS content of the real domestic wastewater had a positive influence on maintaining significantly lower SVI30 and relatively lower effluent SS concentration. Moreover, a higher influent SS content resulted in smaller mature granules during the stable period. Microbial community analyses helped to understand the aerobic granular sludge structure and showed that the sludge retention time and OLR affected the granular sludge population. The high influent SS increased biomass detachment from the granular sludge surface and caused wash-out of some bacteria colonizing the exterior of the granular sludge. (C) 2017 Elsevier Ltd. All rights reserved

Changes in transferrin gene expression in sea bass (Dicentrarchus labrax) challenged with Vibrio anguillarum

Vibrio anguillarum expresses several virulence factors and causes hemorrhagic septicemia accompanied by serious losses in marine fish. Transferrin is a glycoprotein, also known as a multitasking protein, which is mainly synthesized by the liver. It has a fundamental role in the immune system. In the present study, the transferrin gene expression of sea bass (Dicentrarchus labrax) was investigated during an experimental infection with V. anguillarum. Fish samples were examined by hematological and serological methods as well as real-time polymerase chain reaction. The infection was performed via water. The sampled fish displayed vibriosis infection symptoms, both internally and externally. The transferrin saturation in the diseased fish serum decreased dramatically and the transferrin gene expression increased during the first 2 days; however, it decreased in the subsequent days