Stability and reproducibility of low-temperature anaerobic biological wastewater treatment

The reproducibility of low-temperature anaerobic biological wastewater treatment trials was evaluated. Two identical anaerobic expanded granular sludge bed bioreactors were used to treat synthetic volatile fatty acid-based industrial wastewater under ambient conditions (18-20 degrees C) and to investigate the effect of various environmental perturbations on reactor performance and microbial community dynamics, which were assessed by chemical oxygen demand removal or effluent volatile fatty acid determination and terminal restriction fragment length polymorphism analysis, respectively. Methanogenic activity was monitored using specific methanogenic activity assays. Reactor performance and microbial community dynamics were each well replicated between Reactor 1 and Reactor 2. Archaeal dynamics, in particular, were associated with reactor operating parameters. Terminal restriction fragment length polymorphism data suggested dynamic acetoclastic and hydrogenophilic methanogenic populations and were in agreement with temporal specific methanogenic activity data. Putative psychrophilic populations were observed in anaerobic bioreactor sludge for the first time.     

Electromigration, a tool for studies on anaerobic ciliates

Abstract By applying electric current, anaerobic ciliates could be extracted from sludge samples. All the freshwater ciliates tested had one point of optimal current and voltage where they reached the highest net speed of migration in the electric field. The swimming behavior of Metopus es was tested under different current and voltage conditions. During electromigration the freshwater, marine and rumen ciliates moved from the anode to the cathode. In the anaerobic freshwater ciliates high numbersof methanogenic bacteria of different size were present. At the depth in the sludge with the highest number of Metopus minimus (660 cells ·ml⁻¹, a peak of methane production also occurred.     

Effect of seed sludge and operation conditions on performance and archaeal community structure of low-temperature anaerobic solvent-degrading bioreactors

Two laboratory-scale expanded granular sludge bed (EGSB) anaerobic bioreactors (R1 and R2) were inoculated with biomass from different mesophilic (37 °C) treatment plants, and used for the treatment of an organic solvent-based wastewater at 9–14 °C at applied organic loading rates (OLRs) of 1.2–3.6 kg chemical oxygen demand (COD) m⁻³ d⁻¹. Replicated treatment performance was observed at 10–14 °C, which suggested the feasibility of the process at pilot-scale. Stable and efficient COD removal, along with high methane productivity, was demonstrated at 9 °C at an applied OLR of 2.4 kg COD m⁻³ d⁻¹. Clonal libraries and fluorescence in situ hybridization (FISH) indicated that the seed sludges were dominated (>60%) by acetoclastic Methanosaeta-like organisms. Specific methanogenic activity (SMA) profiles indicated shifts in the physiological profiles of R1 and R2 biomass, including the development of psychrotolerant methanogenic activity. Acetoclastic methanogenesis represented the primary route of methane production in R1 and R2, which is in contrast with several previous reports from low-temperature bioreactor trials. A reduction in the abundance of Methanosaeta-like clones (R2), along with the detection of hydrogenotrophic methanogenic species, coincided with altered granule (sludge) morphology and the development of hydrogenotrophic SMA after prolonged operation at 9 °C.     

Quantitative image analysis as a diagnostic tool for monitoring structural changes of anaerobic granular sludge during detergent shock loads

Two shock loads of a commercial detergent (I-150 mg chemical oxygen demand (COD)/L, fed for 56 h; II-300 mg COD/L fed for 222 h) were applied in a lab-scale Expanded Granular Sludge Blanket (EGSB) reactor, fed with 1,500 mg COD/L of ethanol. The impact of the surfactant was assessed in terms of granular sludge morphology, specific methanogenic activity (SMA) in the presence of individual substrates, and reactor performance. COD removal efficiency remained unaffected in the shock I, but 80 h after starting exposure to the shock II, the COD removal efficiency decreased drastically from 75 to 17%. In the first 8 h of operation of shock I, the SMA was stimulated and decreased afterwards, being recovered 5 days after the end of exposure time. Concerning to shock II, the SMA was immediately and persistently reduced during the exposure time, although, the inhibition of SMA in presence of H(2)/CO(2) showed a trend to increase after the exposure time. Acetoclastic bacteria were observed as the most sensitive to the toxic effects of surfactant whereas the hydrogenotrophic bacteria were less affected. The inhibitory effects were dependent on surfactant concentration and exposure time. The ratio filaments length per total aggregates area (LfA) was an early-warning indicator of biomass washout, since it increased 3 and 5 days before effluent volatile suspended solids (VSS) rise, respectively, in shocks I and II.     

Cultivation of low-temperature (15°C), anaerobic, wastewater treatment granules

Aims: Anaerobic sludge granules underpin high‐rate waste‐to‐energy bioreactors. Granulation is a microbiological phenomenon involving the self‐immobilization of several trophic groups. Low‐temperature anaerobic digestion of wastes is of intense interest because of the economic advantages of unheated bioenergy production technologies. However, low‐temperature granulation of anaerobic sludge has not yet been demonstrated. The aims of this study were to (i) investigate the feasibility of anaerobic sludge granulation in cold (15°C) bioreactors and (ii) observe the development of methanogenic activity and microbial community structure in developing cold granules. Methods and Results: One mesophilic (R1; 37°C) and two low‐temperature (R2 and R3, 15°C) laboratory‐scale, expanded granular sludge bed bioreactors were seeded with crushed (diameter <0·4 mm) granules and were fed a glucose‐based wastewater for 194 days. Bioreactor performance was assessed by chemical oxygen demand removal, biogas production, granule growth and temporal methanogenic activity. Granulation was observed in R2 and R3 (up to 33% of the sludge). Elevated hydrogenotrophic methanogenesis was observed in psychrophilically cultivated biomass, but acetoclastic methanogenic activity was also retained. Denaturing gradient gel electrophoresis of archaeal 16S rRNA gene fragments indicated that a distinct community was associated with developing and mature granules in the low‐temperature (LT) bioreactors. Conclusions: Granulation was observed at 15°C in anaerobic bioreactors and was associated with H₂/CO₂‐mediated methanogenesis and distinct community structure development. Significance and Impact of the Study: Granulation underpins high‐rate anaerobic waste treatment bioreactors. Most LT bioreactor trials have employed mesophilic seed sludge, and granulation <20°C was not previously documented.     

Stimulation of methanol degradation in UASB reactors: in situ versus pre-loading cobalt on anaerobic granular sludge

The effect of pre-loading and in situ loading of cobalt onto a cobalt-limited granular sludge on the performance of methanol fed bioreactors was investigated. One upflow anaerobic sludge bed (UASB) reactor was inoculated with cobalt pre-loaded sludge (24h; 30 degrees C; 1 mM CoCl2) and a second UASB with unloaded sludge. The UASB reactors (30 degrees C; pH 7) were operated for 77 days at 8 h hydraulic retention time and organic loading rates ranging from 5 to 20 g COD.L reactor(-1).d(-1). Cobalt pre-loading clearly stimulated the methanogenic activity of the sludge with methanol as the substrate, e.g., after 30 days of reactor operation this activity was 5.8 times higher than that of the cobalt unloaded sludge. During the experiment, part of the cobalt leached from the pre-loaded sludge, i.e., 54% of the cobalt content was lost during the 77 days of reactor operation. Sequential metal extraction showed that losses mainly occurred from the exchangeable and carbonate fraction and in the sludge remaining cobalt was mainly present in the organic/sulfide fraction of the sludge. In situ loading of cobalt in the unloaded UASB reactor on day 57 by adding 31 microM cobalt to the influent for a 24-h period (16% of the cobalt present in the loaded sludge at day 11) resulted in a 4 time increase of the methanogenic activity of the sludge with methanol as the substrate at the end of the reactor experiment, while the accumulated amount of cobalt in the sludge only amounted to 6% of the cobalt accumulated in the loaded sludge (on day 11). This study showed that both pre-loading sludge and in situ loading are adequate for achieving an increased reactor performance of methanol fed UASB reactors operating under cobalt limitation. However, the in situ dosing procedure needs substantially lower amounts of cobalt, while it also gives significantly smaller losses of cobalt with the effluent.     

pH、温度和癸酸对厌氧颗粒污泥产甲烷毒性关系的研究

在间歇培养中研究了癸酸对UASB反应器厌氧颗粒污泥的产甲烷毒性,并考察了pH、温度和癸酸抑制的关系。结果表明,癸酸对厌氧颗粒污泥产甲烷活性有强烈的抑制,对厌氧颗粒污泥比产甲烷活性产生50％抑制的浓度为1．9mmol/L。pH影响癸酸在液相中的存在形式,pH越低,游离癸酸比例越大,对厌氧颗粒污泥的抑制越严重。高温条件下癸酸使厌氧颗粒污泥的结构变得松散,对厌氧颗粒污泥产甲烷毒性较中温和低温条件下严重。     

Biosynthesis of plant hormones during anaerobic digestion of instant coffee waste

A large amount of solid waste remains after the production of instant coffee. This waste has to be moved to dumps, where it poses a threat of environmental pollution. Treatment of this waste by anaerobic methanogenic thermophilic digestion produced, besides biogas, a digested slurry which was used as a growth medium for horticulture, and proved to be a suitable and economical substitute for peat moss. Biological tests with mung bean cuttings and Grevillea plantlets showed promotional effects on rooting of the slurry and its sieved fraction extract, washed with water (Capul). Green coffee beans, instant coffee waste, its anaerobically-digested slurry and Capul were extracted by various methods and the extracts were analyzed by TLC, HPLC and GC/MS. Examinations showed clearly the presence of IAA and IBA in free and bound forms in all the substrates. The values of free and bound IAA were calculated by use of an internal standard and GC/MS. The amount of conjugated IAA was found to be much higher than that of free IAA, in both the coffee beans and instant coffee waste (11.1 vs 2.7 nmol g^–1, respectively). In the digested slurry and Capul, however, most of the IAA was present as the free form and was approximately 23.5–33.0 nmol g^–1, which is almost ten times more than in the waste, and almost twice the total amount of IAA in coffee beans. It is postulated that the high levels of free IAA in the digested instant coffee waste are a result of catabolism of tryptophan by anaerobic bacteria.     

Perturbation‐independent community development in low‐temperature anaerobic biological wastewater treatment bioreactors

The reproducibility and stability of low‐ temperature anaerobic wastewater treatment systems undergoing transient perturbations was investigated. Three identical anaerobic expanded granular sludge bed‐based bioreactors were used to degrade a volatile fatty acid and glucose‐based wastewater under sub‐ambient (15°C) conditions. The effect of a variety of environmental perturbations on bioreactor performance was assessed by chemical oxygen demand removal. Temporal microbial community development was monitored by denaturation gradient gel electrophoresis (DGGE) of 16S rRNA genes extracted from sludge granules. Methanogenic activity was monitored using specific methanogenic activity assays. Bioreactor performance and microbial population dynamics were each well replicated between both experimental bioreactors and the control bioreactor prior to, and after the implementation of most of the applied perturbations. Gene fingerprinting data indicated that Methanosaeta sp. were the persistent, keystone members of the archaeal community, and likely were pivotal for the physical stability and maintenance of the granular biofilms. Cluster analyses of DGGE data suggested that temporal shifts in microbial community structure were predominantly independent of the applied perturbations. Biotechnol. Bioeng. 2010;105: 79–87. © 2009 Wiley Periodicals, Inc.     

Anaerobic biodegradation of oleic and palmitic acids: evidence of mass transfer limitations caused by long chain fatty acid accumulation onto the anaerobic sludge

Palmitic acid was the main long chain fatty acids (LCFA) that accumulated onto the anaerobic sludge when oleic acid was fed to an EGSB reactor. The conversion between oleic and palmitic acid was linked to the biological activity. When palmitic acid was fed to an EGSB reactor it represented also the main LCFA that accumulated onto the sludge. The way of palmitic acid accumulation was different in the oleic and in the palmitic acid fed reactors. When oleic acid was fed, the biomass-associated LCFA (83% as palmitic acid) were mainly adsorbed and entrapped in the sludge that became "encapsulated" by an LCFA layer. However, when palmitic acid was fed, the biomass-associated LCFA (the totality as palmitic acid) was mainly precipitated in white spots like precipitates in between the sludge, which remained "non-encapsulated." The two sludges were compared in terms of the specific methanogenic activity (SMA) in the presence of acetate, propionate, butyrate, and H(2)CO(2), before and after the mineralization of similar amounts of biomass-associated LCFA (4.6 and 5.2 g COD-LCFA/g of volatile suspended solids (VSS), for the oleic and palmitic acid fed sludge, respectively). The "non-encapsulated," sludge exhibited a considerable initial methanogenic activity on all the tested substrates, with the single exception of butyrate. However, with the "encapsulated" sludge only methane production from ethanol and H(2)/CO(2) was detected, after a lag phase of about 50 h. After mineralization of the biomass-associated LCFA, both sludges exhibited activities of similar order of magnitude in the presence of the same individual substrates and significantly higher than before. The results evidenced that LCFA accumulation onto the sludge can create a physical barrier and hinder the transfer of substrates and products, inducing a delay on the initial methane production. Whatever the mechanism, metabolic or physical, that is behind this inhibition, it is reversible, being eliminated after the depletion of the biomass-associated LCFA.     

一种从活性污泥中提取微生物总DNA的方法

对活性污泥的微生物群落进行研究的首要前提是获得大量的高纯度微生物基因组DNA。本文建立了一种高效、简便的提取活性污泥总DNA方法。从提取的核酸总量、纯度、基因组完整性等多方面对所得到的DNA质量进行了评价,结果表明,本法从单位活性污泥中提取的DNA得率为105-823μg/g,结构完整,纯度很高,无需进一步的纯化,可直接进行微生物群落分析及构建文库等后续分子生物学操作。现在实验室使用的提取活性污泥中DNA的方法,纯度普遍都无法达到PCR反应和建立文库的要求,本文建立的活性污泥DNA提取方法则可以克服这一难题。     

Preservation methods for kidney and liver

With the successful testing of the immunosuppressive effects of cyclosporine in transplant patients in 1978, the field of organ transplants began an exponential growth. With that, the field of organ preservation became increasingly important as the need to increase preservation time and improve graft function became paramount. However, for every patient that receives a transplanted organ, there are four more on the waiting list. In addition, a patient dies from the lack of a transplant almost every 1½ hour. To alleviate this donor crisis, there is a need to expand the donor pool to marginal donor organs. The main reason these organs are underutilized is because the current method of static preservation, simple cold storage, is ineffective. This article will provide a general review of the methods of preservation including simple cold storage, hypothermic machine perfusion, normothermic machine perfusion, and oxygen persufflation. In addition, the article will provide a review of how these dynamic preservation methods have improved the recovery and preservation of marginal donor organs including Donation after Cardiac Death and Fatty livers.     

Influence of physico-chemical properties of porous microcarriers on the adhesion of an anaerobic consortium

The ability for biomass colonization of four porous mineral microcarriers (sepiolite, clay, pozzolana and foam glass-Poraver), was studied and related to their surface properties. The surface hydrophobicity of the mineral carriers was a more important factor influencing colonization by the anaerobic consortium than was surface charge. It was possible to correlate linearly the degree of hydrophobicity with the biomass retention capacity. Although the thermodynamic theory did not explain adhesion, an increase in cell attachment was directly related to the decrease of the positive values of the free energy of adhesion. Surface roughness, porosity and the amount of surface Mg²⁺, were also determinant factors in bacterial immobilization. However a great biomass accumulation can originate a decrease in biological activity due to mass transfer limitations. Journal of Industrial Microbiology & Biotechnology (2000) 24, 181–186. Received 09 August 1999/ Accepted in revised form 01 December 1999     

Standardized methods for anaerobic biodegradability testing

The European Community Directive 67/548/EEC (adopted in 1967), Annex V, contains about 85 testing methods, legally binding for all EU-Member States. They are regularly updated, or new methods are introduced in a formal procedure. Until 2002, no anaerobic biodegradability testing methods have been explicitly included either in the EU Directive 67/548/EEC or in the OECD-Guidelines adopted. However, a hidden EU-document with a testing scheme exists which recommends when anaerobic testing may be applied, without specifying in detail which legally binding method is to be used. In order to give an impression of the spectrum available, anaerobic test schemes proposed by ASTM, CEN, DIN, ISO, ECETOC, and OECD, are classified according to temperature, test environment, test purposes, and the equipment used.Some problems with the equipment and the methodology such as permeation phenomena, reproducibility, and the medium composition are addressed. With this, the difficulty to reach a satisfying carbon balance can be better understood. Finally, some recommendations are given on how to improve knowledge on the test equipment and the test procedure.     

Equipment used for testing anaerobic biodegradability and activity

Recently there has been a growing demand for information on the biodegradability and microbial toxicity of xenobiotic compounds. The environmental fate and effect of these chemicals in aerobic and anaerobic conditions has been extensively studied using a variety of different methods. This paper reviews the different protocols, equipment and instruments used in anaerobic biodegradation and activity tests, including the more sophisticated automated techniques recently developed. The different systems for detecting biogas production are given particular attention. As well as gasometric methods, the different instrumental techniques used to follow the fate of compounds under anaerobic conditions, via analysis of substrate consumption and product formation are evaluated.     

High rate treatment of terephthalic acid production wastewater in a two-stage anaerobic bioreactor

The feasibility was studied of anaerobic treatment of wastewater generated during purified terephthalic acid (PTA) production in two-stage upflow anaerobic sludge blanket (UASB) reactor system. The artificial influent of the system contained the main organic substrates of PTA-wastewater: acetate, benzoate, and terephthalate. Three parallel operated reactors were used for the second stage, and seeded with a suspended terephthalate degrading culture, with and without additional methanogenic granular sludge (two different types). The first stage UASB-reactor was seeded with methanogenic granular sludge. Reactors were operated at 37 degrees C and pH 7. During the first 300 days of operation a clear distinction between the biomass grown in both reactor stages was obtained. In the first stage, acetate and benzoate were degraded at a volumetric loading rate of 40 g-COD/L . day at a COD-removal efficiency of 95% within the first 25 days of operation. No degradation of terephthalate was obtained in the first stage during the first 300 days of operation despite operation of the reactor at a decreased volumetric loading rate with acetate and benzoate of 9 g-COD/L . day from day 150. Batch incubation of biomass from the reactor with terephthalate showed that the lag-phase prior to terephthalate degradation remained largely unchanged, indicating that no net growth of terephthalate degrading biomass occurred in the first stage reactor. From day 300, however, terephthalate degradation was observed in the first stage, and the biomass in this reactor could successfully be enriched with terephthalate degrading biomass, resulting in terephthalate removal capacities of 15 g-COD/L . day. Even though no single reason could be identified why (suddenly) terephthalate degradation was obtained after such a long period of operation, it is suggested that the solid retention time as well the prevailing reactor concentrations acetate and benzoate may have played an important role. From day 1 of operation, terephthalate was degraded in the second stage. In presence of methanogenic granular biomass, high terephthalate removal capacities were obtained in these reactors (15 g-COD/L . day) after approximately 125 days of operation. From the results obtained it is concluded that terephthalate degradation is the bottleneck during anaerobic treatment of PTA-wastewater. Pre-removal of acetate and benzoate in staged bioreactor reduces the lag-phase prior to terephthalate degradation in latter stages, and enables high rate treatment of PTA-wastewater.     

Indexes of assessing N availability in sewage sludges

Biological and chemical methods were used in an attempt to estimate N availability in sewage sludges. The two biological methods, i.e. maize plants grown in pots, and soil-sludge mixtures incubated at 2, 4, 6, 8, 12 and 16 weeks, and the four chemical methods, i.e. autoclave, 0.5 M KMnO₄, pepsin and 0.6 M HCl, were compared to determine N availability in twelve sewage sludges in a given soil. In the mineralization test, the aerobically treated sewage sludges gave higher mineralization rates than the anaerobically treated wastes. The simple correlation between available N, estimated from the plant N uptake during 6 weeks and N extracted by chemical methods showed that HCl and pepsin appeared to be the better single indexes. Prediction of availability of N in sewage sludges to plants in the growth chamber improved if N mineralized during the incubation period and extracted by several chemical methods were combined in a multiple regression analysis.     

Mineralization of LCFA associated with anaerobic sludge: Kinetics, enhancement of methanogenic activity, and effect of VFA

Long-chain fatty acids (LCFA) associated with anaerobic sludge by mechanisms of precipitation, adsorption, or entrapment can be biodegraded to methane. The mineralization kinetics of biomass-associated LCFA were established according to an inhibition model based on Haldane's enzymatic inhibition kinetics. A value around 1,000 mg COD-LCFA..g VSS(-1) was obtained for the optimal specific LCFA content that allowed the maximal mineralization rate. For sludge with specific LCFA contents of 2,838 +/- 63 and 4,571 +/- 257 mg COD-LCFA..g VSS(-1), the specific methanogenic activities in the presence of acetate, butyrate, and H(2)/CO(2) were significantly enhanced after the mineralization of the biomass-associated LCFA. For sludge with a specific LCFA content near the optimal value defined by the kinetic model, the effect of adding VFA to the medium was studied during the mineralization of the biomass-associated LCFA. Different patterns were obtained for each individual substrate. Acetate and butyrate were preferentially consumed by the consortium, but in the case of propionate no evidence of a sequential consumption pattern could be withdrawn. It was concluded that LCFA do not exert a bactericidal neither a permanent toxic effect toward the anaerobic consortia. A discussion is addressed to the relative roles of a reversible inhibitory effect and a transport limitation effect imposed by the LCFA surrounding the cells.