Article

Microbial community dynamics reflect reactor stability during the anaerobic digestion of a very high strength and sulfate-rich vinasse: Microbial community dynamics during the anaerobic digestion of vinasse

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  • Universidad de Sancti Spíritus José Martí Pérez (UNISS)
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Abstract

Microbial community dynamics during the anaerobic digestion of vinasse has been little studied. However, having knowledge about it is essential for early detection of reactor operational difficulties to apply preventive actions. This research studies the microbial community dynamics in the anaerobic digestion of vinasse, linking to experimental observations about product yields and organic matter degradation. Methane and sulfide yields decreased with increasing SO42-/COD ratio, while the fraction of organic matter degraded by sulfate reducing bacteria increased from 4.5 ± 0.3% to 27.1 ± 0.6%. The archaeal community showed that acetoclastic Methanosaetaceae were little affected by the increase of the SO42-/COD ratio, in contrast to the Methanomicrobiales and Methanobacteriales population, which decreased during the experiment. The total bacterial diversity was influenced mainly by substrate composition, showing that the increase of the SO42-/COD ratio above 0.10 shifted the bacterial community to a lower richness. These results provide knowledge on the dynamics of the microbial communities, which can be useful to control the anaerobic digestion of sulfate-rich vinasses, showing that reactor stability equates to the higher ratios between total methanogens and total bacteria gene copy numbers, whereas operational difficulties can be associated to lower bacterial richness and higher community organization.

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... So, the lack of knowledge of the microbial communities present in AD of vinasse limits the capacity to maximize the methane production. In AD of vinasse, changes in the structure of microbial communities have been rarely studied (Jiménez et al. 2018). In the next sections, molecular techniques for the microorganism identification in diverse vinasses are summarized. ...
... The double-stranded fragments are separated in polyacrylamide gel with urea-formamide depending on their nucleotide sequence (Myers et al. 1987;Muyzer et al. 1993). In PCR-DGGE, P338f-GC and P518r (Jiménez et al. 2018), 1055F / 1392R-GC (Dias et al. 2016), and 968FGC-1401R (Rodríguez et al. 2012;dos Reis et al. 2015) primers have been used for the study of bacterial communities in the AD of vinasse. For the Domain Archaea, Parch519fGC / Arch915r and A109 (T) -F / 515-GC-R primers have been used (Rodríguez et al. 2012;dos Reis et al. 2015). ...
... For the Domain Archaea, Parch519fGC / Arch915r and A109 (T) -F / 515-GC-R primers have been used (Rodríguez et al. 2012;dos Reis et al. 2015). Also, denaturing gradients ranging from 45% to 60% (Jiménez et al. 2018) and 30% to 70% (Dias et al. 2016) have been used. Other studies have used a gradient of 42% to 67% and 30% to 60% for bacterial and archaeal communities. ...
Article
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Sugars, starches, and cellulose materials are used for ethanol production. When producing a liter of alcohol, 10 to 15 liters of liquid waste are generated. This waste is called vinasse, and it generates negative impacts on the environment. The process of storing and disposing vinasse in soils generates emissions to the atmosphere, mainly methane. Anaerobic treatment allows for the capture and generation of more biogas, therefore allowing mitigation of the environmental impacts. The microbial diversity present in the anaerobic digestion (AD) of vinasse is strongly related to the efficiency and quality of methane production. The gene 16s rDNA-based molecular techniques have been the most commonly used techniques for monitoring microbial communities present in the digesters. However, the identification is not enough. Rather, it is necessary to know the metagenomic functionality in this type of habitat. This review provides a comprehensive overview of methods to identify the microorganisms in the anaerobic digestion of vinasse. In addition, microbial community identification in vinasse reactors and their relationship with methane production are reviewed.
... So, the lack of knowledge of the microbial communities present in AD of vinasse limits the capacity to maximize the methane production. In AD of vinasse, changes in the structure of microbial communities have been rarely studied (Jiménez et al. 2018). In the next sections, molecular techniques for the microorganism identification in diverse vinasses are summarized. ...
... The double-stranded fragments are separated in polyacrylamide gel with urea-formamide depending on their nucleotide sequence (Myers et al. 1987;Muyzer et al. 1993). In PCR-DGGE, P338f-GC and P518r (Jiménez et al. 2018), 1055F / 1392R-GC (Dias et al. 2016), and 968FGC-1401R (Rodríguez et al. 2012;dos Reis et al. 2015) primers have been used for the study of bacterial communities in the AD of vinasse. For the Domain Archaea, Parch519fGC / Arch915r and A109 (T) -F / 515-GC-R primers have been used (Rodríguez et al. 2012;dos Reis et al. 2015). ...
... For the Domain Archaea, Parch519fGC / Arch915r and A109 (T) -F / 515-GC-R primers have been used (Rodríguez et al. 2012;dos Reis et al. 2015). Also, denaturing gradients ranging from 45% to 60% (Jiménez et al. 2018) and 30% to 70% (Dias et al. 2016) have been used. Other studies have used a gradient of 42% to 67% and 30% to 60% for bacterial and archaeal communities. ...
Article
Full-text available
Sugars, starches, and cellulose materials are used for ethanol production. When producing a liter of alcohol, 10 to 15 liters of liquid waste are generated. This waste is called vinasse, and it generates negative impacts on the environment. The process of storing and disposing vinasse in soils generates emissions to the atmosphere, mainly methane. Anaerobic treatment allows for the capture and generation of more biogas, therefore allowing mitigation of the environmental impacts. The microbial diversity present in the anaerobic digestion (AD) of vinasse is strongly related to the efficiency and quality of methane production. The gene 16s rDNA-based molecular techniques have been the most commonly used techniques for monitoring microbial communities present in the digesters. However, the identification is not enough. Rather, it is necessary to know the metagenomic functionality in this type of habitat. This review provides a comprehensive overview of methods to identify the microorganisms in the anaerobic digestion of vinasse. In addition, microbial community identification in vinasse reactors and their relationship with methane production are reviewed.
... When AD is applied to the treatment of sulfur-rich compounds like sulfate, sulfide or thiosulfate wastewater, sulfidogenesis also occurs together with methanogenesis through the activity of sulfate-reducing bacteria (SRB) (Jiménez et al., 2018). In sulfidogenesis, SRB reduce sulfate to sulfide, which in turn is dissolved in the liquid phase (S 2− , HS − , H 2 S) and present in the biogas (H 2 S), causing corrosion, toxicity and release of malodorous (Ahmed and Rodríguez, 2018). ...
Article
The increase in biofuel production by 2030, driven by the targets set at the 21st United Nations Framework Convention on Climate Change (COP21), will promote an increase in ethanol production, and consequently more vinasse generation. Sugarcane vinasse, despite having a high polluting potential due to its high concentration of organic matter and nutrients, has the potential to produce value-added resources such as volatile fatty acids (VFA), biohydrogen (bioH2) and biomethane (bioCH4) from anaerobic digestion. The objective of this paper is to present a critical review on the vinasse treatment by anaerobic digestion focusing on the final products. Effects of operational parameters on production and recovery of these resources, such as pH, temperature, retention time and type of inoculum were addressed. Given the importance of treating sugarcane vinasse due to its complex composition and high volume generated in the ethanol production process, this is the first review that evaluates the production of VFAs, bioH2 and bioCH4 in the treatment of this organic residue. Also, the challenges of the simultaneous production of VFA, bioH2 and bioCH4 and resources recovery in the wastewater streams generated in flex-fuel plants, using sugarcane and corn as raw material in ethanol production, are presented. The installation of flex-fuel plants was briefly discussed, with the main impacts on the treatment process of these effluents either jointly or simultaneously, depending on the harvest season.
... Another study investigated the microbial dynamics in a lab-scale reactor treating sulfate-rich sugarcane vinasse by qPCR and DGGE. The authors concluded that low methane yields could be associated with lower bacterial richness and a specialized community [14]. ...
Article
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The treatment of sugarcane vinasse is still a challenge to develop a sustainable bioethanol production. Anaerobic digestion (AD) is the most promising treatment of vinasse since energy in the form of biogas can be recovered. The aim of this work was to understand the dynamics of the microbial community in a 100-m3 upflow anaerobic sludge blanket (UASB) methanogenic reactor at start-up and during two periods of bioethanol production. An inter-harvest period in which the reactor was not fed was also studied. Metabarcoding analysis of the V4 region of the 16S rRNA gene showed that Firmicutes, Synergistetes, Chloroflexi, and Proteobacteria were the dominant phyla. Firmicutes was abundant, suggesting that this group plays a specific role in the treatment of vinasse. The inoculum adaptation to vinasse was correlated with the microbiota diversity and dynamics. The microbiota diversity was higher during the first harvest and reflected the initial microbial composition. During the second harvest, the increasing organic loading rate (OLR) and the adaptation to the new effluent selected a less diverse community which produced the biogas in the reactor. The qPCR of the mcrA gene and methanogenic activity tests showed that the abundance of methanogens increased over time and remained stable even after the stop period. This work shows the plasticity of the microbial community, which adapted its structure to the changes in the feed and persisted during starvation periods in the reactor. A time-series of microbiological information is necessary to a comprehensive understanding of full-scale reactor maintenance.
... Kiyuna et al. [9] reported that the presence of sulfate (SO 4 2− ) in vinasse, originating from the use of sulfuric acid by sugar mills, is one of the factors that negatively affects the anaerobic digestion process. Sulfate reduction uses hydrogen as an electron donor [10], competes for substrate with methanogenic archaea, and can inhibit methanogenesis through sulfide production [11,12]. According to Fuess et al. [13], SO 4 2− removal in the acidogenic reactor can be an important tool to improve bioenergy production from vinasse. ...
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Currently, the disposal of sugarcane vinasse is one of the greatest issues of sugarcane biorefineries in Brazil because of the large volumes produced. To contribute with an alternative energy recovery process from this by-product, this study proposed a physicochemical pretreatment and adjustment of operating conditions to improve the performance of the acidogenic stage of the anaerobic digestion of sugarcane vinasse. Therefore, this study evaluated the influence of hydraulic retention time (decreasing from 8 to 6, 4, 2, and 1 h) on the bioconversion of pretreated sugarcane vinasse (5000 mg COD L−1) to hydrogen and value-added products. Two anaerobic fluidized bed reactors were operated under mesophilic (AFBR-M, 30 °C) and thermophilic (AFBR-T, 55 °C) conditions. Despite the low similarity between the bacterial populations of AFBR-M and AFBR-T (40% similarity), the maximum hydrogen production rates (0.27 ± 0.07 and 6.42 ± 1.46 L H2 day−1 L−1) and hydrogen yields (0.27 ± 0.07 and 1.06 ± 0.15 mmol H2 g COD−1) occurred at the hydraulic retention time of 2 h by reducing the values from 8 to 2 h. The highest COD/SO42− ratios of 17.4 and 25.1 were also observed in the effluents of the AFBR-M and AFBR-T, respectively, at the hydraulic retention time of 2 h. Under both mesophilic and thermophilic conditions, a similar metabolic distribution was observed at the HRT of 2 h (acetic, propionic, and butyric acids, for AFBR-M and acetic, propionic, butyric acids, and ethanol for AFBR-T). This finding indicates the functional similarity between bacterial populations in both reactors.
... The relative abundance of SRB increased from 5% to 10%, 4% to 10%, and 2% to 4%, in reactors R1, R2, and R3, respectively, while the relative abundance of MPA reduced from 2.4% to 0.7% in R1, 1.7% to 0.7% in R2, and 1.5% to 0.4% in R3. Several studies reported that a reduction in Methanobacterium species occurs due to the fact that SRB outcompete hydrogenotrophic methanogenic archaea for the available hydrogen (Jiménez et al., 2018;Muyzer and Stams, 2008). Concurrently methane yield decreased by 47% in R1, 33% in R2, and 41% in R3 (Fig. 2). ...
Article
This study examined the use of biochar to alleviate sulfide toxicity to methane producing archaea (MPA) and sulfate-reducing bacteria (SRB) during anaerobic treatment of sulfate-rich wastewater with concomitant sulfur recovery. At the sulfate concentration of 6000 mg SO42-/L, the dissolved sulfide (DS) of 131 mg S/L resulted in total volatile fatty acids concentration of 3500 mg/L as acetic acid (HAc) and the reactors were on the verge of failure. Biochar removed >98% of H2S(g), 94% of DS, and 89% of unionized sulfide (H2Saq). 16S rRNA analysis revealed that after sulfide removal the relative abundance of MPA (Methanobacterium and Methanosaeta) increased from 0.7% to 3.7%, while the relative abundance of SRB (Desulfovibrio) decreased from 9.3% to 0.5% indicating that the reactor recovered to stable state. This study showed that biochar could effectively remove H2S from biogas, alleviate sulfide toxicity to MPA and SRB, and promote stability of the anaerobic process.
... indicating that aceticlastic methanogenesis was likely the major pathway for methane production ( Figure 6A). Methanotrichaceae is generally considered indicative of a balanced AD with low residual VFAs [38]. The methanogenic community structures of RM and inoculum, in which Methanosarcinaceae was predominant, were significantly different, although both originated from mesophilic systems ( Figure 6A). ...
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Methanogenesis and sulfidogenesis, the major microbial reduction reactions occurring in the anaerobic digestion (AD) process, compete for common substrates. Therefore, the balance between methanogenic and sulfidogenic activities is important for efficient biogas production. In this study, changes in methanogenic and sulfidogenic performances in response to changes in organic loading rate (OLR) were examined in two digesters treating sulfur-rich macroalgal waste under mesophilic and thermophilic conditions, respectively. Both methanogenesis and sulfidogenesis were largely suppressed under thermophilic relative to mesophilic conditions, regardless of OLR. However, the suppressive effect was even more significant for sulfidogenesis, which may suggest an option for H2S control. The reactor microbial communities developed totally differently according to reactor temperature, with the abundance of both methanogens and sulfate-reducing bacteria being significantly higher under mesophilic conditions. In both reactors, sulfidogenic activity increased with increasing OLR. The findings of this study help to understand how temperature affects sulfidogenesis and methanogenesis during AD.
... Nevertheless, depending on substrate, operational condition, and AD system, some authors reported the dominance of different species such as Methanoculleus, Methanosaeta resp. Methanotrix, Methanosarcina, Methanobacterium, Methanococcus, Methanobrevibacter, and Methanomicrobium (Klocke et al. 2008;Nettmann et al. 2010;Jiménez et al. 2016;Jiménez et al. 2017;Wang et al. 2018). On the other hand, the apparent The original version of this article was revised: After publication of this article, the publisher was notified that Michael Klocke has been listed as an author without his consent. ...
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Microbial metagenome analysis has proven its usefulness to investigate the microbiomes present in technical engineered ecosystems such as anaerobic digestion systems. The analysis of the total microbial genomic DNA allows the detailed determination of both the microbial community structure and its functionality. In addition, it enables to study the response of the microbiome to alterations in technical process parameters. Strategies of functional microbial networks to face abiotic stressors, e.g., resistance, resilience, and reorganization, can be evaluated with respect to overall process optimization. The objective of this paper is to review the main metagenomic tools used for effective studies on anaerobic digestion systems in monitoring the dynamic of the microbiomes, as well as the factors that have been identified so far as limiting the metagenomic studies in this ecosystems.
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Thesis
The production of renewable energy from organic waste streams is one of the most important aspects in the concept of sustainable development. Anaerobic digestion can be considered one of the main techniques to treat organic waste streams, allowing both waste stabilization and renewable energy production in the form of biogas. Its widespread application on full-scale relates to the fact that anaerobic digestion has, apart from biogas production and organic waste stabilization, several other advantages over alternative biological processes, e.g. a low cell yield, a high organic loading rate, limited nutrient demands, and low costs for operation and maintenance of the reactor system. The methanogenic archaea are responsible for the final and critical step of anaerobic digestion, as they produce valuable methane. One of the major drawbacks of anaerobic digestion is, however, the sensitivity of the methanogenic community to different environmental factors or stressors. At this point, our knowledge of the microbial community taking care of the different stages in anaerobic digestion is still limited and, therefore, anaerobic digestion can still be considered a ‘black box’. Indeed, our knowledge of the bacterial community is restricted to the attribution of the first three steps in anaerobic digestion, i.e. hydrolysis, acidogenesis and acetogenesis. Although several key populations have already been identified, the exact contribution of the different bacterial phyla remains, however, to be elucidated. Methanogenesis, the last step, is carried out by archaea. The methanogenic community can be divided into two different groups, related to their main methanogenic pathway, i.e. hydrogenotrophic and acetoclastic methanogens. Thus far, only two genera, Methanosaeta and Methanosarcina, are reported to be able to carry out acetoclastic methanogenesis. Due to a distinct difference in physiology, morphology and metabolic potential, these two genera are expected to occupy different niches in anaerobic digestion. However, up until now, little is known about the specific contribution of both genera to methanogenesis in anaerobic digestion. The main objective of this research was to unravel the ‘black box’ of anaerobic digestion to allow better and more solid process engineering. Several strategies were applied to improve biogas production and process stability, by (in)directly influencing the microbial community. A main focus was placed on the methanogenic community, as methanogenesis can be considered the weak link in the chain, because of the sensitivity of the methanogenic community to different environmental factors. However, to reach stable methane production, a close interaction between the bacterial and methanogenic community is required, hence, the bacterial community was also examined in terms of composition and organization. In Chapter 2, A-sludge originating from the A-stage of the ‘Adsorptions-Belebungsverfahren’, was co-digested with kitchen waste to increase biogas production. This Fe-rich A-sludge appeared to be a suitable co-substrate for kitchen waste, as methane production rate values of 1.15 ± 0.22 and 1.12 ± 0.28 L L-1 d-1 were obtained during mesophilic and thermophilic co-digestion, respectively, of a feed-mixture consisting of 15% kitchen waste and 85% A-sludge. Mono-digestion of kitchen waste resulted in process failure. The thermophilic process led to higher residual volatile fatty acid concentrations, up to 2070 mg COD L-1, hence, the mesophilic process can be considered the most ‘stable’. The optimal combination of A-sludge and kitchen waste served as a basis for the co-digestion of A-sludge with kitchen waste or molasses at mesophilic conditions in Chapter 3. In this chapter the objective was to evaluate the exact stabilizing mechanism of A-sludge as co-substrate in anaerobic digestion. Co-digestion of kitchen waste and molasses with A-sludge resulted in stable methane production, as values up to 1.53 L L-1 d-1 for kitchen waste and 1.01 L L-1 d-1 for molasses were obtained. The stabilizing effect of A-sludge in anaerobic digestion could not be attributed to bioaugmentation, despite its indigenous methanogenic activity, and therefore was dominated by nutrient addition. Methanosaetaceae maintained high copy numbers, between 109 and 1010 copies g-1 sludge, as long as optimal conditions were maintained, irrespective of the selected (co-)substrates. However, an increase in volatile fatty acids and a decrease in pH resulted in a decreased abundance of Methanosaetaceae. In Chapter 4, a different feeding pattern was applied to obtain a higher degree of functional stability by (in)directly changing the evenness, dynamics and richness of the bacterial community. A short-term stress test revealed that pulse feeding leads to a higher tolerance of the digester to an organic shock load of 8 g COD L-1 and total ammonia levels up to 8000 mg N L-1. The bacterial community showed a high degree of dynamics over time, yet the methanogenic community remained constant. These results suggest that the regular application of a limited pulse of organic material and/or a variation in the substrate composition might promote higher functional stability in anaerobic digestion. In Chapter 2-4, the anaerobic sludge originating from the same sludge digester was used as inoculum. The contribution of the inoculum to stable methane production and stress tolerance was investigated in Chapter 5. A different response in terms of start-up efficiency and ammonium tolerance was observed between the different inocula. Methanosaeta was the dominant acetoclastic methanogen, yet Methanosarcina increased in abundance at elevated ammonium concentrations. A shift from a Firmicutes to a Proteobacteria dominated bacterial community was observed in failing digesters. Methane production was strongly positively correlated with Methanosaetaceae, but with several bacterial populations as well. Overall, these results indicated the importance of inoculum selection to ensure stable operation and stress tolerance in anaerobic digestion. In several studies, the positive effect of a bioelectrochemical system on biogas production in anaerobic digestion is described, however, the main mechanism behind this remained unsolicited, and primary controls were not executed. In Chapter 6, the stabilizing ability of a bioelectrochemical system for molasses digestion was evaluated in a 154 days experiment. A high abundance of Methanosaeta was detected on the electrodes, however, irrespective of the applied cell potential. This study demonstrated that, in addition to other studies reporting only an increase in methane production, a bioelectrochemical system can also remediate anaerobic digestion systems that exhibited process failure. However, the lack of difference between current driven and open circuit systems indicates that the key impact is through biomass retention, especially Methanosaetaceae, rather than electrochemical interaction with the electrodes. Anaerobic membrane bioreactors with different fouling prevention strategies, i.e. biogas recirculation or membrane vibration, were applied to increase the retention of slow growing methanogens in Chapter 7. Biogas recirculation was the best mechanism to avoid membrane fouling, while the trans membrane pressures in the vibrating membrane bioreactor increased over time, due to cake layer formation. Stable methane production, up to 2.05 L L-1 d-1 and a concomitant COD removal of 94.4%, were obtained, only when diluted molasses were used, since concentrated molasses resulted in process failure. Real-time PCR results revealed a clear dominance of Methanosaetaceae over Methanosarcinaceae as the main acetoclastic methanogens in both anaerobic membrane bioreactor systems. In Chapter 8, an extensive evaluation of 38 samples from 29 full-scale anaerobic digestion plants was carried out to relate operational parameters to microbial community composition and organization. The bacterial community was dominated by representatives of the Firmicutes, Bacteroidetes and Proteobacteria, covering 86.1 ± 10.7% of the total bacterial community. Acetoclastic methanogenesis was dominated by Methanosaetaceae, yet, only Methanobacteriales significantly positively correlated to biogas production. Three potential clusters, that could be considered as ‘AD-types’, were identified. These so-called ‘AD-types’ were determined by total ammonia concentration, free ammonia concentration and temperature, and characterized by an increased abundance of the Bacteroidales, Clostridiales and Lactobacillales, respectively. The identification of these three potential AD-types may serve as a basis for directly engineering the microbial community in anaerobic digestion. However, further research will be required to validated the actual existence of these three clusters in AD. This research demonstrated the potential of several operational and technological strategies to improve biogas production and process stability in anaerobic digestion. Stable anaerobic digestion hosts a static methanogenic community, as long as evolving operational parameters or substrate composition do not influence the optimal conditions for methanogenesis, and an ever dynamic bacterial community. Methanosaetaceae are the uncontested dominant methanogens in anaerobic digestion, irrespective of the substrate, operational conditions or reactor configuration. However, increasing ammonium, salt and volatile fatty acid concentrations cause a shift from acetoclastic methanogenesis by Methanosaetaceae to hydrogenotrophic methanogenesis. Comparison of the lab-scale reactor results with full-scale plant microbial community analysis results showed a high similarity on bacterial level. However, at ‘deteriorating’ conditions at lab-scale a transition to a Methanosarcinaceae dominated methanogenesis was observed, while this shift could not be observed in full-scale plants. Hence, instead of Methanosarcinaceae, the Methanobacteriales are to be considered as the main drivers of so-called high-rate anaerobic digestion. The identification of the three AD-types can serve as a basis for unravelling the anaerobic digestion microbiome. Further in-depth research, however, will be required to determine the exact role of the core micro-organisms in each cluster to allow microbial community based engineering of anaerobic digestion ecosystems. The application of RNA, protein and metabolite based methods will be essential to estimate the effective metabolic activity of the microbial community in anaerobic digestion, thus, allowing more in-depth process control and further unravelling of the anaerobic digestion ‘black box’.
Article
In view of the difficulty in treating the bioleaching solutions, the selective precipitation of metals using H 2S produced biologically by sulfate reducing bacteria (SRB) was as an alternative process. At first, the initial COD/SO 42- ratio and pH value were determined, and then the capability of different immobilized carriers was investigated by batch experiments. The results showed that high removal rate of SO 42- was achieved when the initial COD/SO 42- ratio was 3 and the initial pH value was 7. In the experiments, it was found that two factors were important for influencing the formation of biofilm and the reducing capability of SRB, the roughness of the immobilized carriers and the pore volume. There was no biofilm found when glass beads were immobilized carrier, and the reducing capability of SRB was low, the removal rate of SO 42- was only 50%. Furthermore, the removal rate of SO 42- increased with the pore volume of immobilized carriers. So, in view of the stability of process and the removal rate of SO 42-, polyurethane foam was top priority as the immobilized carrier in the experiments, the removal rate of SO 42- with it reached 95%, and the process was easily operated.
Article
The anaerobic biological treatment of volatile fatty acid (VFA) – and sucrose – based wastewaters was investigated in two anaerobic bioreactors, R1 and R2, over a 300-day trial period. During the trial, the operating temperature of both reactors was lowered, in a stepwise fashion, from 37 to 16 °C. The VFA-fed reactor maintained an excellent level of performance, regardless of operating temperature, reaching COD removal efficiencies of 95% at 18 °C, and a biogas methane content in excess of 70% at 16 °C, at an imposed OLR of 20 kg COD m−3 d−1. However, an increase in the applied liquid upflow velocity to the bottom chamber of the reactor from 5 to 7.5 m h−1on day 236 resulted in a considerable decline in reactor performance. COD removal efficiencies in excess of 80% were achieved by the sucrose-fed reactor at 18 °C, at an imposed OLR of 20 kg COD m−3 d−1. An increase in the liquid upflow velocity applied to the sucrose-fed reactor resulted in enhanced reactor performance and stability, with respect to decreasing temperature. The different responses of both reactors to increased upflow velocity was associated with variations in the microbial population structure of the sludges, as determined by culture-independant molecular approaches, specifically the presence of high levels of δ-Proteobacteria and hydrogenotrophic methanogens in the VFA-fed biomass. High levels of Methanomicrobiales sp., in particular Methanocorpusculum parvum sp., were observed in both R1 and R2 during the trial. There was a distinct shift from acetoclastic methanogenic dominance to hydrogenotrophic dominance in both reactors in response to a decrease in the operating temperature.
Article
This research presents the modeling of the anaerobic digestion of cane-molasses vinasse, hereby extending the Anaerobic Digestion Model No. 1 with sulfate reduction for a very high strength and sulfate rich wastewater. Based on a sensitivity analysis, four parameters of the original ADM1 and all sulfate reduction parameters were calibrated. Although some deviations were observed between model predictions and experimental values, it was shown that sulfates, total aqueous sulfide, free sulfides, methane, carbon dioxide and sulfide in the gas phase, gas flow, propionic and acetic acids, chemical oxygen demand (COD), and pH were accurately predicted during model validation. The model showed high (±10%) to medium (10%–30%) accuracy predictions with a mean absolute relative error ranging from 1% to 26%, and was able to predict failure of methanogenesis and sulfidogenesis when the sulfate loading rate increased. Therefore, the kinetic parameters and the model structure proposed in this work can be considered as valid for the sulfate reduction process in the anaerobic digestion of cane-molasses vinasse when sulfate and organic loading rates range from 0.36 to 1.57 kg m−3 d−1 and from 7.66 to 12 kg COD m−3 d−1, respectively.
Article
Linkage between reactor performance and microbial community dynamics was investigated during mesophilic anaerobic co-digestion of restaurant grease waste (GTW) with municipal wastewater sludge (MWS) using 10L completely mixed reactors and a 20day SRT. Test reactors received a mixture of GTW and MWS while control reactors received only MWS. Addition of GTW to the test reactors enhanced the biogas production and methane yield by up to 65% and 120%, respectively. Pyrosequencing revealed that Methanosaeta and Methanomicrobium were the dominant acetoclastic and hydrogenotrophic methanogen genera, respectively, during stable reactor operation. The number of Methanosarcina and Methanomicrobium sequences increased and that of Methanosaeta declined when the proportion of GTW in the feed was increased to cause an overload condition. Under this overload condition, the pH, alkalinity and methane production decreased and VFA concentrations increased dramatically. Candidatus cloacamonas, affiliated within phylum Spirochaetes, were the dominant bacterial genus at all reactor loadings.
Article
Vinasse is a sulfate-rich liquid substrate, from which high levels of hydrogen sulfide in biogas can be obtained due to the sulfate reduction process under anaerobic conditions. Hydrogen sulfide is corrosive and toxic and must be removed for any utilization of the biogas. Mathematical models have been developed to study separately sulfate reduction in anaerobic digestion and sulfide removal from biogas streams. However, the levels of hydrogen sulfide produced in the anaerobic digestion stage have an effect on the sulfide removal processes in the next stage. As a method to study both processes and their interaction, a new approach is introduced and reviewed in the present article: the sulfur chain in biogas production. The necessity of studying the sulfate reduction processes in vinasse as a typical sulfate-rich substrate to predict hydrogen sulfide concentrations in the gas phase, as well as the best model approach to that aim are established here. In addition, the approaches to model sulfide removal based on direct conversion processes, the models' capability to predict the removal of hydrogen sulfide from the biogas (at levels between 20 000 and 30 000 ppmv) as well as the concentration profile of the reactants in this removal processes are discussed. © 2013 Society of Chemical Industry
Article
An expanded granular sludge bed reactor, inoculated with acclimated sulfidogenic granular sludge, was operated at 33 °C and fed with acetic acid as COD source and sulfate as electron acceptor. The bioreactor had a sulfate conversion efficiency of 80–90% at a high sulfate loading rate of 10.4 g SO42--S/l.d after only 60 days of start-up. This was achieved by implementing a dual operational strategy. Firstly acetic acid was dosed near stoichiometry (COD over sulfur ratio = 2.0 to 2.2) which allowed almost complete sulfate removal. Secondly the pH in the bioreactor was kept slightly alkaline (7.9 ± 0.1) which limited the concentration of the inhibitory undissociated hydrogen sulfide H2S (pKa = 7). This allowed the acetotrophic sulfate reducing bacteria to predominate throughout the long term experiment. The limitations of the EGSB technology with respect to the sulfate conversion rate appeared to be related to the biomass wash-out and granule deterioration occurring at superficial upflow velocities above 10 m/h. Increasing the recirculation flow caused a drop in the sulfate reduction rate and efficiency, an increase of the suspended sludge fraction and a considerable loss of biomass into the effluent, yielding bare mainly inorganic granules. Elemental analysis revealed that a considerable amount of the granular sludge dry matter at the end of the experiment, at an upflow velocity of 20 m/h, consisted of calcium (32%), mainly in the form of carbonate deposits, while organic matter only represented 7%.
Article
Anaerobic processes have been widely used for the treatment of various high-strength industrial wastewaters. However, application has been limited for the treatment of sulfate-rich industrial wastewaters, such as those from the petrochemical, and mining industries. Wastewaters containing benzoate and sulfate were treated in two upflow anaerobic sludge blanket (UASB) reactors at 34--37 C for 320 d. The sulfate concentration was increased stepwise in Reactor-A up to 7,500 mg/L, and was kept mostly constant at 3,000 mg/L in Reactor-B. Both reactors removed over 98% of organic chemical-oxygen demand (COD) for sulfate up to 6,000 mg/L, despite the fact that the mixed liquor contained up to 769 mg S/L of total sulfides and up to 234 mg S/L of dissolved HâS. Sulfate0reducing efficiency decreased with the increase in sulfate concentration, but increased with time at each sulfate concentration. Reactor-B consistently reduced 89% of sulfate. However, both organic COD removal and sulfate-reducing efficiencies of Reactor-A dropped drastically at 7,500 mg SOâ⁻²/L, and showed no sign of recovery after 50 d. The system failure was likely due to the increased sulfate, instead of sulfide, toxicity. From the COD balance, 93.4% of COD removed was converted to methane instead of sulfides, with a net sludge yield of 0.047 g volatile suspended solids (VSS)/g COD. The sulfur balance was over 97%.
Article
In this study, a novel approach was developed for sulfate - containing wastewater treatment via dosing FeO in a two - stage anaerobic reactor (A1, S1). The addition of FeO in its second stage i.e. acidogenic sulfate-reducing reactor (S1) resulted in microbial reduction of Fe (III), which significantly enhanced the biological sulfate reduction. In reactor S1, increasing influent sulfate concentration to 1400 mg/L resulted in a higher COD removal (27.3%) and sulfate reduction (57.9%). In the reference reactor without using FeO (S2), the COD and sulfate removal were 15.6% and 29%, respectively. The combined performance of the two-stage anaerobic reactor (A1, S1) also showed a higher COD removal of 74.2%. Denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis showed that the dominant bacteria with high similarity to IRB species as well as sulfate reducer Desulfovibrio and acidogenic bacteria (AB) were enriched in S1. Quantitative Polymerase Chain Reaction (qPCR) analysis presented a higher proportion of sulfate reducer Desulfovibrio marrakechensis and Fe (III) reducer Iron-reducing bacteria HN54 in S1.
Article
Glucose-fed high-rate UASB reactors were tested at three COD/SO4 ratios and hydraulic retention times to promote sulfate reducing activity and observe the effects on reactor performance. Different COD/SO4 ratios (20, 10, and 5) resulted in changes in organic matter removal, methane production, alkalinity, dissolved sulfide and biomass concentrations and profile. The COD removal dropped from 95 to 80–84 % at the lowest COD/SO4 ratio. Sulfate was removed at 79 to 89 % at the highest ratio and dropped to 72 to 74 % with increasing sulfate loading. Alkalinity was produced at higher levels with increasing sulfate loading. Specific methane production dropped with decreasing hydraulic retention times. Sulfate-reducing activity used a maximum of 11.7 % of organic matter at the highest sulfate loading level, producing a slight shift to sulfate-reducing activity in the substrate competition between sulfate-reducing bacteria and methanogens. Increased sulfate loading at COD/SO4 ratios of 10 and 5 caused deterioration of the concentration profile of the sludge, resulting in biomass washout and decreased volatile fraction of biosolids in the reactors.
Article
The influence of parameter changes on the bacterial community of a laboratory-scale anaerobic digester fed with glucose was investigated using a culture-independent approach based on single-strand conformation polymorphism (SSCP) analysis of total 16S rDNA and 16S rRNA amplification products. With the digester operating at steady state, the 16S rDNA SSCP patterns of the bacterial community showed eight peaks, whereas the 16S rRNA patterns showed six peaks with a very prominent one corresponding to a Spirochaetes-related bacterium. An acidic shock at pH 6 caused an increase in the 16S rRNA level of two Clostridium-related bacteria. After a 1 week starvation period, the major bacteria present reverted to a basal 16S rRNA level proportional to their 16S rDNA level. Starvation revealed the presence of a previously undetected peak whose corresponding sequence was deeply branched into the low G+C Gram-positive bacteria phylum. Twenty-four hours after a spiked addition to the starved digester community of starch, glucose, lactate or sulphate, an upsurge in several new 16S rRNA-derived peaks was observed. Thus, the perturbation approach combined with 16S rRNA analysis revealed bacteria that had not been detected through 16S rDNA analysis.
Article
Until recently, biological treatment of sulphate-rich wastewater was rather unpopular because of the production of H2S under anaerobic conditions. Gaseous and dissolved sulphides cause physical-chemical (corrosion, odour, increased effluent chemical oxygen demand) or biological (toxicity) constraints, which may lead to process failure. Anaerobic treatment of sulphate-rich wastewater can nevertheless be applied successfully provided a proper treatment strategy is selected. The strategies currently available are discussed in relation to the aim of the treatment: i) removal of organic matter, ii) removal of sulphate or iii) removal of both. Also a whole spectrum of new biotechnological applications (removal of organic chemical oxygen demand, sulphur, nitrogen and heavy metals), recently developed based on a better insight in sulphur transformations, are discussed.
Article
During anaerobic treatment of sulphate-containing wastewaters, sulphate-reducing bacteria (SRB) compete with methane-producing bacteria (MPB) for the available electron-donors. In this work, the anaerobic treatment of a synthetic wastewater, consisting of a mixture of acetate, propionate and butyrate and high concentrations of sulphate (COD: sulphate ratio 0·5) was studied in an upflow anaerobic granular sludge bed reactor. The influence of the superficial upward liquid velocity (), the influent composition and reactor pH on the competition between SRB and MPB was investigated. At a of 2 m h−1 and pH 8, 93–97% of the COD was degraded by SRB. With increasing , COD removal efficiencies decreased, while at a of 6 m h−1 the fraction of COD removed by MPB rose to 23%. Elevation of the influent acetate concentrations, by decreasing the (lower recirculation) or by the use of an influent volatile fatty acid mixture with a higher acetate content, resulted in an increase of methanogenesis up to 41% of the total COD removal. In contrast, elevated levels of propionate and butyrate in the influent favoured the sulphate reducing process. A decrease of pH from 8 to 7 resulted in free hydrogen sulphide concentrations higher than 200 mg litre−1. This strongly inhibited methanogenesis while SRB were hardly affected, with a subsequent decrease of the COD removed by MPB from 41 to 7% as a result.
Article
Effect of sulfate on the anaerobic degradation of benzoate was investigated by using the chemostat-type reactors at 35°C. The benzoate concentrations were equivalent to 1250–10000 mg.l−1 in COD (chemical oxygen demand) and the sulfate concentrations were equivalent to 167–1670 mg.l−1 in sulfur (S). Interactions between the methane-producing bacteria (MPB) and sulfate-reducing bacteria (SRB) were dependent strongly on the ratio of COD/S in wastewater. The MPB consumed 99% of the available electron donors at COD/S ratio of 60, but consumed only 69% at ratio of 1.5, and 13% at 0.75. The biochemical reactions and the bacterial composition in the biomass were also governed by the COD/S ratio. At high COD/S ratios (3.0 or higher), benzoate was degraded mainly to methane via acetate and hydrogen/formate. The degradation of benzoate required the syntrophic association between the hydrogen-producing acetogens such as Syntrophus buswellii and hydrogen-consuming MPB, plus Methanothrix-like MPB. On the other hand, at low COD/S ratio (1.5 or lower), benzoate was consumed mainly by SRB, converting sulfate into sulfide and suppressing the methane production. The anaerobic degradation of benzoate was partially inhibited when sulfide concentration was high.
Article
The effect of sulfate reduction on the acidogenic phase was investigated using anaerobic chemostat systems at 35°C. Sucrose (10,000 mg COD/l) was used as the sole organic substrate. Chemostat systems were maintained at hydraulic retention times (HRTs) of 2, 4, 6, 8 and 10 h. The sulfate concentration in the substrate was increased to 0 (control), 600, 1200 and 2400 mg/l. Sulfate reduction occurred even at an HRT of 2 h, while the hydrogen production rate evidently decreased with increasing sulfate concentration at HRTs of 2 and 4 h. Hydrogen was a key electron donor for the sulfate-reducing bacteria (SRB). The sulfate removal efficiencies were over 90% at sulfate concentrations of 600 and 1200 mg/l at HRTs of longer than 8 h. No inhibition in the degradation of sucrose was observed although the free-H2S concentration was up to 99 mg S/l. Hydrogen-consuming and lactate-consuming SRB were maximally enumerated at 108 and 109 MPN/ml at a sulfate concentration of 2400 mg/l and an HRT of 2 h. The SRB were enumerated at 105 to 107 MPN/ml even in the absence of sulfate. The results of this study showed that SRB could grow under acidogenic condition, and almost sulfate could be removed in the acidogenic phase.
Article
In the present study, thermophilic anaerobic digestion of wheat straw stillage was investigated. Methane potential of stillage was determined in batch experiments at two different substrate concentrations. Results showed that higher methane yields of 324 ml/g-(volatile solids) VSadded were obtained at stillage concentrations of 12.8 g-VS/L than at 25.6 g-VS/l. Continuous anaerobic digestion of stillage was performed in an up-flow anaerobic sludge blanket (UASB) reactor at 55 °C with 2 days hydraulic retention time. Results showed that both substrate concentration and organic loading rate (OLR) influenced process performance and methane yields. Maximum methane yield of 155 ml CH4/g-COD was obtained at stillage mixtures with water of 25% (v/v) in the feed and at an OLR of 17.1 g-COD/(l.d). Soluble chemical oxygen demand (SCOD) removal at this OLR was 76% (w/w). Increase in OLR to 41.2 g-COD/(l.d) and/or stillage concentration in the feed to 33–50% (v/v) resulted in low methane yields or complete process failure. The results showed that thermophilic anaerobic digestion of wheat straw stillage alone for methane production is feasible in UASB reactor at an OLR of 17.1 g-COD/(l.d) and at substrate concentration of 25% in the feed. The produced methane could improve the process energy and economics of a bioethanol plant and also enable to utilize the stillage in a sustainable manner.
Article
A study of the effect of organic loading rate (OLR) and hydraulic retention time (HRT) on the performance, stability and microbial communities of a laboratory-scale completely stirred tank anaerobic reactor treating two-phase olive mill solid residue (OMSR) was carried out at mesophilic temperature (35 °C). The reactor operated at a fixed influent substrate concentration of 162 g total chemical oxygen demand (COD)/L and 126 g volatile solids (VS)/L. The OLR and HRT varied in the ranges of 0.8–11.0 g COD/L day and 108–15 days, respectively. COD removal efficiencies in the range of 97–77% were achieved for OLRs and HRTs in the ranges of 1.5–9.2 g COD/L day and 108–17 days, respectively. The maximum methane production rate was found to be 1.7 L CH4 STP/L day and it was achieved for an OLR of 9.2 g COD/L day and HRT of 17 days. The methane yield coefficient was 0.244 ± 0.005 L methane at STP conditions/g COD removed. The results obtained demonstrated that an OLR of 11.0 g COD/L day and a HRT of 15 days brought about a decrease in the pH and total volatile fatty acids (TVFA)/alkalinity ratios up to values of 5.3 and 1.5 (mequiv. acetic acid/mequiv. CaCO3), respectively, causing the destabilization of the reactor and process failure. Microbial communities, both Bacteria and Archaea, were studied by molecular fingerprinting methods, cloning and sequencing. Molecular fingerprints of bacterial communities showed higher number of major bands at increasing OLR. Firmicutes, mostly represented by the genus Clostridium, were the predominant bacteria at low OLR. Other bacterial communities such as Gammaproteobacteria, Actinobacteria, Bacteroidetes and Deferribacteres were the most abundant at high OLR. The Archaea were mainly represented by four phylotypes belonging to the genus Methanosaeta independently of the OLR. This study remarks the interest of relating OMSR decomposing bioreactor performance with the microbial communities carrying out the process in order to better understand and monitor this anaerobic digestion.
Article
The mutual interaction between sulfate-reducing bacteria (SRB) and methane-producing bacteria (MPB) in anaerobic sludge consortia was investigated using three identical laboratory-scale UASB reactors. Reactors were fed in parallel with a synthetic low strength waste (starch and sucrose, 500 mg COD l−1), but with different levels of sulfate (30, 150, 600 mg SO42- l−1, respectively). The mass balances of COD and sulfur over the experimental period of 180 days operation indicated that the higher the level of sulfate the less methane production caused since a greater electron flow was distributed to the SRB. Namely, at the last stage of the experiment in which the highest sulfate level was imposed, 75% of the total COD removal was performed by SRB. The specific methanogenic activities (SMAs) of the respective sludges were evaluated using the serum vial test using different substrates and by setting different sulfate levels. SMA was not affected by the presence of sulfate in the vials when acetate was used as the vial substrate. In the case of glucose as the test substrate, SMA increased with an increase in sulfate level. On the other hand, SMA decreased with increasing sulfate level when hydrogen was employed as the test substrate. A large amount of propionate accumulation was observed during vial tests, in which glucose was fed to the sludge grown at higher levels of sulfate, when zero or low levels of sulfate were added to the vials. This result suggests that SRB played an important role in the breakdown of propionate either through direct utilization or through a so-called interspecies hydrogen transfer.
Article
The feasibility of thermophilic (55°C) anaerobic treatment of an alcohol distillery wastewater (cane molasses vinasse) was studied using a 140 l upflow anaerobic sludge blanket (UASB) reactor for a period of 430 days. Organic loading rates were applied up to 28 kg chemical oxygen demand (COD) m−3 d−1 by reducing hydraulic retention time (HRT) at a fixed influent concentration of 10 g COD l−1. Chemical oxygen demand removals during the entire experimental period were relatively low (39–67%), while biochemical oxygen demand (BOD) removals were more satisfactory (more than 80%). The biodegradability of the vinasse used in this study was assessed by a serum vial test. The test revealed that methane production rates from the vinasse were only , , and as great as those from , acetate, glucose and another type of vinasse (malt), respectively. The poor performance of the reactor for COD elimination can probably be attributed to the low degradability of the waste itself. Methanogenic activity of the retained sludge increased 24 times, for acetate, and 13 times, for , as much as that of the inoculum sludge. The optimum temperature of methanogenic activity of the sludge depended on the substrate used; i.e. 60°C for acetate, 60–65°C for , and around 55°C for the waste.
Article
A technical evaluation of stillage characterization, treatment, and by-product recovery in the ethanol industry was performed through a review of the scientific literature, with particular emphasis on solutions pertinent to a cellulosic-based ethanol production system. This effort has generated substantial information supporting the viability of anaerobic digestion for stillage treatment followed by land application on biomass crops for nutrient recovery. Generally, the characteristics of stillage from cellulosic materials appear comparable to those of conventional sugar- and starch-based feedstocks. However, the data on cellulosic stillage characteristics and treatment parameters are extremely limited and highly variable. This has significant impacts on the capital costs and biogas recovery of anaerobic treatment systems predicted from these data. In addition, technical questions remain unanswered with regard to stillage toxicity from untested feedstocks and the impact of heavy metal leaching when acid hydrolysis reactors are fabricated from corrosion-resistant alloys. Thermophilic anaerobic digestion of ethanol stillage achieves similar treatment efficiencies and methane yields compared to mesophilic treatment, but at almost twice the organic loading rate. Therefore, application of thermophilic anaerobic digestion would improve process economics, since smaller digesters and less stillage cooling are required. Downstream processes for stillage utilization and by-product recovery considered worthy of continued investigation include the production of feed (from single cell protein and/or algae production), color removal, and production of calcium magnesium acetate. This study finds that sustainable and economically viable solutions are available for mitigating the environmental impacts which result from large-scale biomass-to-ethanol conversion facilities. However, further research in some areas is needed to facilitate successful implementation of appropriate technology options.
Article
During the anaerobic treatment of high sulfate content wastewater, large amounts of sulfide are produced and cause unfavorable conditions. The start-up of such a digestion is often a critical step. This study provides key parameters for a proper start-up. The effect of the sludge and the influence of trace metals were investigated. Using discontinuous cultures, it was shown that an addition of iron followed by a cobalt supply increased the Total Organic Carbon (TOC) — and acetate — removal rates, while an input of one metal alone remained inefficient. Nickel seemed not to be limiting.This treatment was successfully applied to Continuous Stirred Tank Reactors (CSTR). Finally, after modifying the design from a chemostat to an anaerobic contact reactor, the digestion was immediately stabilized. This sudden behavior shift was unexpected. It was suggested that metal complexing agents produced by bacteria were recycled with the sludge and contributed to an increase of essential metals in the broth.
Article
The biomass of industrially grown Phaeodactylum tricornutum was subjected in a novel way to bio-methanation at 33°C, i.e., in an anaerobic membrane bioreactor (AnMBR) at a hydraulic retention time of 2.5 days, at solid retention times of 20 to 10 days and at loading rates in the range of 2.6-5.9 g biomass-COD L(-1) day(-1) with membrane fluxes ranging from 1 to 0.8 L m(-2) h(-1). The total COD recovered as biogas was in the order of 52%. The input suspension was converted to a clear effluent rich in total ammonium nitrogen (546 mg TAN L(-1)) and phosphate (141 mg PO(4)-P L(-1)) usable as liquid fertilizer. The microbial community richness, dynamics, and organization in the reactor were interpreted using the microbial resource management approach. The AnMBR communities were found to be moderate in species richness and low in dynamics and community organization relative to UASB and conventional CSTR sludges. Quantitative polymerase chain reaction analysis revealed that Methanosaeta sp. was the dominant acetoclastic methanogen species followed by Methanosarcina sp. This work demonstrated that the use of AnMBR for the digestion of algal biomass is possible. The fact that some 50% of the organic matter is not liquefied means that the algal particulates in the digestate constitute a considerable fraction which should be valorized properly, for instance as slow release organic fertilizer. Overall, 1 kg of algae dry matter (DM) could be valorized in the form of biogas ( euro 2.07), N and P in the effluent (euro 0.02) and N and P in the digestate (euro 0.04), thus totaling about euro 2.13 per kilogram algae DM.
Article
Shifts in bacterial and archaeal communities, associated with changes in chemical profiles, were investigated in an anaerobic batch reactor treating dairy-processing wastewater prepared with whey permeate powder. The dynamics of bacterial and archaeal populations were monitored by quantitative real-time PCR and showed good agreement with the process data. A rapid increase in bacterial populations and a high rate of substrate fermentation were observed during the initial period. Growth and regrowth of archaeal populations occurred with biphasic production of methane, corresponding to the diauxic consumption of acetate and propionate. Bacterial community structure was examined by denaturing gel gradient electrophoresis (DGGE) targeting 16S rRNA genes. An Aeromonas-like organism was suggested to be mainly responsible for the rapid fermentation of carbohydrate during the initial period. Several band sequences closely related to the Clostridium species, capable of carbohydrate fermentation, lactate or ethanol fermentation, and/or homoacetogenesis, were also detected. Statistical analyses of the DGGE profiles showed that the bacterial community structure, as well as the process performance, varied with the incubation time. Our results demonstrated that the bacterial community shifted, reflecting the performance changes and, particularly, that a significant community shift corresponded to a considerable process event. This suggested that the diagnosis of an anaerobic digestion process could be possible by monitoring bacterial community shifts.
Article
In this study, the microbial community characteristics in continuous lab-scale anaerobic reactors were correlated to reactor functionality using the microbial resource management (MRM) approach. Two molecular techniques, denaturing gradient gel electrophoresis (DGGE) and terminal-restriction fragment length polymorphism (T-RFLP), were applied to analyze the bacterial and archaeal communities, and the results obtained have been compared. Clustering analyses showed a similar discrimination of samples with DGGE and T-RFLP data, with a clear separation between the meso- and thermophilic communities. Both techniques indicate that bacterial and mesophilic communities were richer and more even than archaeal and thermophilic communities, respectively. Remarkably, the community composition was highly dynamic for both Bacteria and Archaea, with a rate of change between 30% and 75% per 18 days, also in stable performing periods. A hypothesis to explain the latter in the context of the converging metabolism in anaerobic processes is proposed. Finally, a more even and diverse bacterial community was found to be statistically representative for a well-functioning reactor as evidenced by a low Ripley index and high biogas production.
Article
Qualitative and quantitative molecular analysis techniques were used to determine associations between differences in methanogenic microbial communities and the efficiency of batch anaerobic digesters. Two bioreactors were initially seeded with anaerobic sludge originating from a local municipal wastewater treatment plant and then supplemented with swine wastewater. Differences were observed in the total amount of methane produced in the two bioreactors (7.9L/L, and 4.5L/L, respectively). To explain these differences, efforts were taken to characterize the microbial populations present using a PCR-based DGGE analysis with methanogenic primer and probe sets. The groups Methanomicrobiales (MMB), Methanobacteriales (MBT), and Methanosarcinales (MSL) were detected, but Methanococcales (MCC) was not detected. Following this qualitative assay, real-time PCR was used to investigate quantitative differences in the populations of these methanogenic orders. MMB was found to be the dominant order present and its abundance patterns were different in the two digesters. The population profiles of the other methanogenic groups also differed. Through redundancy analysis, correlations between the concentrations of the different microbes and chemical properties such as volatile fatty acids were calculated. Correlations between MBT and MSL populations and chemical properties were found to be consistent in both digesters, however, differences were observed in the correlations between MMB and propionate. These results suggest that interactions between populations of MMB and other methanogens affected the final methane yield, despite MMB remaining the dominant group overall. The exact details of why changes in the MMB community caused different profiles of methane production could not be ascertained. However, this research provides evidence that microbial behavior is important for regulating the performance of anaerobic processes.
Article
The competition of acetotrophic methanogenic bacteria (AMB) and acetotrophic sulfidogenic bacteria (ASRB) was studied by assessing growth rates, activities and acetate and sulfate affinities at different pH levels and sulfide concentrations in batch reactors. Both anaerobic granular sludge and suspended anaerobic sludge were tested. The results indicate that at pH-levels below 6.9 AMB will outcompete ASRB, whereas above a pH of 7.7, ASRB will win the competition. If ASRB and AMB are present in granular sludge growth will be found in a wider pH-range than if they are present as suspended sludge. The affinities for acetate and the sulfide toxicity are dependent on the sludge form as well. In granular sludge the acetate affinities of ASRB and AMB are comparable, whereas in suspended sludge ASRB show a lower affinity than AMB. With respect to sulfide toxicity, the results indicate that above pH 7 sulfide inhibition in granular sludge is caused by the total sulfide concentration, while in suspended sludge the free H2S-concentration determines the toxicity. At high pH-levels growth is stronger inhibited than the activity.
Article
In the anaerobic treatment of sulfate containing wastewater sulfate reducing bacteria (SRB) will compete with methanogenic- (MB) and acetogenic bacteria (AB) for the available substrates such as hydrogen, acetate, propionate and butyrate. The outcome of this competition will determine the endproduct of the anaerobic mineralisation proces: methane or sulfide.The occurrence of the sulfate reduction proces is often considered unwanted due to the problems associated with the sulfide formed in the proces. These problems are: malodour, corrosion, toxicity, reduced removal of COD, reduced methane formation and higher levels of H <sub>2</sub> S in the biogas. More recently the sulfate reduction proces is used in biological processes that aim at the removal of oxidised sulfur compounds from different waste streams. In the competition between the SRB and AB this study shows that butyrate degrading AB can effectively compete with the SRB. The growth rates of both bacteria was found in the same range. On the contrary, propionate degrading AB are outcompeted by the SRB due to the better growth kinetic properties of the latter.Concerning the competition between the SRB and MB for hydrogen the present study clearly shows that in anaerobic reactors hydrogenotrophic MB are outcompeted by the SRB. This apply both for mesophilic (30 °C) as for thermophilic (55 °C) conditions. However, the hydrogenotrophic MB are not expelled from the biomass but remain present in relative high number.The competition between the acetotrophic MB (AMB) and acetotrophic SRB (ASRB) depends on several conditions. In this research the following items were investigated:* The kinetic growth properties of AMB and ASRB under different conditions with respect to the pH and sulfide concentration.* The ability of the ASRB and AMB to attach to granular sludge or a biofilm.* The competition between the bacteria at higher temperatures (55 °C)The results of these studies are:* At neutral or acidic pH values the AMB can compete with the ASRB. The growth rates and acetate affinities for both bacteria are than in the same range. Moreover, at these pH values the AMB and ASRB are more or less equally inhibited by the toxic sulfide. At more alkalic pH values (PH>7.5) the ASRB likely will outcompete the AMB. At these pH values the growth rates of the ASRB are significant higher then for the AMB and the ASRB are much less inhibited by the produced sulfide.* Granulation experiments shows that the ASRB can maintain in granular sludge, resulting in the formation of sulfidogenic granular sludge. They are also effectively able to form a biofilm on pumice as a carrier. No significant difference between the attachment capacity of AMB or ASRB could be detected.* Under thermophilic (55 °C) conditions the ASRB can compete with the AMB. At higher pH values than 7.5 the ASRB even become predominant. At more neutral pH values there exist an equilibrium between the ASRB and AMB.
Article
In the anaerobic treatment of sulfate containing wastewater sulfate reducing bacteria (SRB) will compete with methanogenic- (MB) and acetogenic bacteria (AB) for the available substrates such as hydrogen, acetate, propionate and butyrate. The outcome of this competition will determine the endproduct of the anaerobic mineralisation proces: methane or sulfide.The occurrence of the sulfate reduction proces is often considered unwanted due to the problems associated with the sulfide formed in the proces. These problems are: malodour, corrosion, toxicity, reduced removal of COD, reduced methane formation and higher levels of H 2 S in the biogas. More recently the sulfate reduction proces is used in biological processes that aim at the removal of oxidised sulfur compounds from different waste streams. In the competition between the SRB and AB this study shows that butyrate degrading AB can effectively compete with the SRB. The growth rates of both bacteria was found in the same range. On the contrary, propionate degrading AB are outcompeted by the SRB due to the better growth kinetic properties of the latter.Concerning the competition between the SRB and MB for hydrogen the present study clearly shows that in anaerobic reactors hydrogenotrophic MB are outcompeted by the SRB. This apply both for mesophilic (30 °C) as for thermophilic (55 °C) conditions. However, the hydrogenotrophic MB are not expelled from the biomass but remain present in relative high number.The competition between the acetotrophic MB (AMB) and acetotrophic SRB (ASRB) depends on several conditions. In this research the following items were investigated:* The kinetic growth properties of AMB and ASRB under different conditions with respect to the pH and sulfide concentration.* The ability of the ASRB and AMB to attach to granular sludge or a biofilm.* The competition between the bacteria at higher temperatures (55 °C)The results of these studies are:* At neutral or acidic pH values the AMB can compete with the ASRB. The growth rates and acetate affinities for both bacteria are than in the same range. Moreover, at these pH values the AMB and ASRB are more or less equally inhibited by the toxic sulfide. At more alkalic pH values (PH>7.5) the ASRB likely will outcompete the AMB. At these pH values the growth rates of the ASRB are significant higher then for the AMB and the ASRB are much less inhibited by the produced sulfide.* Granulation experiments shows that the ASRB can maintain in granular sludge, resulting in the formation of sulfidogenic granular sludge. They are also effectively able to form a biofilm on pumice as a carrier. No significant difference between the attachment capacity of AMB or ASRB could be detected.* Under thermophilic (55 °C) conditions the ASRB can compete with the AMB. At higher pH values than 7.5 the ASRB even become predominant. At more neutral pH values there exist an equilibrium between the ASRB and AMB.
Article
Until recently, biological treatment of sulphate-rich wastewater was rather unpopular because of the production of H2S under anaerobic conditions. Gaseous and dissolved sulphides cause physical-chemical (corrosion, odour, increased effluent chemical oxygen demand) or biological (toxicity) constraints, which may lead to process failure. Anaerobic treatment of sulphate-rich wastewater can nevertheless be applied successfully provided a proper treatment strategy is selected. The strategies currently available are discussed in relation to the aim of the treatment: i) removal of organic matter, ii) removal of sulphate or iii) removal of both. Also a whole spectrum of new biotechnological applications (removal of organic chemical oxygen demand, sulphur, nitrogen and heavy metals), recently developed based on a better insight in sulphur transformations, are discussed.
Article
The anaerobic biological treatment of volatile fatty acid (VFA) - and sucrose - based wastewaters was investigated in two anaerobic bioreactors, R1 and R2, over a 300-day trial period. During the trial, the operating temperature of both reactors was lowered, in a stepwise fashion, from 37 to 16 degrees C. The VFA-fed reactor maintained an excellent level of performance, regardless of operating temperature, reaching COD removal efficiencies of 95% at 18 degrees C, and a biogas methane content in excess of 70% at 16 degrees C, at an imposed OLR of 20 kg COD m(-3) d(-1). However, an increase in the applied liquid upflow velocity to the bottom chamber of the reactor from 5 to 7.5 m h(-1)on day 236 resulted in a considerable decline in reactor performance. COD removal efficiencies in excess of 80% were achieved by the sucrose-fed reactor at 18 degrees C, at an imposed OLR of 20 kg COD m(-3) d(-1). An increase in the liquid upflow velocity applied to the sucrose-fed reactor resulted in enhanced reactor performance and stability, with respect to decreasing temperature. The different responses of both reactors to increased upflow velocity was associated with variations in the microbial population structure of the sludges, as determined by culture-independant molecular approaches, specifically the presence of high levels of delta-Proteobacteria and hydrogenotrophic methanogens in the VFA-fed biomass. High levels of Methanomicrobiales sp., in particular Methanocorpusculum parvum sp., were observed in both R1 and R2 during the trial. There was a distinct shift from acetoclastic methanogenic dominance to hydrogenotrophic dominance in both reactors in response to a decrease in the operating temperature.
Article
The diversity of bacterial groups of activated sludge samples that received wastewater from four different types of industry was investigated by a nested PCR-DGGE (denaturing gradient gel electrophoresis) approach. Specific 16S rRNA primers were chosen for large bacterial groups (Bacteria and alpha-Proteobacteria in particular), which dominate activated sludge communities, as well as for actinomycetes, ammonium oxidisers and methanotrophs (types I and II). In addition primers for the new Acidobacterium kingdom were used to observe their community structure in activated sludge. After this first PCR amplification, a second PCR with bacterial primers yielded 16S rRNA gene fragments that were subsequently separated by DGGE, thus generating 'group-specific DGGE patterns'. The community structure and diversity of the bacterial groups from the different samples was further analysed using different techniques, such as statistical analysis and Shannon diversity index evaluation of the band patterns. By combining the seven DGGE gels, cluster analysis, multidimensional scaling and principal component analysis clearly clustered two of the four activated sludge types separately. It was shown that the combination of molecular and statistical methods can be very useful to differentiate microbial communities.