A kinetic model incorporating energy spilling for substrate removal in substrate-sufficient batch culture of activated sludge

Batch assays are currently used to study the kinetic behavior of microbial growth. However, it has been shown that the outcome of batch experiments is greatly influenced by the initial ratio of substrate concentration (S _o) to biomass concentration (X _o). Substrate-sufficient batch culture is known to have mechanisms of spilling energy that lead to significant nongrowth-associated substrate consumption, and the Monod equation is no longer appropriate. By incorporating substrate consumption associated with energy spilling into the balance of the substrate oxidation reaction, a kinetic model for the observed specific substrate consumption rate was developed for substrate-sufficient batch culture of activated sludge, and was further verified by experimental data. It was demonstrated that the specific substrate consumption rate increased with the increase of the S _o/X _o ratio, and the majority of substrate was consumed through energy spilling at high S _o/X _o ratios. It appears that the S _o/X _o ratio is a key parameter in regulating metabolic pathways of microorganisms. Received: 18 January 1999 / Received revision: 7 May 1999 / Accepted: 28 May 1999     

Kinetics of product formation in batch and continuous culture of Clostridium barkeri

Summary The main fermentation end products in batch culture (unlimited glucose supply) of Clostridium barkeri were butyrate and lactate. The specific rate of butyrate production was linearly proportional to the growth rate while the specific rate of lactate production increased at low growth rates. In a glucose limited chemostat culture butyrate production was partly growth associated while acetate and lactate production was growth associated. Lactate was, however, only produced at high dilution rates. By varying the glucose concentration in the inflowing medium it was shown that lactate production was stimulated by a high feeding rate of the carbon source. These results are discussed in view of the fructose-1,6-diphosphate dependent lactate dehydrogenase activity in many other organisms.     

A kinetic model for energy spilling-associated product formation in substrate-sufficient continuous culture

It has been demonstrated that excess substrate can cause uncoupling between anabolism and catabolism, which leads to energy spilling. However, the Luedeking-Piret equation for product formation does not account for the energy spilling-associated product formation due to substrate excess. Based on the growth yield and energy uncoupling models proposed earlier, a kinetic model describing energy spilling-associated product formation in relation to residual substrate concentration was developed for substrate-sufficient continuous culture and was further verified with literature data. The parameters in the proposed model are well defined and have their own physical meanings. From this model, the specific productivity of unit energy spilling-associated substrate consumption, and the maximum product yield coefficient, can be determined. Results show that the majority of energy spilling-associated substrate consumption was converted to carbon dioxide and less than 6% was fluxed into the metabolites, while it was found that the maximum product yield coefficients varied markedly under different nutrient limitations. The results from this research can be used to develop the optimized bioprocess for maximizing valuable product formation.     

Formation of soluble microbial products by activated sludge under anoxic conditions

In this work, both experimental and modeling approaches are used to explore the formation of soluble microbial products (SMP) by activated sludge under anoxic conditions. With substrate consumption, the SMP concentration increases gradually. Utilization associated products (UAP) are the main fraction of SMP when substrate is present; whereas biomass associated products (BAP) are the major content of SMP as substrate is completely consumed. The fraction of the accumulated SMP accounts for 3-4% of initial organic substrate. Three dimensional excitation emission matrix analysis results indicate that the SMP concentration increases in the denitrification process. The accumulation of nitrite up to 22.6 mg/l under anoxic conditions has no significant effect on the SMP formation. With a consideration of SMP formation under anoxic conditions, an ASM3-based denitrification model is developed. The results show that the developed model is able to capture the relationship between the SMP formation and the substrate consumption by activated sludge in the denitrification process.     

Molecular identification of the microbial diversity in two sequencing batch reactors with activated sludge

The diversity of the microbial community was identified in two lab-scale, ideally mixed sequencing batch reactors which were run for 115 days. One of the reactors was intermittently aerated (2 h aerobically/2 h anaerobically) whereas the other was consistently aerated. The amount of biomass as dry matter, the degradation of organic carbon determined by chemical oxygen demand and nitrogen-degradation activity were followed over the operation of the two reactors and did not show significant differences between the two approaches at the end of the experiment. At this point, the composition of the microbial community was determined by a terminal restriction fragment length polymorphism approach using multiple restriction enzymes by which organisms were retrieved to the lowest taxonomic level. The microbial composition was then significantly different. The species richness was at least five-fold higher in the intermittently aerated reactor than in the permanently kept aerobic approach which is in line with the observation that ecosystem disturbances result in higher diversity.     

Formation of soluble microbial products (SMP) by activated sludge at various salinities

Soluble microbial products (SMP) present a significant component of effluent organic matter from biological wastewater treatment reactors, and can affect the membrane fouling and formation of disinfection by-products. Thus, SMP have attracted increasing concerns in wastewater treatment and reclamation. In this work, the formation of SMP by activated sludge at various NaCl concentrations is investigated by using fluorescence excitation–emission matrix (EEM) spectroscopy with parallel factor analysis (PARAFAC) and fluorescence regional integration (FRI). The results show that a high level of salinity decreases substrate removal efficiency and leads to an accumulation of SMP, especially proteins. Three components of SMP, one protein-like and two humic-acid-like components, are identified by PARAFAC, which exhibit different trends with the variation of NaCl concentration. FRI analysis reveals that the majority of protein fluorescence is attributed to tryptophan and tryptophan-like proteins, rather than tyrosine and tyrosine-like proteins. With an increase in NaCl concentration, the normalized volume percentages of tyrosine and tryptophan region increase, while those of humic- and fulvic-acid-like region decrease significantly. This work demonstrates that salinity affects the formation of SMP, and that EEM with PARAFAC in combination with FRI analysis is a useful tool to get insight into the formation of SMP by activated sludge.     

Comparison of soluble microbial products released from activated sludge and aerobic granular sludge systems in the presence of toxic 2,4-dichlorophenol

Bioprocess and Biosystems Engineering - The objective of this study was to compare the release of soluble microbial products (SMP) from activated sludge (AS) and aerobic granular sludge (AGS) in...     

Kinetics of formation of humic substances in activated sludge systems and their effect on flocculation

Humic substances were isolated during batch aeration studies with activated sludge and a complex waste source, by using concentration and separation techniques that employ reverse osmosis, ultrafitration, and gel permeation chromatography. The study suggests that the formation of high molecular weight humic substances may occur after the removal of the readily available carbon source. The amount of refractory material finally present in the solution will depend on its adsorptive properties toward bacterial cells. The adsorptive characteristics may be determined by the magnitude of the carbohydrate fraction present in the humic substances. If the carbohydrate content decreases, adsorption onto the cells may decrease resulting in an impairment of both the floe formation and settleability of the sludge floes. Decreased adsorption will result in a higher total organic carbon content and an increase in color bearing materials in the effluent.     

Model of energy uncoupling for substrate-sufficient culture

The growth yields (Y(obs)) are greater under substrate-limited conditions than those under substrate-sufficient conditions in continuous cultures. This indicates that the excess substrate should cause uncoupling between anabolism and catabolism, which leads to energy spilling. Although the uncoupling between anabolism and catabolism has already been recognized in the microbiology literature, how to quantitatively describe such uncoupling remains unclear. Based on a balance on substrate reaction, a growth yield model was developed in relation to residual substrate concentration for substrate-sufficient continuous cultures. On the basis of that yield model, the concept of an uncoupling coefficient between anabolism and catabolism is defined in this work. A model describing the effect of the residual substrate concentration on the uncoupling coefficient of anabolism to catabolism is proposed. This model agrees very well with literature data. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 571-576, 1997.     

Reduction in excess sludge production by addition of chemical uncouplers in activated sludge batch cultures

AIMS: To investigate the possibility of reducing excess sludge production in activated sludge processes by the addition of chemical uncouplers to greatly dissociate anabolism from catabolism. METHODS AND RESULTS: Ortho-chlorophenol (oCP), 2,4-dichlorophenol (DCP), 3,3',4',5-tetrachlorosalicylanilide (TCS), para-dinitrophenol (pNP) and 2,4-dinitrophenol (DNP) were chosen for short-term tests for their ability to reduce sludge yield by shaking bottle test. The most effective chemicals, DNP and pNP, together with TCS were tested for various uncoupler concentrations and biomass concentrations. TCS was tested in a lab-scale completely mixed activated sludge batch culture. The model (demonstrated by Liu) was verified with experimental data in completely mixed activated sludge batch test, but was inconsistent with the results from the shaking bottle batch test. The observed growth yield (Yobs) decreased with increasing of the ratio of initial uncoupler concentration to initial biomass concentration (Cu/X0). CONCLUSIONS: We suggest that the uncouplers oCP, DCP, TCS, pNP and DNP can cause a significant decrease in sludge production, the metabolism of which can explain the decline in sludge yield. SIGNIFICANCE AND IMPACT OF THE STUDY: The real strength of chemical uncoupler imposing on biomass should be Cu/X0, not initial uncoupler concentration (Cu) alone. Chemical uncouplers can be used to develop the activated sludge processes for minimizing excess sludge production.     

Modeling of typical microbial cell growth in batch culture

A mathematical model was developed, based on the time dependent changes of the specific growth rate, for prediction of the typical microbial cell growth in batch cultures. This model could predict both the lag growth phase and the stationary growth phase of batch cultures, and it was tested with the batch growth ofTrichoderma reesei andLactobacillus delbrucckii.     

Kinetics of microbial product formation and its consequences for the optimization of fermentation processes

Different types of product formation kinetics are discussed with respect to their significance for fermentation process economics. Microbial products belonging to various classes are formed in a growth-coupled manner. It is often found that the specific rate of product formation increases with the specific growth rate, approaching a maximum at higher growth rates. It is illustrated that for such types of relationship between the product formation rate and the growth rate process conditions are optimal when the specific rate of product formation is about half-maximal.     

活性污泥微生物群落宏组学研究进展

活性污泥是全球最常用的废水生物处理人工生态系统,微生物是驱动其污染净化能力的关键。活性污泥微生物群落所有物种与基因(简称"微生物组")的研究先后经历了"显微镜观察和纯菌培养分离"(1915)、"PCR扩增-测序"(1994)和"高通量测序-宏组学分析"(2006)三个重要阶段的发展变迁。相应地,我们对活性污泥微生物组的认知经历了从最早对微型动物(如钟虫和轮虫)及其他微生物的形貌观察和纯种培养鉴定到今天对整个微生物组的全局多样性认识的飞跃。近13年来,基于高通量测序的宏组学方法被广泛应用于揭示活性污泥微生物群落组成结构和功能,我们现在充分意识到活性污泥微生物组蕴藏着大量不可培养新物种和基因多样性,驱动着各类污染物的降解与转化。目前,特异性分子标记基因的扩增子测序技术已经被广泛应用于揭示城市和工业废水处理活性污泥微生物组和典型功能种群(如硝化细菌和聚磷菌)的时空多样性和群落构建机制,进而为未来实现活性污泥微生物组功能的精准调控奠定理论基础。宏基因组学研究在群落、种群和个体基因组水平全面解析了活性污泥微生物组驱动的碳、氮、磷元素循环过程,以及有机微污染物的生物降解和转化机理。将来活性污泥微生物组学研究需要在"标准化的组学分析方法和绝对定量""高通量培养组学""高通量功能基因组学"和"多组学方法的结合及多种方法并用"4个方面取得实现精准生态基因组学所需的技术突破,以最大限度发掘活性污泥微生物组在污水处理与资源回收领域的生态学与工程学价值。     

Entropy balances of microbial product formation

Considering the approach of Bermudez and Wagensberg (1986) devoted to the entropy balance of growing microorganisms some equations were developed which describe particularly the entropy balance of microbial product formation. The formula allows to determine the coefficients of resistance R(mn) and of coupling L(mn) according to rates of growth, product formation, maintenance metabolism and heat evolution assuming a linear relationship between thermodynamic fluxes and forces.In order to check the usefulness of the derived model appropriate experimental data of two microbial batch processes concerning production of L-lysine and the antibiotic nourseothricine were taken into account. The results showed similar courses of entropy balances despite different pathways of product formation which were characterized by an overshoot of entropy production at the beginning of biosynthesis of the primary and secondary metabolite. This fact was interpreted as a more general phenomenon for microorganisms under inbalanced nutritional conditions.     

Determination of microbial respiratory and redox activity in activated sludge

The tetrazolium salt 5-cyano-2,3-ditolyltetrazolium chloride (CTC) was used for the determination of metabolically active bacteria in active sludge. The method was adapted and optimized to the conditions of activated sludge. The colorless and nonfluorescent tetrazolium salt is readily reduced to a water-insoluble fluorescent formazan product via the microbial electron transport system and indicates mainly dehydrogenase activity. After more than 2 h incubation, no further formation of new formazan crystals was observed, although the existing crystals in active cells continued to grow at the optimal CTC-concentration of 4 mM. The dehydrogenase activity determined by direct epifluorescence microscopic enumeration did not correlate with cumulative measured activity as determined by formazan extraction. The addition of nutrients did not lead to an increase of CTC-active cells. Sample storage conditions such as low temperature or aeration resulted in a significant decrease in dehydrogenase activity within 30 min. The rapid and sensitive method is well suited for the detection and enumeration of metabolically active microorganisms in activated sludge. Extracellular redox activity was measured with the tetrazolium salt 3′-{1-[phenylamino-) carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate (XTT), which remains soluble in its reduced state, after extraction of extracellular polymeric substances (EPS) with a cation exchange resin. Received 12 August 1996/ Accepted in revised form 29 May 1997     

Characterization of soluble microbial products (SMP) derived from glucose and phenol in dual substrate activated sludge bioreactors

Soluble microbial products (SMP) generated by activated sludge cultures receiving a mixed feed of phenol and glucose were characterized with respect to molecular weight (MW) distribution, octanol-water partition coefficient (K(ow)), and Microtox toxicity. Short-term batch reactor tests using 14C-labeled substrates were performed to collect SMP derived from each substrate, while long-term tests were performed with SMP accumulated over multiple feed cycles using fed-batch reactors receiving non-labeled substrates. Yield of SMP in the batch tests, 10%-20% for phenol and 2%-5% for glucose, differed for each substrate and was independent of initial concentration. The MW distribution (MWD) of SMP was independent of feed composition, and was bimodal in the < 1 kDa and 10-100 kDa MW ranges for phenol-derived SMP and predominantly < 1 kDa for glucose-derived SMP. In the non-labeled tests, the fraction of SMP of MW > 100 kDa increased with the proportion of glucose in the feed. The K(ow) of phenol-derived SMP was higher compared to glucose-derived SMP, indicating that the phenol-derived SMP were more hydrophobic. This was particularly true at an acidic pH, where the K(ow) was 4.2 +/- 1.0 for phenol-derived SMP versus 0.13 +/- 0.13 for glucose-derived SMP. Toxicity testing indicated that phenol-derived SMP, exerting a mean Microtox inhibition of 1%, were less toxic than phenol itself, and showed little correlation between toxicity and concentration. However, glucose-derived SMP were generally more toxic than glucose itself (a non-toxic substrate), and the toxicity increased linearly with the concentration of SMP.