Granular Biomass Research Articles

Interactions between anaerobic ammonium- and methane-oxidizing microorganisms in a laboratory-scale sequencing batch reactor

The reject water of anaerobic digestors still contains high levels of methane and ammonium that need to be treated before these effluents can be discharged to surface waters. Simultaneous anaerobic methane and ammonium oxidation performed by nitrate/nitrite-dependent anaerobic methane-oxidizing(N-damo) microorganisms and anaerobic ammonium-oxidizing(anammox) bacteria is considered a potential solution to this challenge. Here, a stable coculture of N-damo archaea, N-damo bacteria, and anammox bacteria was obtained in a sequencing batch reactor fed with methane, ammonium, and nitrite. Nitrite and ammonium removal rates of up to 455 mg N-NO2− L−1 day−1 and 228 mg N-NH4+ L−1 were reached. All nitrate produced by anammox bacteria (57 mg N-NO3− L−1 day−1) was consumed, leading to a nitrogen removal efficiency of 97.5%. In the nitrite and ammonium limited state, N-damo and anammox bacteria each constituted about 30–40% of the culture and were separated as granules and flocs in later stages of the reactor operation. The N-damo archaea increased up to 20% and mainly resided in the granular biomass with their N-damo bacterial counterparts. About 70% of the nitrite in the reactor was removed via the anammox process, and batch assays confirmed that anammox activity in the reactor was close to its maximal potential activity. In contrast, activity of N-damo bacteria was much higher in batch, indicating that these bacteria were performing suboptimally in the sequencing batch reactor, and would probably be outcompeted by anammox bacteria if ammonium was supplied in excess. Together these results indicate that the combination of N-damo and anammox can be implemented for the removal of methane at the expense of nitrite and nitrate in future wastewater treatment systems.

Treatment of poultry slaughterhouse wastewater using a down-flow expanded granular bed reactor

Abstract This study evaluated the performance of a novel high rate anaerobic bioreactor system for the treatment of poultry slaughterhouse wastewater (PSW). The new system consisted of a granule-based technology operated in a down-flow configuration, with the assistance of medium-sized pumice stones used as packing materials for the retention of the anaerobic granules, to avoid challenges associated with the use of the three-phase separator of up-flow systems and the washout of the anaerobic biomass. Furthermore, a recycling stream was applied to the system to improve the mixing inside the Down-flow Expanded Granular Bed Reactor (DEGBR), i.e. the influent distribution to the granular biomass, and the implementation of intermittent fluidization when required to alleviate the effects of pressure drop in such systems. The DEGBR was operated under mesophilic conditions (30–35 °C) and achieved total chemical oxygen demand (tCOD), five-day biological oxygen demand and total suspended solids average removal percentages >95%, and a fats, oils and grease average removal percentage of 93.67% ± 4.51, for an organic loading rate varying between 1.1 to 38.9 gCOD/L.day.

Performance and bacterial characteristics of aerobic granular sludge in response to alternating salinity

Salt concentration often fluctuates in influent industrial wastewater. The behavior of aerobic granular sludge (AGS) in response to the stress of alternating salt concentrations remains unclear. In this study, we ran six identical sequencing batch reactors to follow the performance and microbial characteristics of AGS under 1%, 2%, 4% salinity and corresponding alternating salinity, respectively. The results demonstrated that when compared with the control, alternating salinity not only increased the COD removal efficiency of the reactor significantly (p < 0.001) but also generated high concentration of granular biomass with good settling ability. Pearson correlation analysis indicated that the microorganism mostly used an intracellular second messenger molecule c-di-GMP to regulate extracellular polymer contents. Specifically, the increase of extracellular polysaccharide was conducive to the cohesive strength of the granules, resulting in higher biomass concentration and carbon removal efficiency. Additionally, microbial community analysis confirmed that the relative abundance of c-di-GMP pathway-dependent community, in particular Propionibacterium (24.4%–50.9%), was significantly more dominant under alternating salt conditions.

Performance and microbial community structure of an aerobic granular sludge system at different phenolic acid concentrations.

The present work aims to use aerobic granular sludge technology for the treatment of wastewater containing high organic matter loads and a mixture of phenolic compounds normally present in olive washing water. The physicochemical performance of five bioreactors treating different concentrations of mixture of phenolic acid was monitored to observe the response of the systems. The bioreactors that operated at 50, 100 and 300 mg L-1 did not show relevant changes in terms of performance and granules properties, showing high ratio of phenolic compound removal ratio. However, the bioreactors operated with high phenolic compound concentrations showed low rates of organic matter, nitrogen and phenolic acid removal. In the same way, high concentrations of phenolic compounds determined the disintegration of the granular biomass. Next-generation sequencing studies showed a stable community structure in the bioreactors operating with 50, 100 and 300 mg L-1 of phenolic acids, with the genera Lampropedia and Arenimonas, family Xanthobacteraceae and Fungi Pezizomycotina as the dominant phylotypes. Conversely, the reactors operated at 500 and 600 mg L-1 of phenolic substances promoted the proliferation of Oligohymenophorea ciliates. Thus, this study suggests that aerobic granular sludge technology could be useful for the treatment of wastewaters such as olive washing water.

Mechanical characteristics of pine biomass of different sizes and shapes

In the present study, the mechanical properties of granular biomass of pine origin (sawdust, shavings, long shavings and pellets) were determined. The bulk and tapped densities were determined in a cylindrical chamber. The compacted density was measured, and the influence of the moisture content on this quantity was examined in a vane tester. The flowability and strength properties were determined using a direct shear tester (Jenike box) 210 mm in diameter and standard Schulze ring shear tester. The tests in the Jenike tester complied with the Eurocode 1 procedure for normal pressure (σn) of 15 and 30 kPa and speed of shearing of 0.17 mm s−1. Ring shear testing was conducted using a Schulze annular shear cell of 195 mm outer diameter. Tests were performed for 10 and 20 kPa pre-shear σn. A prototype vane tester was constructed for determining the torque, shear strength, and relaxation of a consolidated sample of granular biomass. σn was exerted by a pneumatic system with a rubber air spring and yoke. The torque and density were measured for moisture content in the range of 10–50% and for σn in the range of 5–30 kPa. The torque was observed to be affected by σn and the moisture content, whereas no relationship between the torque and the rotational speed was observed.

Substrate inhibition and pH effect on denitritation with granular biomass

Abstract Undissociated HNO2 (up to 2 mg dm−3) was confirmed as substrate inhibitor for granular biomass from a denitritation upflow sludge bed reactor used for biological removal of nitrite. On the contrary, total nitrite nitrogen (N-NO2 up to 500 mg dm−3) and methanol (COD up to 2000 mg dm−3) were not proven to be inhibitors. pH also affected the denitritation efficiency (optimal pH was 5.9). Reduction of HNO2 concentration in the reactor by effluent recycling is recommended.

Does the feeding strategy enhance the aerobic granular sludge stability treating saline effluents?

The development and stability of aerobic granular sludge (AGS) was studied in two Sequencing Batch Reactors (SBRs) treating fish canning wastewater. R1 cycle comprised a fully aerobic reaction phase, while R2 cycle included a plug-flow anaerobic feeding/reaction followed by an aerobic reaction phase. The performance of the AGS reactors was compared treating the same effluents with variable salt concentrations (4.97–13.45 g NaCl/L) and organic loading rates (OLR, 1.80–6.65 kg CODs/(m3·d)). Granulation process was faster in R2 (day 34) than in R1 (day 90), however the granular biomass formed in the fully aerobic configuration was more stable to the variable feeding composition. Thus, in R1 solid retention times (SRT), up to 15.2 days, longer than in R2, up to 5.8 days, were achieved. These long SRTs values helped the retention of nitrifying organisms and provoked the increase of the nitrogen removal efficiency to 80% in R1 while it was approximately of 40% in R2. However, the presence of an anaerobic feeding/reaction phase increased the organic matter removal efficiency in R2 (80–90%) which was higher than in R1 with a fully aerobic phase (75–85%). Furthermore, in R2 glycogen-accumulating organisms (GAOs) dominated inside the granules instead of phosphorous-accumulating organisms (PAOs), suggesting that GAOs resist better the stressful conditions of a variable and high-saline influent. In terms of AGS properties an anaerobic feeding/reaction phase is not beneficial, however it enables the production of a better quality effluent.

Long-term stability of aerobic granular sludge for the treatment of very low-strength real domestic wastewater

Aim of the present study was to assess the feasibility of cultivating aerobic granular sludge for the treatment of real very low-strength (COD <120 mg L−1) municipal wastewater by the application of a strict metabolic selective pressure. The feasibility was evaluated in terms of long-term physical stability of the granular biomass as well as COD and phosphate removal efficiencies. A laboratory-scale sequencing batch reactor was inoculated with conventional activated sludge and operated for 175 d. Complete granulation of conventional activated sludge (SVI5/SVI30 = 1) was achieved within 45 d at an average influent COD concentration of 290 ± 44 mg L−1 obtained by the addition of an external source of acetate. At day 51, acetate dosage was stopped and the reactor was then operated for other four months with only real wastewater as carbon source, resulting in an influent COD concentration as low as 115 ± 23 mg L−1. Besides the decrease of the influent COD concentration and of the organic load, the long term stability of the granular sludge was not affected and granular size continuously increased until it reached a maturation phase with an average diameter of 1.5 mm, with more than 30% of the aggregates being larger than 2 mm and marginal presence of floccular biomass (about 5%). The applied metabolic selective pressure based on the complete biodegradable COD uptake under anaerobic conditions (85 ± 7% biodegradable COD removal efficiency obtained) was shown to be able to stimulate the formation of stable and large granules, indicating the other commonly applied selective pressure based on settling velocity (commonly > 8 m h−1 versus 1.9 m h−1 applied in this study), as not strictly required. The identification of the key factors to preserve biomass long-term stability and functionality, poses the basis to allow the application of aerobic granular sludge technology in the context of combined sewers systems and facilitate the retrofitting of existing wastewater treatment plants excluding the need for a plug-flow distribution system for the influent wastewater.

Microbial ecology dynamics of a partial nitritation bioreactor with Polar Arctic Circle activated sludge operating at low temperature

A lab-scale partial nitritation SBR was operated at 11 °C for 300 days used for the treatment of high-ammonium wastewater, which was inoculated with activated sludge from Rovaniemi WWTP (located in Polar Arctic Circle) in order to evaluate the influence the temperature on the performance, stability and dynamics of its microbial community. The partial nitritation achieved steady-state long-term operation and granulation process was not affected despite the low temperature and high ammonia concentration. The steady conditions were reached after 60 days of operation where the granular biomass was fully-formed and the 50%–50% of ammonium-nitrite effluent was successful achieved. Inoculation with cold adapted inoculum showed to yield bigger, denser granules with faster start-up without necessity of low temperature adaptation period. Next-generation sequences techniques showed that Trichosporonaceae and Xanthomonadaceae were the dominant OTUs in the mature granules. Our study could be useful in the implementation of full-scale partial nitritation reactors in cold regions such as Nordic countries for treating wastewater with high concentration of ammonium.

Denitrification performance and microbial versatility in response to different selection pressures

This study investigated functional dynamics of microbial community in response to different selection pressures, with a focus on denitrification. Suspended-biomass experiments demonstrated limited aerobic and relatively higher anoxic nitrate and nitrite reduction capabilities; the highest NO2-N and NO3-N removal rates were 1.3 ± 0.1 and 0.74 ± 0.01 in aerobic and 1.4 ± 0.05 and 3.4 ± 0.1 mg/L.h in anoxic media, respectively. Key potential denitrifiers were identified as: (i) complete aerobic denitrifiers: Dokdonella, Flavobacterium, and Ca. Accumulibacter; (ii) complete anoxic denitrifiers: Acinetobacter, Pseudomonas, Arcobacter, and Comamonas; (iii) incomplete nitrite denitrifier: Diaphorobacter (aerobic/anoxic), (iv): incomplete nitrate denitrifiers: Thauera (aerobic/anoxic) and Zoogloea (strictly-aerobic). Granular biomass removed 72 mg/L NH4-N with no NOx− accumulation. Heterotrophic nitrification and aerobic denitrification were proposed as the principal nitrogen removal pathway in granular reactors, potentially performed by two key organisms Thuaera and Flavobacterium. Biodiversity analysis suggested that the selection pressure of nourishment condition was the decisive factor for microbial selection and nitrogen removal mechanism.

Effect of anaerobic COD utilization on characteristics and treatment performance of aerobic granular sludge in anaerobic/anoxic/oxic SBRs

Influence of anaerobic COD consumption on the properties and nutrient removal performance of aerobic granules was studied. Three identical sequencing batch reactors (SBR) were operated under two operation strategies over four months. Results indicated that, the applied anaerobic contact provided better mass transfer during the anaerobic phase and improved the nutrient removal efficiency of the granules. Anaerobic contact increased anaerobic COD utilization from 17–24% to 45–53% and improved phosphorus removal from 63 to 93%. The anaerobic contact also decreased effluent solids from 87 to 46 mg/L VSS with preventing the growth of biomass in suspension associated with rapidly-growing organisms. With the application of anaerobic contact, slowly growing organisms dominated the system and solid retention time (SRT) increased from 15 to 32 days. Alerting anaerobic continuous feeding with anaerobic contact worsened settling properties of the granular biomass. However, good-settling granules were obtained after acclimation of the biomass to the new condition. The application of anaerobic contact also affected granules’ size and extracellular polymeric substance (EPS) content. More compact granules with less net EPS content and lower protein-to-carbohydrate ratio were obtained under increased anaerobic contact condition compared with operating with anaerobic continuous feeding. Results suggested that appropriate anaerobic contact could enhance the nutrient removal efficiency and effluent quality of mature aerobic granules treating low-strength wastewater. Therefore, design considerations should be taken into account to provide sufficient anaerobic contact in granular sequencing batch reactors (SBRs) when applied for the treatment of low-strength wastewater.

Analysis and Modeling of the Hydraulic Behavior of EGSB Reactors with Presence and Absence of Granular Biomass at Different Hydraulic Retention Times

The efficiency of biological wastewater treatment systems is linked fundamentally to the hydraulic performance of each treatment unit. These units should guarantee an adequate contact between the microorganisms and the residual water, and the compliance with the hydraulic retention time established, in order to decrease the number of dead zones or short circuits that may exist inside the reactors. In this work, hydraulic performance was evaluated in seven Expanded Granular Sludge Bed (EGSB) reactors with a useful volume of 3,4 L and constructed in acrylic. The analysis was carried out through the stimulus-response test, using bromide as tracer. Two hydraulic retention times (8 and 24 h) and the effect of the presence of granular biomass were considered. Results were analyzed qualitatively through the construction of curves C, E and F, and quantitatively through the construction of a mathematical model of axial dispersion. The results of the hydraulic performance of the reactors revealed a marked tendency to a complete mix flow pattern, with a low effect of HRT or the presence of granular biomass on their operation.

Biohydrogen production using a granular sludge membrane bioreactor

Biohydrogen was produced using a granular biomass reactor coupled to a submerged internal membrane. The reactor performance was evaluated under different organic loading rates (OLR) ranging from 5 to 60 g L−1 d−1 and hydraulic retention times (HRT) from 5.5 to 1.25 h. The UASB reactor was operated at 35 °C and pH 4.5. It was observed that the membrane introduction to the reactor does not affect the granule size or integrity. The maximum hydrogen production rate was obtained at 30 g L−1 d−1 and 4 h of HRT (475 ± 15 mLH2 L−1 h−1). A further increase of the OLR resulted in a lower hydrogen production due to a shift of the metabolism to solvent production. The use of membranes allowed the application of relatively low HRT; however, HRT lower than 2 h promoted the homoacetogenic metabolism, decreasing the hydrogen production. The results indicate that the membrane fouling is not only affected by the total EPS formed but also by the operational flux applied.

Effect of light intensity on the characteristics of algal-bacterial granular sludge and the role of N-acyl-homoserine lactone in the granulation

The effects of light intensity on the development of algal-bacterial granular sludge (ABGS) were investigated over a period of 12 weeks. The ABGS developed at low light intensity (142 ± 10 μmol m−2·s−1) exhibited excellent settling ability (SVI30 of 30.9 mL/g), COD and TN removal efficiencies (97.6% and 60.4%, respectively). High light intensity (316 ± 12 μmol m−2·s−1) accelerated granular biomass growth (5.3 g/L) and enhanced the TP removal efficiency (83.7%). Extracellular polymeric substance (EPS) analysis revealed that low light intensity induced more large weight distribution protein production (9–12 kDa and 50–150 kDa), predominantly tryptophan and aromatic proteins. Furthermore, N-acyl-homoserine lactones (AHLs) with a side chain ≤ C10 were commonly shared in the ABGS, and the ABGS developed at low light intensity had a higher C6- and 3OC8-HSL content, which effectively promoted the biofilm formation. The add-back studies showed that the AHLs facilitated the regulation of EPS synthesis. Statistical analysis indicated that the AHLs content had a close correlation with the EPS production, the 50th percentile of the particle size distribution and microbial community assembly, suggesting that AHLs-mediated quorum sensing have an important ecological role in EPS expression and algal-bacterial granulation. Overall, this study describes the ABGS development at different light intensities and the mechanisms of ABGS formation treating synthetic domestic wastewater.

Adaptation of granular sludge microbial communities to nitrate, sulfide, and/or p-cresol removal.

Effluents from petroleum refineries contain a toxic mixture of sulfide, nitrogen, and phenolic compounds that require adequate treatment for their removal. Biological denitrification processes are a cost-effective option for the treatment of these effluents, but the knowledge on the microbial interactions in simultaneous sulfide and phenol oxidation in denitrifying reactors is still very limited. In this work, microbial community structure and macrostructure of granular biomass were studied in three denitrifying reactors treating a mixture of inorganic (sulfide) and organic (p-cresol) electron donors for their simultaneous removal. The differences in the available substrates resulted in different community assemblies that supported high removal efficiencies, indicating the community adaptation capacity to the fluctuating compositions of industrial effluents. The three reactors were dominated by nitrate reducing and denitrifying bacteria where Thiobacillus spp. were the prevalent denitrifying organisms. The toxicity and lack of adequate substrates caused the endogenous decay of the biomass, leading to release of organic matter that maintained a diverse although not very abundant group of heterotrophs. The endogenous digestion of the granules caused the degradation of its macrostructure, which should be considered to further develop the denitrification process in sulfur-based granular reactors for treatment of industrial wastewater with toxic compounds.

Preliminary analysis of Chloroflexi populations in full-scale UASB methanogenic reactors.

The phylum Chloroflexi is frequently found in high abundance in methanogenic reactors, but their role is still unclear as most of them remain uncultured and understudied. Hence, a detailed analysis was performed in samples from five up-flow anaerobic sludge blanket (UASB) full-scale reactors fed different industrial wastewaters. Quantitative PCR show that the phylum Chloroflexi was abundant in all UASB methanogenic reactors, with higher abundance in the reactors operated for a long period of time, which presented granular biomass. Both terminal restriction fragment length polymorphism and 16S rRNA gene amplicon sequencing revealed diverse Chloroflexi populations apparently determined by the different inocula. According to the phylogenetic analysis, the sequences from the dominant Chloroflexi were positioned in branches where no sequences of the cultured representative strains were placed. Fluorescent insitu hybridization analysis performed in two of the reactors showed filamentous morphology of the hybridizing cells. While members of the Anaerolineae class within phylum Chloroflexi were predominant, their diversity is still poorly described in anaerobic reactors. Due to their filamentous morphology, Chloroflexi may have a key role in the granulation in methanogenic UASB reactors. Our results bring new insights about the diversity, stability, dynamics and abundance of this phylum in full-scale UASB reactors which aid in understanding their function within the reactor biomass. However, new methodological approaches and analysis of bulking biomass are needed to completely unravel their role in these reactors. Combining all this knowledge with reactor operational parameters will allow to understand their participation in granulation and bulking episodes and design strategies to prevent Chloroflexi overgrowth.

Development of aerobic granular sludge under tropical climate conditions: The key role of inoculum adaptation under reduced sludge washout for stable granulation

Aerobic granular sludge (AGS) is a promising technology for wastewater treatment. However, the success of the process depends on the formation of stable granular biomass, which is associated with the microbiological aspects of the sludge and reactor operating conditions. In this study, the development of AGS from a poor nitrifying flocculent sludge obtained in a sewage treatment plant designed only for organic matter removal was assessed in a sequencing batch reactor (SBR) under tropical climate conditions (temperatures of 28 ± 4 °C). The results showed that, despite the alternating anaerobic-aerobic conditions during the granules selection phase under high sludge washout rates (low settling time), readily biodegradable organic matter was mainly removed aerobically. The formed granules were unstable, exhibiting a substantial amount of filaments and pasty consistency. The biomass characteristics (e.g., sludge volume index, density, diameter and settling velocity) were negatively impacted as complete granulation was reached, while biomass loss and degranulation became inevitable. Poor nitrification and no enhanced biological phosphate removal (EBPR) were observed. Implementation of a new operational strategy incorporating an adaptation of the seed sludge under reduced washout conditions (high settling time) prior to the granules selection stage enabled most of the influent organics to be removed anaerobically. Besides allowing a feast-famine regime to be established in the reactor, the sludge acclimation phase favoured the development of slow-growing organisms and suppressed the appearance of filamentous-like structures. Fast-settling granules with regular shape remained stable in the long-term, while high ammonium (>95%) and total nitrogen removal (>90%) was obtained. However, EBPR activity was very unstable, most likely due to the high temperatures. The findings of this study are important for the spreading of the AGS technology worldwide, especially in developing countries where the conditions are different in all aspects.

A novel storage driven granular post denitrification process: Long-term effects of volume reduction on phosphate recovery

Anoxic granular biomass with enhanced biological phosphorus (P) removal was used in a post-denitrification configuration to concentrate P in wastewater. The study examined the use of anoxic granules to facilitate application of volume reduction to create a P-enriched stream (>100 mg-P/L). The results indicated the importance of maintaining a food to microorganism (F/M) ratio of ∼0.124 g-COD/g-MLSS.d to achieve P and nitrogen (N) removal close to 100%. While granulation required a short settling time and a high-volume exchange ratio, biomass wasting was essential to control the F/M ratio to maintain a suitable microbial diversity and abundance. Diversity and abundance were also impacted by volume reduction, but the effect of this was marginal compared with the effect of decreasing F/M ratio. Furthermore, a decrease in the F/M ratio enhanced sedimentation (SVI5 decreased from 55.5 to 32.0 mL/g-MLSS) but decreased dewaterability (capillary suction time increased from 15.5 s to 19.4 s). Recovery of P as a concentrated liquor had minimal impact on the bacterial diversity.

Novel system configuration with activated sludge like-geometry to develop aerobic granular biomass under continuous flow

A novel continuous flow system with “flat geometry” composed by two completely mixed aerobic tanks in series and a settler was used to promote the formation of aerobic granular sludge. Making similarities of this system with a typical sequencing batch reactor (SBR), for aerobic granules cultivation, the value of the tank 1/tank 2 vol ratio and the biomass recirculation rate would correspond with the feast/famine length ratio and the length of the operational cycle, respectively, while the settler upflow liquid velocity imposed would be related to the settling time. From the three experiments performed the best results were obtained when the tank 1/tank 2 vol ratio was of 0.28, the sludge recycling ratio of 0.25 and the settler upflow velocity of 2.5 m/h. At these conditions the aggregates had settling velocities between 29 and 113 m/h, sludge volume index at 10 min (SVI10) of 70 mL/g TSS and diameters between 1.0 and 5.0 mm.

Strategies to re-establish stable granulation after filamentous outgrowth: Insights from lab-scale experiments

Abstract Aerobic granular sludge (AGS) is a promising technology for wastewater treatment. However, maintaining the granular biomass stability is not trivial. During the long-term operation of AGS systems, we observed that uncontrolled sludge retention time led to the appearance of substantially large and fluffy granules characterized by a dark anaerobic inner core. Such conditions favoured the proliferation of filamentous bacteria and deterioration of AGS properties. Consequently, granules disintegration became inevitable. We decided to assess two operational strategies to supress filamentous overgrowth and recover the granular biomass stability. Strategy 1 involved the enhancement of shear stress by increasing the aeration intensity (from 1.34 to 1.87 cm s−1), while strategy 2 relied on the combination of enhanced aeration and iron (10 mg L−1) addition. The implementation of strategy 1 contributed to decrease the average diameter of AGS (4.7–4.1 mm) and sludge volume index (SVI) (300–110 mL g−1). Nevertheless, filamentous bacteria remained intact. Conversely, prolonged shear stress combined with iron supplementation (strategy 2) led to significant improvements in the physical characteristics of the granules. Very small particles acting as precursors of mature AGS were observed while big-size granules (>4 mm) were no longer detected. Moreover, filamentous overgrowth was controlled and the growth of new granules was stimulated. No adverse effect on the conversion processes (COD and nitrogen removal) was observed in the AGS system and stable granulation was achieved from this period onwards. To better explain the role of the implemented strategies, some hypothesis are presented and discussed.