Combined removal of sulfur compounds and nitrate by autotrophic denitrification in bioaugmented activated sludge system

An autotrophic denitrification process using reduced sulfur compounds (thiosulfate and sulfide) as electron donor in an activated sludge system is proposed as an efficient and cost effective alternative to conventional heterotrophic denitrification for inorganic (or with low C/N ratio) wastewaters and for simultaneous removal of sulfide or thiosulfate and nitrate. A suspended culture of sulfur-utilizing denitrifying bacteria was fast and efficiently established by bio-augmentation of activated sludge with Thiobacillus denitrificans. The stoichiometry of the process and the key factors, i.e. N/S ratio, that enable combined sulfide and nitrogen removal, were determined. An optimum N/S ratio of 1 (100% nitrate removal without nitrite formation and low thiosulfate concentrations in the effluent) has been obtained during reactor operation with thiosulfate at a nitrate loading rate (NLR) of 17.18 mmol N L(-1) d(-1). Complete nitrate and sulfide removal was achieved during reactor operation with sulfide at a NLR of 7.96 mmol N L(-1) d(-1) and at N/S ratio between 0.8 and 0.9, with oxidation of sulfide to sulfate. Complete nitrate removal while working at nitrate limiting conditions could be achieved by sulfide oxidation with low amounts of oxygen present in the influent, which kept the sulfide concentration below inhibitory levels.     

Isolation and characterization of Pseudomonas stutzeri QZ1 from an anoxic sulfide-oxidizing bioreactor

Bacterial strain QZ1 was isolated from sludge of anoxic sulfide-oxidizing (ASO) reactor. Based on 16S rDNA sequence analysis and morphological characteristics, the isolate was identified as Pseudomonas stutzeri. The isolate was found to be a facultative chemolithotroph, using sulfide as electron donor and nitrite as electron acceptor. The strain QZ1 produced sulfate as the major product of sulfide oxidation, depending on the initial sulfide and nitrite concentrations. The isolate was capable of growth under strictly autotrophic conditions. The growth and substrate removal of Pseudomonas stutzeri QZ1 were optimal at an initial pH of 7.5–8.0 at 30 °C. The specific growth rate (μ) was found as 0.035 h⁻¹ with a doubling time of 21.5 h. For isolate QZ1, the EC₅₀ values both for sulfide and nitrite were found to be 335.95 mg S L⁻¹ and 512.38 mg N L⁻¹, respectively, showing that the sulfide oxidation into sulfate by Pseudomonas stutzeri QZ1 was badly affected beyond these substrate concentrations.     

亚硝酸盐型同步厌氧生物脱氮除硫工艺的运行性能

采用上流式厌氧污泥床(UASB)反应器研究了亚硝酸盐型同步厌氧生物脱氮除硫工艺的性能。该工艺具有很高的硫化物和亚硝酸盐转化潜能,最大容积硫化物去除率和容积硝酸盐去除率分别为13.4kg/(m3·d)和2.3kg/(m3·d);所能耐受的最大进水硫化物和亚硝酸盐浓度分别为880mg/L和252.7mg/L;最适进水硫化物和亚硝酸盐浓度分别为460mg/L和132.3mg/L,最适水力停留时间为4h。硫化物和亚硝酸盐的表观半抑制浓度分别为403.9mg/L和120.8mg/L,两者之间的联合毒性为拮抗作用。     

Assessment of the positive effect of salinity on the nitrogen removal performance and microbial composition during the start-up of CANON process

In this study, a non-woven rotating biological contactor reactor was operated for the start-up of completely autotrophic nitrogen removal over nitrite (CANON) process. In this perfectly attached growth system, nitrite oxidizing was identified, which interfered with the nitrogen removal performance. Batch tests indicated that 10 g NaCl per liter salinity was a preferable definite level to stand out ammonium-oxidizing activity and anammox activity, and selectively suppress nitrite-oxidizing activity under oxygen-limited conditions. Reactor operation showed that the maximum TN removal rate was increased from 425 mg N l(-1) day(-1) to 637 mg N l(-1) day(-1) after the addition of 10 g NaCl per liter salinity on analogous technological parameters. Microbiological community analysis revealed that bacteria strains similar to the genus Nitrospira sp. were specialized nitrite oxidizers existing in CANON reactor, which were then eliminated under salinity exposure for their no salinity-tolerant relative. However, anammox bacteria belonging to Planctomycetes and some aerobic ammonium oxidizers belonging to Nitrosomonas could be highly enriched under this oxygen-limited salinity conditions. Salinity-contained high ammonium wastewater will be so considered as suitable influent for CANON process in further industrial application.     

Lead removal through biological sulfate reduction process

The feasibility of lead removal through biological sulfate reduction process with ethanol as electron donor was investigated. Sulfide-rich effluent from biological process was used to remove lead as lead sulfide precipitate. The experiments were divided into two stages; Stage I startup and operation of sulfidogenic process in a UASB reactor and Stage II lead sulfide precipitation. In Stage I, the COD:S ratio was gradually reduced from 15:1 to 2:1. At the COD:S ratio of 2:1, sulfidogenic condition was achieved as identified by 80-85% of electron flow by sulfate reducing bacteria (SRB). COD and sulfate removal efficiency were approximately 78% and 50%, respectively. In Stage II, the effluent from UASB reactor containing sulfide in the range of 30-50 mg/L and lead-containing solution of 45-50 mg/L were fed continuously into the precipitation chamber in which the optimum pH for lead sulfide precipitation of 7.5-8.5 was maintained. It was found that lead removal of 85-95% was attained.     

Effects of pH adjustment by parawood ash and effluent recycle ratio on the performance of anaerobic baffled reactors treating high sulfate wastewater

The objective of this study was to compare the performance of anaerobic baffled reactor (ABR) treating concentrated rubber latex wastewater under different pH adjustment substances and recycling ratios (R). Two ABRs, one received wastewater pretreated with NaOH and the other with ash, were operated at 35 degrees C under identical HRTs from 10 to 1.25d. Results show that both ABRs had highest COD and sulfate removal efficiencies at HRT 10d (averaged 82.71% and 96.16% of ABR-NaOH, and 80.77% and 96.60% of ABR-Ash, respectively), where majority of the influent COD and sulfate were removed by the first compartment of the ABR at all conditions tested. Increasing R (0, 0.3 and 0.5) raised the hydraulic loading on the system and resulted in a drop of organic removal efficiency and methane yield. Translocation of sulfate reducing bacteria and methanogens in the ABRs caused by increased organic loading and effluent recycle is discussed. The results show great potential of parawood ash as a pH adjustment substance for acidic wastewaters.     

Hydrogen sulfide removal by immobilized Thiobacillus novellas on SiO2 in a fluidized bed reactor

The removal of hydrogen sulfide (H2S) from aqueous media was investigated using Thiobacillus novellas cells immobilized on a SiO2 carrier (biosand). The optimal growth conditions for the bacterial strain were 30 degrees C and initial pH of 7.0. The main product of hydrogen sulfide oxidation by T. novellus was identified as the sulfate ion. A removal efficiency of 98% was maintained in the three-phase fluidized-bed reactor, whereas the efficiency was reduced to 90% for the two-phase fluidized-bed reactor and 68% for the two-phase reactor without cells. The maximum gas removal capacity for the system was 254 g H2S/m3/h when the inlet H2S loading was 300 g/m3/h (1,500 ppm). Stable operation of the immobilized reactor was possible for 20 days with the inlet H2S concentration held to 1,100 ppm. The fluidized bed bioreactor appeared to be an effective means for controlling hydrogen sulfide emissions.     

Neural network prediction of thermophilic (65 degrees C) sulfidogenic fluidized-bed reactor performance for the treatment of metal-containing wastewater

The performance of a fluidized-bed reactor (FBR) based sulfate reducing bioprocess was predicted using artificial neural network (ANN). The FBR was operated at high (65 degrees C) temperature and it was fed with iron (40-90 mg/L) and sulfate (1,000-1,500 mg/L) containing acidic (pH = 3.5-6) synthetic wastewater. Ethanol was supplemented as carbon and electron source for sulfate reducing bacteria (SRB). The wastewater pH of 4.3-4.4 was neutralized by the alkalinity produced in acetate oxidation and the average effluent pH was 7.8 +/- 0.8. The oxidation of acetate is the rate-limiting step in the sulfidogenic ethanol oxidation by thermophilic SRB, which resulted in acetate accumulation. Sulfate reduction and acetate oxidation rates showed variation depending on the operational conditions with the maximum rates of 1 g/L/d (0.2 g/g volatile solids (VS)/d) and 0.3 g/L/d (0.06 g/g VS/d), respectively. This study presents an ANN model predicting the performance of the reactor and determining the optimal architecture of this model; such as best back-propagation (BP) algorithm and neuron numbers. The Levenberg-Marquardt algorithm was selected as the best of 12 BP algorithms and optimal neuron number was determined as 20. The developed ANN model predicted acetate (R=0.91), sulfate (R=0.95), sulfide (R=0.97), and alkalinity (R=0.94) in the FBR effluent. Hence, the ANN based model can be used to predict the FBR performance, to control the operational conditions for improved process performance.     

A new method of autotrophic denitrification with spent sulfidic caustic as substrate and alkalinity source

A new method based on sulfide utilizing autotrophic denitrification was adopted to remove nitrate from wastewater and to reuse spent sulfidic caustic containing high sulfide and alkalinity levels. The experiments were performed using a bench-scale upflow anoxic hybrid growth reactor (UAHGR) and an upflow anoxic suspended growth reactor (UASGR) to characterize the stoichiometric relationship between sulfur and nitrate in the process as well as the performance of the reactors. The level of nitrate removal from the UAHGR and UASGR were maintained at over 90% at a nitrate loading rate ranging from 0.15∼0.40 kgNO₃ ⁻/m³·d and no significant nitrite accumulation was observed in either reactor. Although the influent pH values were higher than the optimum range of autotrophic denitrification at 8.7∼10.1, the effluent pH was stable at 7.2∼7.9 due to the production of hydrogen ions during operation. The stoichiometric ratio of sulfate production to nitrate removal was 1.5∼2.1 mgSO₄ ²⁻/mgNO₃ ⁻ in both reactors. A comparison of the reactor performance revealed that the chemical parameters of the UAHGR operation corresponded to a plug flow like type reactor while the chemical parameters of the UASGR operation corresponded to a completely stirred tank reactor like type reactor. UAHGR did not require sludge recycling due to the packed media while UASGR required 300∼700% sludge recycling. Therefore, spent sulfidic caustic could be used in the sulfur utilizing autotrophic denitrification processes as substrate and alkalinity sources.     

Study of a combined sulfur autotrophic with proton-exchange membrane electrodialytic denitrification technology: sulfate control and pH balance

A novel combined system established for nitrate removal from aqueous solution consisted of two parts: sulfur autotrophic denitrification and bio-electrochemical denitrification based on proton-exchange membrane electrodialysis (PEMED). The system was operated at various hydraulic retention times (HRT) and current intensities. Its optimum operation condition was also determined. The combined process had pH adjustment thus generating less nitrite than PEMED process. The denitrification rate of sulfur autotrophic part was dependent on HRT, while shorter HRT could reduce the sulfate generated by the sulfur autotrophic process. The denitrification rate of PEMED process depended on the applied current. For 32 ± 1 mg-N/L nitrate in influent, the optimum operation parameters of combined process were: HRT 2 h; applied current 350 mA. The combined reactor could achieve 95.8% nitrate removal without nitrite accumulation, the pH of effluent kept neutral and the sulfate of effluent was 202.1 mg/L, lower than the drinking water standard in China.     

Removal of highly elevated nitrate from drinking water by pH-heterogenized heterotrophic denitrification facilitated with ferrous sulfide-based autotrophic denitrification

The performance of acetic acid-supported pH-heterogenized heterotrophic denitrification (HD) facilitated with ferrous sulfide-based autotrophic denitrification (AD) was investigated in upflow activated carbon-packed column reactors for reliable removal of highly elevated nitrate (42 mg NO₃-N l⁻¹) in drinking water. The use of acetic acid as substrate provided sufficient internal carbon dioxide to completely eliminate the need of external pH adjustment for HD, but simultaneously created vertically heterogenized pH varying from 4.8 to 7.8 in the HD reactor. After 5-week acclimation, the HD reactor developed a moderate nitrate removal capacity with about one third of nitrate removal occurring in the acidic zone (pH 4.8–6.2). To increase the treatment reliability, acetic acid-supported HD was operated under 10% carbon limitation to remove >85% of nitrate, and ferrous sulfide-based AD was supplementally operated to remove residual nitrate and formed nitrite without excess of soluble organic carbon, nitrite or sulfate in the final effluent.     

Effects of temperature on simultaneous nitrification and denitrification via nitrite in a sequencing batch biofilm reactor

A laboratory scale experiment was described in this paper to enhance biological nitrogen removal by simultaneous nitrification and denitrification (SND) via nitrite with a sequencing batch biofilm reactor (SBBR). Under conditions of total nitrogen (TN) about 30 mg/L and pH ranged 7.15–7.62, synthetic wastewater was cyclically operated within the reactor for 110 days. Optimal operation conditions were established to obtain consistently high TN removal rate and nitrite accumulation ratio, which included an optimal temperature of 31 °C and an aeration time of 5 h under the air flow of 50 L/h. Stable nitrite accumulation could be realized under different temperatures and the nitrite accumulation ratio increased with an increase of temperature from 15 to 35 °C. The highest TN removal rate (91.9%) was at 31 °C with DO ranged 3–4 mg/L. Process control could be achieved by observing changes in DO and pH to judge the end-point of oxidation of ammonia and SND.     

Consortium diversity of a sulfate‐reducing biofilm developed at acidic pH influent conditions in a down‐flow fluidized bed reactor

Sulfate reduction is an appropriate approach for the treatment of effluents with sulfate and dissolved metals. In sulfate‐reducing reactors, acetate may largely contribute to the residual organic matter, because not all sulfate reducers are able to couple the oxidation of acetate to the reduction of sulfate, limiting the treatment efficiency. In this study, we investigated the diversity of a bacterial community in the biofilm of a laboratory scale down‐flow fluidized bed reactor, which was developed under sulfidogenic conditions at an influent pH between 4 and 6. The sequence analysis of the microbial community showed that the 16S rRNA gene sequence of almost 50% of the clones had a high similarity with Anaerolineaceae. At second place, 33% of the 16S rRNA phylotypes were affiliated with the sulfate‐reducing bacteria Desulfobacca acetoxidans and Desulfatirhabdium butyrativorans, suggesting that acetotrophic sulfate reduction was occurring in the system. The remaining bacterial phylotypes were related to fermenting bacteria found at the advanced stage of reactor operation. The results indicate that the acetotrophic sulfate‐reducing bacteria were able to remain within the biofilm, which is a significant result because few natural consortia harbor complete oxidizing sulfate‐reducers, improving the acetate removal via sulfate reduction in the reactor.     

Impacts of reduced sulfur components on active and resting ammonia oxidizers

While there has been significant research on the nature and extent of the impact of inhibitory reduced sulfur with respect to anaerobic (e.g., methanogenic and sulfidogenic) microbial systems, only limited study has yet been conducted on the comparable effects of soluble sulfides which might occur within aerobic wastewater treatment systems. Admittedly, aerobic reactors would not normally be considered conducive to the presence of reduced sulfur constituents, but there do appear to be a number of processing scenarios under which related impacts could develop, particularly for sensitive reactions like nitrification. Indeed, the following scenarios might well involve elevated levels of reduced sulfur within an aerobic reactor environment: (1) mixed liquor recycle back through sulfide-generating anaerobic zones (e.g., in conjunction with biological nutrient removal processes, etc.), (2) high-level side-stream sulfide recycle via sludge digestion, etc., back to aerobic reactors, and (3) high-level influent sulfide inputs to wastewater treatment facilities via specific industrial, septage, etc., streams. The objective of this study was, therefore, to determine the subsequent metabolic impact of soluble sulfide under aerated and unaerated conditions, focusing in particular on ammonia-oxidizing bacteria due to their critical first-step role with nitrification. The obtained results indicated that, under catabolically active conditions, cultures of ammonia oxidizers were extremely sensitive to the presence of sulfide. At total soluble sulfide concentrations of 0.25 mg l^–1 S, active ammonia oxidation was completely inhibited. However, immediately following the removal of this soluble sulfide presence, ammonia oxidation started to recover; and it continued to improve over the next 24 h. Similar sulfide impact tests conducted with inactive ammonia oxidizers exposed during anaerobic conditions, albeit at higher dosage levels, also revealed that their subsequent aerobic activity would correspondingly be retarded. These results indicated that, after sulfide exposure under unaerated conditions, subsequent aerobic oxidative activity rates rapidly decreased as the soluble sulfide exposure was increased from 0.5 gm l^–1 S to 5 mg l^–1 S and that further reductions in this activity progressively developed as the concentration was increased to 200 mg l^–1 S. The recovery following unaerated exposure to sulfide was significantly higher at pH 7, as compared with pH 8, and although the specific nature of this variation was not established, a hypothetical explanation appeared warranted.     

The effect of pH on the efficiency of an SBR processing piggery wastewater

To treat piggery wastewater efficiently, the hydrolysis of urea (mainly derived from swine urine) in piggery wastewater with the change of sewage pH must be considered. Using activated sludge, piggery wastewater was treated in a sequencing batch reactor (SBR), and the effects of influent pH on SBR processing efficiency, sludge settle ability, and sludge activity were investigated. The results showed that a high influent pH value contributed to the improvement of the removal rate of ammonia nitrogen and reduction of the chemical oxygen demand (COD). When the influent pH was between 9.0 and 9.5, the removal rate of ammonia nitrogen was higher than 90%, and the reduction of COD from its original value was 80%. The influent pH had a greater influence on sludge concentration and sludge activity. When the influent pH increased from 7.0 to 9.5, the sludge concentration increased from 2,350 to 3,947 mg/L in the reactor, and the activities of ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) first increased and then decreased. When the influent pH was 9.0 and 8.0, the maximum values (0.48 g O₂/(g MLSS/day) and 0.080 g O₂/(g MLSS/day)) were reached, and the sludge settling ratio was nearly steady between 20 and 35% in each reactor.     

Effects of nitrite inhibition on anaerobic ammonium oxidation

In order to assess the stability of nitrogen removal systems utilizing anaerobic ammonium oxidation (anammox), it is necessary to study the toxic effects of nitrite on these biochemical reactions. In this study, the effects of nitrite on anammox bacteria entrapped in gel carriers were investigated using batch and continuous feeding tests. The results showed that the nitrite concentration in a reactor must be less than 274-mg N/L in order to prevent a decrease in the anammox activity, which occurred when the gel carriers were soaked in nitrite solutions with concentrations greater than 274-mg N/L in a batch test. In a continuous feeding test, nitrite inhibition was not observed at low concentrations of nitrite. However, the anammox activity decreased to 10% when the nitrite concentration increased to 750-mg N/L over a 7-day period in the reactor. In addition, it was shown that the effects of nitrogen on the anammox reaction were reversible because the anammox activity completely recovered within 3 days when the influent nitrite concentration was decreased to less than 274-mg N/L.     

Monitoring the denitrification of wastewater containing high concentrations of nitrate with methanol in a sulfur-packed reactor

Biological denitrification of high nitrate-containing wastewater was examined in a sulfur-packed column using a smaller amount of methanol than required stoichiometrically for heterotrophic denitrification. In the absence of methanol, the observed nitrate removal efficiency was only about 40%, and remained at 400 mg NO(3)(-)-N/l, which was due to an alkalinity deficiency of the pH buffer and of CO(2) as a carbon source. Complete denitrification was achieved by adding approximately 1.4 g methanol/g nitrate-nitrogen (NO(3)(-)-N) to a sulfur-packed reactor. As the methanol concentration increased, the overall nitrate removal efficiency increased. As influent methanol concentrations increased from 285 to 570, 855, and 1,140 mg/l, the value of Delta mg alkalinity as CaCO(3) consumed/Delta mg NO(3)(-)-N removed increased from -1.94 to -0.84, 0.24, and 0.96, and Delta mg SO(4)(2-) produced/Delta mg NO(3)(-)-N removed decreased from 4.42 to 3.57, 2.58, and 1.26, respectively. These results imply the co-occurrence of simultaneous autotrophic and heterotrophic denitrification. Sulfur-utilizing autotrophic denitrification in the presence of a small amount of methanol is very effective at decreasing both sulfate production and alkalinity consumption. Most of methanol added was removed completely in the effluent. A small amount of nitrite accumulated in the mixotrophic column, which was less than 20 mg NO(2)(-) -N/l, while under heterotrophic denitrification conditions, nitrite accumulated steadily and increased to 60 mg NO(2)(-) -N/l with increasing column height.     

Anaerobic decolorisation of simulated textile wastewater containing azo dyes

The effects of the addition of powered particles of kaolin to nitrifying activated sludge systems were studied. Kaolin was added to a nitrifying activated sludge reactor, during the operational phase, to observe the effects of this clay on reactor performance. The results were compared to those obtained from a similar unit operated without kaolin. The settling properties of the sludges from both units were similar (sludge volume index (SVI) of 14.5 ml/g VSS; zone settling velocity (ZSV) of 7.5 m/h), but the specific nitrifying activities of ammonia and nitrite oxidizing processes were enhanced up to 75% and 50%, respectively, when kaolin was added. The mechanism of action of kaolin was not clear. Additional ammonia, nitrite and nitrate adsorption tests showed that these compounds were not adsorbed by kaolin. This demonstrated that no beneficial effect was caused by adsorption of either substrates or products. Short-term activity tests also showed that the stimulating effects of kaolin on specific activity were not immediate. The effects of kaolin when nitrifying units were operated under unfavorable conditions were also evaluated: In a second set of experiments, a nitrifying unit was operated with low levels of dissolved oxygen (DO), with and without kaolin. The presence of kaolin exerted practically no effect on ammonia oxidation but nitrite oxidation slightly diminished. In a third set of experiments, a nitrifying unit was subjected to pH shocks (9, 10 and 11) over 3 h with pH then restored to 7.8. A pH shock of 11 caused a decrease of 60% in nitrifying activity for 12 days. When kaolin was added to this unit the efficiency of the system was completely restored in 4 days. Therefore, kaolin might be useful to restore damaged units.     

Novel microbial nitrogen removal processes

The present-day wastewater treatment practices can be significantly improved through the introduction of new microbial treatment technologies. Recently, several new processes for nitrogen removal have been developed. These new nitrogen removal technologies provide practicable options for treating nitrogen-laden wastewaters. The new processes are based on partial nitrification of ammonium to nitrite combined with anaerobic ammonium oxidation. These processes include the single reactor system for high ammonia removal over nitrite (SHARON) process, which involves part conversion of ammonium to nitrite; the anaerobic ammonium oxidation (ANAMMOX) process, which involves anaerobic ammonium oxidation; and the completely autographic nitrogen removal over nitrite (CANON) process, which involves nitrogen removal within one reactor under oxygen-limited conditions. These new processes target the removal of nitrogen from wastewaters containing significant quantities of ammonium.     

Sulfide oxidation at halo-alkaline conditions in a fed-batch bioreactor

A biotechnological process is described to remove hydrogen sulfide (H(2)S) from high-pressure natural gas and sour gases produced in the petrochemical industry. The process operates at halo-alkaline conditions and combines an aerobic sulfide-oxidizing reactor with an anaerobic sulfate (SO(4) (2-)) and thiosulfate (S(2)O(3) (2-)) reducing reactor. The feasibility of biological H(2)S oxidation at pH around 10 and total sodium concentration of 2 mol L(-1) was studied in gas-lift bioreactors, using halo-alkaliphilic sulfur-oxidizing bacteria (HA-SOB). Reactor operation at different oxygen to sulfide (O(2):H(2)S) supply ratios resulted in a stable low redox potential that was directly related with the polysulfide (S(x) (2-)) and total sulfide concentration in the bioreactor. Selectivity for SO(4) (2-) formation decreased with increasing S(x) (2-) and total sulfide concentrations. At total sulfide concentrations above 0.25 mmol L(-1), selectivity for SO(4) (2-) formation approached zero and the end products of H(2)S oxidation were elemental sulfur (S(0)) and S(2)O(3) (2-). Maximum selectivity for S(0) formation (83.3+/-0.7%) during stable reactor operation was obtained at a molar O(2):H(2)S supply ratio of 0.65. Under these conditions, intermediary S(x) (2-) plays a major role in the process. Instead of dissolved sulfide (HS(-)), S(x) (2-) seemed to be the most important electron donor for HA-SOB under S(0) producing conditions. In addition, abiotic oxidation of S(x) (2-) was the main cause of undesirable formation of S(2)O(3) (2-). The observed biomass growth yield under SO(4) (2-) producing conditions was 0.86 g N mol(-1) H(2)S. When selectivity for SO(4) (2-) formation was below 5%, almost no biomass growth was observed.