WO2014154559A1 - Nitrification process for the treatment of wastewater at high temperature - Google Patents

Abstract

The present invention relates to the treatment of wastewater at temperatures higher than 40°C. More specifically, the present invention relates to microbial consortia capable to oxidize ammoniacal nitrogen to nitrite and/or nitrate. Indeed, the present invention discloses: 1) mesophilic nitrifying consortia which are rendered thermophilic via increased osmolarity, and, 2) nitrifying consortia enriched from one or more thermophilic environments. Both of the latter consortia are shown to efficiently oxidize ammoniacal nitrogen to nitrite and/or nitrate in waste water at high temperature.

Description

Nitrification process for the treatment of wastewater at high temperature

Technical field of invention

The present invention relates to the treatment of wastewater at temperatures higher than 40 °C. More specifically, the present invention relates to microbial consortia capable to oxidize ammoniacal nitrogen to nitrite and/or nitrate. Indeed, the present invention discloses: 1 ) mesophilic nitrifying consortia which are rendered thermophilic via increased osmolarity, and, 2) nitrifying consortia enriched from one or more thermophilic environments. Both of the mentioned consortia are shown to efficiently oxidize ammoniacal nitrogen to nitrite and/or nitrate in wastewater at high temperatures.

Background art

Nitrogen is a major wastewater component that can cause severe problems to global ecology and society when released into the environment. Physicochemical processes such as stripping and struvite precipitation can be used to remove the nitrogen, but biological nitrogen removal is the most cost-efficient option for domestic and industrial wastewaters (Mulder, 2003). Nitrification/denitrification and partial nitritation/anammox are the most important biological nitrogen removal applications (Vlaeminck et al., 2012). Nitrification consists out of subsequent nitritation, i.e. aerobic ammonium-oxidizing bacteria and/or archaea (AerAOB/AOA) producing nitrite, and nitratation, i.e. aerobic nitrite-oxidizing bacteria (NOB) producing nitrate. Anammox is performed by anoxic ammonium-oxidizing bacteria (AnAOB) combining the ammonium with nitrite to mainly nitrogen gas and some nitrate. Temperature is a crucial environmental parameter for nitrogen removal, particularly for the aerobic processes. Reactors with nitrification or nitritation are typically run in the range of 5-35 °C (Guo et al., 2010; Hies & Mavinic, 2001 ). Nevertheless, at the higher end of the temperature spectrum some nitrogen oxidizing organisms have been detected and/or enriched in batch cultures from a natural thermophilic environment, such as deep-sea hydrothermal vents and hot springs (Golovacheva, 1976; Lebedeva et al., 201 1 ). At this moment however, there is no information on nitrifying reactors operated above 40 °C. Until now, thermophilic nitrogen removal has mainly been investigated as a part of studies focusing on thermophilic activated sludge. Oxidation of organic material was the main goal in those studies, and the exact fate of nitrogen in these systems remained unclear. The main nitrogen removal mechanisms in these thermophilic environments are assumed to be ammonia stripping and nitrogen assimilation into biomass. For example, Couillard et al. (1988, Water Research, 23-5) disclose a low-efficient treatment of slaughterhouse effluent by a thermophilic process and indicate that the major ammonium removal mechanism is nitrogen assimilation into biomass and ammonia stripping. Also Simstich et al. (2012, Bioresource Technology 122, 1 1 -16) discuss the treatment of paper mill deinking wastewater under thermophilic conditions and indicate that, despite the occurrence of ammonium- oxidizing bacteria, no nitrification could be measured. Furthermore, Abeynayaka and Visvanathan (201 1 ) observed some minor nitrification (20%) at both 47 and 60 °C. In that case, the 80% nitrogen loss was composed of about 60% ammonia stripping and 20% nitrogen assimilation. Kurian et al. (2005) did not observe any nitrification at 45°C and ammonia stripping and nitrogen assimilation accounted for respectively 75-83% and 16-25% of the nitrogen loss. Shore et al. (2012) could not show an efficient removal of ammonium from high temperature industrial wastewater at temperatures above 40 °C in a moving bed biofilm reactor comprising bacteria belonging to the genera Nitrosomonas and Nitrospira. (Shore et al., 2012).

However, thermophilic aerobic processes are known to be more stable, to achieve higher rates (smaller bioreactors can be used), to produce less biological sludge and to achieve better hygienization (Lapara & Alleman, 1999). Additionally, elimination of cooling requirements would result in both lower investment and operating costs. Thermophilic nitrification could thus be of interest for the removal of nitrogen from high-temperature industrial wastewater, municipal wastewater with high seasonal temperatures, livestock manure, supernatant from thermophilic anaerobic digesters or from so-called 'autothermal aerobic digestion' processes.

Microorganisms are known to adapt to higher temperatures. Adaptation of mesophilic sludges to thermophilic temperatures already have been observed in several processes such as anaerobic digestion (Bouskova et al., 2005; Ortega et al., 2008) and activated sludge systems (Suvilampi & Rintala, 2003). Acclimatization periods are mostly needed, but some factors can accelerate the adaptation process. For example, high osmolarity by salt shocking or salt cultivation induced increased thermotolerance in both Salmonella and Bacillus species (den Besten et al., 2010; Fletcher & Csonka, 1998). As a response to osmotic stress, microorganisms accumulate compatible solutes, small molecules also known as osmoprotectantia, acting as osmolytes. The role of those compatible solutes is however not limited to osmoprotection but can also protect the cell or cell components from other environmental constraints such as freezing, dessication, oxygen radicals, and high temperature (Welsh, 2000). Indeed, the production of compatible solutes glycine beta^'ine and glutamate acted both as an osmo- and thermoprotectant (Caldas et al., 1999; Holtmann & Bremer, 2004). However and although increased thermotolerance by salt stress was already observed in relation with food related pathogens as indicated above, salt is considered as a common stress factor inhibiting the autotrophic nitrification (Moussa et al., 2006). Increasing salt concentrations were indeed shown to linearly reduce both AerAOB and NOB activity, NOB being more sensitive (Dincer & Kargi, 1999; Sanchez et al., 2004).

Taken together, there is still a need to design and develop a biological nitrification process to efficiently remove nitrogen from wastewater at a temperature higher than 40 °C. Indeed, and as discussed above, Shore et al. (2012) indicated that no nitrification above 40 °C by the bacterial genera Nitrosomonas and Nitrospira was possible, and, Abeynayaka & Visvanathan (201 1 a) demonstrated that only minor nitrification is possible at temperatures higher than 40 °C when using an aerobic activated sludge. Thus, the art gives not a single, straightforward hint to use osmoprotected, mesophilic consortia or enriched thermophilic consortia to remove high quantities of ammonium from waste water at high temperatures via biological nitrification as is disclosed by the present invention.

Brief description of figures

Figure 1 : Effect of salt addition (5 g NaCI L^" ) on the specific AerAOB and NOB activities of the OLAND-biomass at different temperatures (34, 40 and 45 °C). Both the direct specific activity (top) as the specific activity after 48h (bottom) were determined. All experiments were performed in triplicate and statistically significant differences (p<0.05) between the control and the salt addition treatment are indicated with an asterisk.

Figure 2: Operation and performance characteristics of control (left) and salt reactor (right). A. Temperature and influent NaCI dosage. B. Influent nitrogen concentrations and volumetric loading rates. C. Effluent nitrogen concentrations. D. Specific AerAOB and NOB activities.

Figure 3: qPCR abundance of AerAOB, NOB- Nitrospira, AnAOB, expressed as copies ng^" DNA, in the control and salt reactor. AOA were under the detection limit of 120 copies ng^"1 DNA). Figure 4: DGGE for β-proteobacterial AerAOB. Biomass samples were taken at the end of a temperature period (34, 40, 42.5 and 45°C). Similarities were calculated using the Pearson correlation coefficient. C: control reactor; S: salt reactor Figure 5: Ammonium, nitrite and nitrate concentrations during incubation of compost sample A at 50° C with ammonium as substrate after 2 dilutbns.

Figure 6: Ammonium, nitrite and nitrate concentrations during incubation of compost sample B at 50 °C with ammonium as substrate after 2 dilutbns. Figure 7: Ammonium, nitrite and nitrate concentrations during incubation of compost sample C at 50 °C with nitrite as substrate after 2 dilutions.

Figure 8: Ammonium, nitrite and nitrate concentrations during incubation of compost sample D at 50 °C with nitrite as substrate after 2 dilutions.

Figure 9: Performance of thermophilic nitrification enrichment reactor operating at 50 °C. Figure 10: Lowest common Ancestors and their abundance in biomass sample of thermophilic nitrification enrichment reactor operating at 50 °C (lllumina sequencing 16S rRNA).

Figure 1 1 : Specific AerAOB and NOB activities at 30°C and at different salt concentrations after an overnight incubation at 30 °C (control) and40°C (T-shock) prior to the actual activity measurement.

Figure 12: Salt inhibition effect on AerAOB and NOB activities at 30 °C after an overnight incubation at 30 °C (control) and 40 °C (T-shock) prcbr to the actual activity measurement.

Description of invention

The present invention discloses an alternative and efficient biological nitrification process for the treatment of ammoniacal nitrogen in wastewater at temperatures higher than 40 °C in a reactor. Indeed, the present invention discloses: 1 ) mesophilic nitrifying microbial consortia which are rendered thermophilic via increased osmolarity such as salt stress, and 2) specific thermophilic nitrifying microbial consortia enriched from one or more thermophilic environments. Both consortia are capable to efficiently oxidize ammoniacal nitrogen to nitrite and/or nitrate at high temperatures and can thus be used in the biological process of the present invention. Indeed each of both consortia is capable to remove at least 80% of all ammonium present in wastewater via oxidizing at least 80% of said removed ammonium into nitrite and/or nitrate. The latter implies that maximally 20% of said 80% removed ammonium may be removed by nitrogen assimilation and/or ammonia stripping, and that max 20% of said ammonium present in waste water is not removed. The latter mesophilic consortia which are rendered thermophilic will oxidize at temperatures between 40 and 45°C; the latter thermophilic consortia enriched from thermophilic environments will oxidize at temperatures above 50 °C.

The present invention thus relates to a process to remove at least 80% of ammonium from ammoniacal waste water having a temperature higher than 40°C in a reactor comprising: a) subjecting at least one mesophilic nitrifying consortium to an increased osmolarity of at least factor 10 compared to the osmolarity which is present when subsequently contacting said consortium with said ammoniacal waste water having a temperature higher than 40 °C, in order to obtain a consortium comprising bacteria belonging to the phylum Proteobacteria and the genus Nitrospira that oxidizes at least 80% of the total amount of removed ammonium to nitrite and/or nitrate at a temperature between 40 °C and 45°C, or b) incubating samples from a thermophilic environment at a temperature of at least 50 °C at a pH below 7 for a period of at least 30 days during which ammonia and nitrite are given as only substrate at a concentration of maximum 20 mg N/L in order to obtain a thermophilic consortium comprising archaea belonging to the phylum Thaumarchaeota and bacteria belonging to the genus Nitrospira that oxidizes at least 80% of the total amount of removed ammonium to nitrite and/or nitrate at a temperature above 50 °C.

With the term 'a biological nitrification process' is meant any biological process to treat nitrogen in wastewater wherein the biological oxidation of ammonia with oxygen into nitrite (eq 1 ) occurs and which is possibly followed by the oxidation of the formed nitrite into nitrate (eq 2).

NH₃ + 1 .5 0₂ -» N0₂- + H₂0 + H⁺ (eq 1 )

N0₂- + 0.5 0₂ -» N0₃ ^" (eq 2) Hence, the present invention relates to a process involving microbial consortia and comprising the oxidation of ammonia in wastewater, and, to a process involving microbial consortia and comprising the oxidation of ammonia followed by the removal of nitrite from wastewater.

The term 'wastewater having a temperature higher than 40 °C refers for example to industrial wastewater, municipal wastewater, livestock manure and/or anaerobic digestates and the like which have a temperature higher than 40 °C. Witi the terms 'a temperature higher than 40 °C is meant having a temperature of 41 , 42, 43,44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60 °C or even higher than 60 °C.

The term 'ammoniacal wastewater' refers to wastewater as indicated above containing ammonia sensu lato, i.e. ammonia sensu strictu (NH₃), ammonium (NH₄ ⁺) and/or organic nitrogen.

The term 'a reactor' refers to any bioreactor known to skilled person operating in a batch, semi-continuous or continuous mode: i.e. a reactor wherein ammonia is fed into the reactor so that nitrogenous products such as nitrite, nitrate, nitric oxide (NO), nitrous oxide (N₂0) and/or nitrogen gas (N₂ emerge in said bioreactor by activity of the microbial consortium.

The term 'consortium' relates to an association of two or more microorganisms with the objective of participating in a common activity or pooling/coupling their substrates/products for achieving a common goal.

The terms 'at least one mesophilic nitrifying microbial consortium' refers to a microbial consortium growing at mesophilic temperatures (i.e. at temperatures between 5 and 40 °C) which contains at least ammonia-oxidizing bacteria (AerAOB=AOB) and/or ammonia- oxidizing archaea (AOA) and/or heterotrophic ammonia-oxidizing bacteria (HetAOB) and which are capable to oxidize ammonia into nitrite (nitritation). In addition, the latter consortium may also contain nitrite-oxidizing bacteria (NOB) which are capable to oxidize nitrite into nitrate (nitratation). Hence, the present invention relates to consortia comprising at least one of AerAOB, AOA or HetAOB so that oxidation of ammonia occurs (in order to remove ammonia from wastewater as indicated above), or relates to consortia comprising at least one of AerAOB, AOA or HetAOB, and NOB so that the oxidation of ammonia is followed by oxidation or removal of nitrite from said wastewater (as indicated above).

The terms 'subjecting to increased osmolarity' refers to the addition of: a) NaCI or other mono-, di- or trivalent salts including but not limited to CaCI₂, MgCI₂, AICI₃..., or b) sucrose or other components including but not limited to sorbitol, mannitol, amino acids... in the influent of said bioreactor so that the osmolarity in the reactor increases. The latter addition should result in an increase -compared to the osmolarity in the reactor before or after said addition- in osmolarity of ideally 0.17 Osm/L (minimum 0.03 and maximum 1 .2 Osm/L). Preferably, the latter addition should occur some days (i.e. 1 , 2, 3... days) before a planned temperature increase, and removed once temperature transition was successfully done. In this regard said increased osmolarity preferably means an increase by at least factor 10 (i.e. factor 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20...) compared to the osmolarity which is present when subsequently contacting said consortium with said ammoniacal waste water having a temperature higher than 40 °C. The latter increase h osmolarity will transform said mesophilic nitrifying microbial consortia into thermophilic nitrifying microbial consortia which will efficiently oxidize ammoniacal nitrogen into nitrite and/or nitrate at temperatures higher than 40°C, preferably between 40°C and 45°C. As ammonia xidizing bacteria contain the genes for sucrose production (i.e. the genes encoding for a sucrose phosphate synthase/phosphatase and/or a sucrose synthase), the increased thermotolerance may be due to the production of the compatible solute 'sucrose'.

The present invention specifically relates to a biological nitrification process as described above wherein said increased osmolarity corresponds to an increased osmolarity of at least factor 15 compared to the osmolarity which is present when subsequently contacting said consortium with said ammoniacal waste water having a temperature higher than 40 ° .

With the term 'oxidizes at least 80% of the total amount of removed ammonium to nitrite and/or nitrate' is particularly meant that at least 80% of all ammonia in said waste water is removed and that biological ammonia oxidation is the major mechanism for ammonia removal (i.e. a minimum 80% (i.e. 80, 81 , 82, 83, 84, 85...,90,..., 95%...) of the total of removed ammonia is removed by biological ammonia oxidation) and that other mechanisms - such as ammonia stripping and/or nitrogen assimilation into biomass- can be only minor mechanisms for ammonia removal (i.e. maximum 20% (i.e. 20, 19, 18, 17, 16, 15..., 10,..., 5%) of the total of removed ammonia).

The present invention more specifically relates to a process as described above wherein said removal of at least 80% of ammonium from ammoniacal waste water is 85, 90 or 95%. The terms 'a thermophilic nitrifying consortium enriched from thermophilic environments' refer to any technique known in the art which allows a particular type of microorganisms - such as thermophilic AerAOB, AOA, HetAOB and/or NOB- to grow faster than any other microorganism in a given sample taken from thermophilic environments so that said particular type of microorganisms can be enriched. For example -and as disclosed further in detail- thermophilic AerAOB, AOA, HetAOB and/or NOB can be enriched from thermophilic environments such as green waste, rabbit manure and green waste, digested organic waste or cow manure by incubating samples from said thermophilic environments aerobically at a temperature above 40 °C (for example at least 50 °C)at a pH below 7 for a period of at least 30 days (i.e. 30, 40, 50, 60,..., 100, 120..., 200, 300 or more days...) wherein ammonia or nitrite is given as only substrate at a concentration of maximum 20 mg N/L (i.e. 20, 19, 18, 17, 16, 15, 10, ...5N/L...). The enriched thermophilic nitritation (when ammonia is give as only substrate) and nitratation (when nitrite is given as only substrate) enrichments can thereafter be combined to create a 'synthetic', thermophilic, nitrifying consortium.

The term 'incubating' specifically refers to maintaining a chemical or biochemical system under specific conditions in order to promote a particular reaction.

The latter thermophilic consortia are capable to efficiently oxidize ammoniacal nitrogen to nitrite and/or nitrate at a temperature above 40 °C, preferably above 50 °C (i.e. 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60°C...).

Hence, the present invention further relates to a biological nitrification process as described above wherein said mesophilic nitrifying microbial consortium or said enriched thermophilic nitrifying consortium comprises autotrophic or heterotrophic, ammonia and/or nitrite oxidizing, bacteria or archaea.

The term 'AOB' comprises ammonium oxidizing bacteria catalyzing the aerobic conversion from ammoniacal nitrogen into nitrite autotrophically (see eq 1 above). Most common examples in biological nitrogen removal systems are bacteria belonging to the phylum Proteobacteria such as the species Nitrosomonas europaea, Nitrosomonas eutropha, Nitrosomonas halophila and Nitrosomonas mobilis. The term 'AOA' comprises ammonium oxidizing archaea, catalyzing the aerobic conversion from ammoniacal nitrogen into nitrite (see eq 1 above). Examples are archaea belonging to the phyla Crenarchaeota or Thaumarchaeota such as Nitrosopumilus maritimus, Nitrosocaldus yellowstonii, Nitrososphaera gargensis and Nitrososphaera viennensis. The term 'HetAOB' comprises heterotrophic ammonium oxidizing bacteria, catalyzing the aerobic conversion from ammonia into nitrite heterotrophically. Examples are Pseudomonas and Bacillus.

The term 'NOB' comprises nitrite oxidizing bacteria catalyzing the aerobic conversion from nitrite into nitrate (see eq 2 above). Examples are Nitrospira marina, Nitrospira moscoviensis,. Nitrobacter winogradskyi, Nitrobacter hamburgensis, Nitrobacter vulgaris, Nitrococcus mobilis, Nitrospina gracilis, Candidatus Nitrotoga arctica and members belonging to the Chloroflexi phylum.

The present invention more specifically relates to consortia comprising bacteria belonging to the phylum Proteobacteria and the genus Nitrospira that oxidizes at least 80% of the total amount of removed ammonium to nitrite and/or nitrate at a temperature between 40 °C and 45°C, or specifically relates to consortia comprishg archaea belonging to the phylum Thaumarchaeota and bacteria belonging to the genus Nitrospira that oxidizes at least 80% of the total amount of removed ammonium to nitrite and/or nitrate at a temperature above 50 °C.The present invention further relates to a bidogical nitrification process as described above wherein said temperature in step a) is between 40 °C and 45 °C, or, wherein said temperature as indicated in step b) is above 50 °C.

Hence, the present invention specifically relates to a process as described above wherein said consortium obtained by increased osmolarity comprises bacteria belonging to the taxa Proteobacteria, Deinococcus-Thermus, Nitrospira, NC10, Chloroflexi, Firmicutes and Acidobacteria.

Hence, the present invention specifically also relates to a process as described above wherein said consortium obtained from a thermophilic environment comprises archaea belonging to the taxa Thaumarchaeota and Crenarchaeota and bacteria to the taxa Proteobacteria, Deinococcus-Thermus, Nitrospira, NC10, Chloroflexi, Firmicutes and Acidobacteria.

The present invention relates to a consortium comprising bacteria belonging to the phylum Proteobacteria and the genus Nitrospira that oxidizes at least 80% of the total amount of removed ammonium that is present in waste water to nitrite and/or nitrate at a temperature between 40 °C and 45 °C and that is obtainable by a pocess as described above.

The present invention also relates to a thermophilic consortium comprising archaea belonging to the phylum Thaumarchaeota and bacteria belonging to the genus Nitrospira that oxidizes at least 80% of the total amount of removed ammonium that is present in waste water to nitrite and/or nitrate at a temperature above 50 °C and that is obtainable by a process as indicated above.

The present invention further relates to the usage of a mesophilic nitrifying consortium as described above and/or a thermophilic nitrifying consortium as described above to nitrify ammoniacal wastewater in a reactor at a temperature higher than 40 °C wherein the concentration of free ammonia in said wastewater is controlled through controlling the pH of said wastewater and/or through controlling the ammoniacal nitrogen concentration in the reactor so that the concentration of said free ammonia is below 0.5 mg N/L.

Data from the thermophilic enrichments show that the nitritation is inhibited by high free ammonia (FA, NH₃, sensu stricto) concentrations. In order to prevent inhibition in a reactor the FA should therefore be controlled. FA can directly be calculated with measurements of the pH, temperature and the ammoniacal concentration in the reactor.

FA should ideally be kept below 0.3 mg N/L and should certainly not exceed 0.5 mg N/L.

The pH should ideally be kept between pH 6.4 and pH 7 and should certainly be kept within the pH window pH 6 - pH7.5.

A possible control manner of pH and ammoniacal concentration is the following: from the moment FA approaches the maximum values, an acid can be dosed to the reactor in order to lower the pH. Alternatively, the influent flow can temporarily be lowered in order to prevent accumulation of the ammonical concentrations in the reactor. In addition, the present invention relates to employing the above-described temperature- salinity shock linkage in the reversed way, i.e. the usage of a transient temperature shock on a mesophilic nitrifying consortium to facilitate the functioning of a mesophilic nitrification process to a waste water higher in salinity.

With the term 'transient temperature shock' is meant subjecting said consortium to an environment wherein the temperature is at least 2.5°C and maximum 30°C higher (i.e. 2.5, 3, 5, 6, 7, 8, 9, 10,...,15,...,20,...30 °C) than the waste water treatment temperature for a limited period of time. Ά limited period of time' is for example 'overnight' or about 8-12 hours. Following the transient temperature shock, the operational waste water treatment temperature is again used. The term 'higher salinity' refers to a salinity which is at least 3 g/L NaCI equivalents and which is maximum 19 g/L NaCI equivalents (i.e. 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 g/L NaCI equivalents) higher than the environment the consortium originated from. The latter term preferably refers to a salinity higher than 7 g/L NaCI equivalents but lower than 20 g/L NaCI equivalents (such as 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 g/L NaCI equivalents). The term 'NaCI equivalents' refers any combination of salts that constitutes the total salinity of the wastewater with the salts present in such concentrations that they create an electrical conductivity equivalent to the one created by the mentioned NaCI concentrations.

Hence, the present invention relates to a process to increase the resistance of mesophilic nitrifiers (= a mesophilic ammoniacal nitrogen consortium) towards higher salinity comprising subjecting said consortium originating from a lower salinity environment to a transient temperature increase of at least 2.5°C and maximum 30°C above the waste water treatment temperature for a subsequent exposure of said consortium to a salinity increase of at least 3 g/L NaCI equivalents and maximum 19g/L NaCI equivalents.

The present invention further relates to a process as described above wherein said transient temperature increase is at least 7°C and maximum 30°C and wherein the salinity increase of the environment is higher than 7 g/L NaCI equivalents and lower than 20g/L NaCI equivalents.

The present invention more specifically relates to a process as described above wherein said transient temperature increase is 10°C and wherein the said salinity increase of the environment is 10 g/L NaCI equivalents. The present invention will now be illustrated by the following non-limiting examples.

Examples

Example 1 : Subjecting mesophilic consortia to increased osmolarity

Materials and methods

Batch activity tests Aerobic batch tests were performed according to (2007). Biomass with AerAOB and NOB activity was used, originated from an oxygen-limited autotrophic nitrification/denitrification (OLAND) rotating biological contactor (Pynaert et al., 2003). A dosage of 5 g NaCI L^" above the salinity of the cultivation reactor was tested at three different temperatures (31 , 40 and 45°C) and both the direct activity as the activity after 48h was determined. The aerobic tests were performed in open Erienmeyer flasks with ammonium as substrate (50 mg N L^" ) and a buffering solution (final concentrations 1 g NaHC0₃ L^"1 , 4.2 g KH₂P0₄ L^"1 , 5.8 g K₂HP0₄ L^" and 5 g NaCI L^" ). Liquid samples were taken over time for ammonium, nitrite and nitrate analysis. All tests were performed in triplicate on a shaker, including a control without NaCI addition and a control for ammonia stripping (without biomass) for each temperature. Reactor set up and operation

The two parallel reactors (salt and control) had an effective liquid volume of 2 L and an inner diameter of 12 cm. The reactors were operated in a semi-continuous mode. Besides suspended biomass growth, attached was facilitated through providing 20% (v/v) K1 carrier material (Anoxkaldnes, Sweden). Reactors were inoculated with OLAND biomass resulting in an initial biomass concentration of 3.3 ± 0.1 g VSS L-1 . The influent of the salt reactor was supplemented with NaCI until a final electrical conductivity of 17 mS cm-1 was reached. In that way, an additional salinity of 5 g NaCI L-1 was dosed, compared with the OLAND inoculum cultivation reactor, a rotating biological contactor (Pynaert et al., 2003). After a start-up period of 14 days at 34 °C, the temperature of both was gradually increased to 40, 42.5, 45, 47.5 and 50°C on days 14, 88, 130, 180 and 193, respectively. At 40 and 42.5°C, a period where no NaCI was added to the feed of the salt reactor was also included. Both reactors were fed with synthetic wastewater consisting of (NH4)2S04 (50 mg N L-1 ), 7 g NaHC03 g N-1 and KH2P04 (10 mg P L-1 ). In principle the influent nitrogen concentrations were kept stable. However, from the moment that AerAOB activity decreased, the influent NH4+ concentration was lowered from 50 to 20 mg N L-1 to avoid higher free ammonia (FA) concentrations. Average FA concentrations of 0.27 ± 0.21 and 0.18 ± 0.29 mg N L-1 were reached in the control and salt reactor, respectively. When no nitrite accumulation occurred, nitrite was additionally dosed in the influent as NaN02 (50 mg N L-1 ) as done on day 23 in the control reactor and on day 58 in the salt reactor.

The reactor vessels were jacketed, and the temperature was controlled with a circulating thermostatic water bath. The reactor pH was controlled between pH 6.8 and 7.0 by dosage of 0.1 M NaOH/HCI, resulting in averages values of 6.88 ± 0.18 and 6.91 ± 0.16 for the control and salt reactor, respectively. High pressure air pumps provided aeration through a diffuser stone at an average superficial air flow rate of 1 .33 m3 m-2 h-1 , resulting in the dissolved oxygen (DO) concentrations of 3.94 ± 1 .23 and 4.06 ± 1 .14 mg 02 L-1 for the control and salt reactor, respectively. Flow rates were 7.5 ± 1 .0 and 7.8 ± 1 .2 L d-1 , for control and salt reactor respectively, resulting in hydraulic retention times (HRT) of 6.51 ± 0.80 and 6.12 ± 0.60 h, respectively. Specific AerAOB and NOB activities were calculated from the concentrations of different nitrogen compounds in the effluent according to the OLAND stoichiometry at stable periods of minimum 5 days (Vlaeminck et al., 2012).

Chemical analyses Ammonium (Nessler method) and volatile suspended solids (VSS) were measured according to standard methods (Greenberg et al., 1992). Nitrite and nitrate were determined on a 761 Compact Ion Chromatograph (Metrohm, Switzerland) equipped with a conductivity detector. DO and pH were measured with, respectively, a HQ30d DO meter (Hach Lange, Germany) and an electrode installed on a R305 pH-controller (Consort, Belgium) Molecular analyses of the microbial communities.

Fluorescent in-situ hybridization (FISH) was used to determine the NOB genus, i.e. distinguish between Nitrobacter and Nitrospira. Inoculum and endpoint samples were examined by FISH as described by Vlaeminck et al. (2010).

Quantitative polymerase chain reaction (qPCR) was used to quantify the abundance of AOA, AerAOB, NOB and AnAOB over time. Biomass samples were taken from the inoculum and before each change in temperature or salt concentration. DNA extraction and qPCR were performed according to De Clippeleir et al. (2012) targeting the functional amoA gene for AerAOB and AOA, and the 16S rRNA genes of the AnAOB (Kuenenia and Brocadia), and Nitrospira sp. Denaturing gradient gel electrophoresis (DGGE) was used to evaluate the AerAOB community evolution. An inoculum sample was compared with samples of both reactors at 45°C, the temperature at which no AerAOB activity was observed anymore (day 148). Nested PCR, and denaturing gradient gel electrophoresis (DGGE) were performed based on the primers CT0189ABf, CT0189Cf, and CT0653r for β-proteobacterial AerAOB (Pynaert et al., 2003). The obtained DGGE patterns were subsequently processed with BioNumerics software (Applied Maths, Belgium). Results

Batch activity tests

Batch activity tests were used to determine the effect of salt addition on the specific microbial activities in OLAND biomass at three different temperatures (Figure 1 ). In both the control and salt treatment of 5 g L-1 NaCL, specific activities decreased with increasing temperature. The lowest temperature tested was 34 °C, the temperature corresponding to the temperature of the OLAND inoculum cultivation reactor. At 34°C, salt addition induced no significant immediate changes for AerAOB and NOB activity. Also after 48 h, no salt effect could be detected. At 40 °C, salt addition increased the immediate AerAOB activity with 21 % compared to the control treatment at 40 °C. This effect was however not maintained on the longer term. The same phenomenon was observed at 45 °C, where the salt addition increased the immediate AerAOB activity with 20%. While for the AerAOB salt addition had a positive effect on the activity, for the NOB salt addition decreased the direct activity at 45 °C with 83%. After 48h at 45 °C, for both bacterial groups, no activity could be detected anymore. Reactor performance

A control reactor and a salt reactor were set up in parallel to investigate the effect of salt addition on the adaptation of AerAOB and NOB towards higher temperatures. Nitrification was the main process involved, as always >87% of the removed ammonium was recovered as nitrate in the effluent. After a start-up period of 14 days, stable nitrification rates of 229 ± 40 and 134 ± 10 mg N L-1 d-1 were reached for the control and the salt reactor, respectively (Figure 2D).

Increasing the temperature from 34 to 40 °C initialy decreased the specific AerAOB and NOB activities in the control reactor by respectively 90 and 88%. The salt reactor was more resistant to this temperature shock as the immediate decrease in activities for AerAOB and NOB were only 25 and 51 %, respectively. The nitrification in the salt reactor restored nearly completely after 20 days. However, in the control reactor, only the NOB could restore entirely, while the AerAOB only regained 54% of their activity present before the temperature increase.

Raising the temperature from 40 to 42.5 °C on day 88 showed a similar trend. No significant changes in activity were observed in the salt reactor while in the control reactor, AerAOB and NOB activity decreased with respectively 36 and 21 % (Figure 2D).

The further temperature increase to 45°C was fatalfor the AerAOB in both reactors rendering 42.5 °C the maximum cultivation temperature for AerAOB in this study with an ammonium oxidation rate of 1 13 ± 13 mg N L-1 d-1 in the control reactor and 184 ± 9 mg N L-1 d-1 in the salt reactor (Figure 2D). Nevertheless, NOB activity could be maintained until 47.5 °C with a sixfold higher activity in the salt reactor than in the control reactor. A nitrite oxidation rate of 302 ± 21 mg N L-1 d-1 was namely reached in the salt reactor while the NOB activity in the control reactor decreased until 49 ± 7 mg N L-1 d-1 (Figure 2D).

In order to investigate whether the salt was essential in the functioning of the salt reactor, a period without salt addition was included, both at 40 and 42.5°C. At 40°C, removing the salt had no effect on the AerAOB activity but did increase the NOB activity (Figure 2D). At 42.5 °C, removal of the salt did not affect the NOB, but affected the AerAOB activity with a decrease of 19% (Figure 2D). Restarting salt addition, however, increased AerAOB activity to the original level. Salt thus seemed important during the temperature transitions, but was not essential for a good performance after the temperature raise. Fatty acid methyl ester (FAME) composition

FAME profiles allow to study the adaptation of microorganisms to a particular condition. No major differences could be observed in the relative FAME of the inoculum and the biomass from the control and salt reactor at day 148. Over time, some clear changes occurred in the reactors compared to the inoculum. The saturated fatty acid 16:0 almost doubled in abundance while some unsaturated fatty acids such as 14:1 (n-5) and 16:1 (n-7) decreased, suggesting hydrogenation of unsaturated fatty acids (S1 ). This phenomenon is clearly illustrated in the evolution of the ratio saturated upon unsaturated fatty acids (sat/unsat) over time. The ratio evolved similarly in both reactors, starting at 1 and gradually increasing until 2, and thus doubling the level of saturation. Molecular analyses of the microbial communities

FISH revealed that only Nitrospira NOB was present in the inoculum and both reactors at day 148, and that Nitrobacter could not be retrieved.

For qPCR, AerAOB, AO A, AnAOB and the NOB Nitrospira were targeted (Figure 3). AOA were not detected in the inoculum, nor in one of the reactors. AerAOB, AnAOB and NOB evolved similarly in both reactors. AerAOB and Nitrospira copies ng-1 DNA increased with respectively one and two log units. The AnAOB copies ng-1 DNA however decreased with one log unit. When the last qPCR result is compared with the corresponding specific activities (Figure 2D), it has to be noted that although no AerAOB activity was detected for almost one week, still 3 ^χ 105 and 1 ^χ 106 AerAOB copies ng-1 DNA were measured.

The DGGE profiles for AerAOB confirmed that adaptation had occurred at the species level, suggesting that salinity did not induce a strong selection towards specialized bacteria (Figure 4).

Example 2: Thermophilic consortia enriched from thermophilic environments

The present invention discloses a thermophilic nitrifying consortium composed of a 'synthetic consortium' of separate AOB/AOA and NOB thermophilic enrichments from one or more thermophilic environments. As oxygen, nitrogen and high temperatures are present in aerobic compost and several studies show the presence of both AOA and AOB(Yamamoto et al., 2010; Zeng et al., 201 1 ), compost is taken as a suitable inoculum for thermophilic nitrifying enrichments. Four different aerobic compost were sampled for AOB/AOA and NOB enrichments at 50 °C with addition of respectively ammonium and nitrite as only substrate. The four aerobic composts are presented in Table 1 together with the concentrations of ammonium, nitrite and nitrate in the compost solutions (20g compost/200 ml_ water).

Table 1 : Overview of samples aerobic compost and nitrogen concentrations in compost solution (20g compost/200 ml_ water)

Sample Aerobic compost NH₄ ⁺ N0₂ ^" N0₃ ^"

(mg N L¹) (mg N L¹) (mg N L¹)

A Green waste 0 2 18

B Rabbit manure/green 0 4 50

waste

C Digested organic waste 1 14 0 1

D Cow manure 26 0 1 First ammonium oxidation was observed after one month of incubation in samples A and B. These are the composts where nitrite and nitrate, but no ammonium was detected in the initial compost solution (Table 1 ).

Although sample C and D did not show any ammonium oxidation in the enrichments where ammonium was added, nitrate formation was observed in the enrichments where nitrite was added as substrate. This nitrite oxidation occurred after about 120 and 200 days of incubation.

The four different compost inoculums could thus be divided in two groups. Sample A and B that could be enriched in ammonium oxidizing organisms and sample C and D in nitrite oxidizing organisms. After one year of incubation and several dilution steps two coupled nitritation/nitratation (Figure 5 and 6) and two highly active nitratation enrichments were obtained (Figure 7 and 8).

A mix of sample A and B enrichments was used as a inoculum for the start-up of a fully controlled (pH and DO) fixed bed biofilm reactor operating at 50 °C with synthetic wastewater. Average ammonium and nitrite oxidizing volumetric rates of 4 and 13 mg N/L/d were reached during first 100 days of operation (Figure 9), considered as the start-up period. For a good start-up and performance of the thermophilic nitrification reactor, the synthetic influent based on tap water was supplemented with a trace element solution (Fe, Zn, Co, Mn, Cu, Mo, Ni, Se, B). From day 100 on, a sharp increase in both activities was measured, reaching up to 128 and 200 mg N/L/d for ammonium and nitrite oxidation, respectively. Once those rates were reached, for the last 50 days an average ammonium removal efficiency of 96% was reached and an average nitrogen loss of 4% (stripping and/or assimilation) was observed, indicating that nitrification is the major process involved in the nitrogen removal.

Illumina sequencing (16S rRNA) was performed on the biomass of the thermophilic nitrification reactor and revealed a highly enriched community of Thaumarchaeota and Nitrospira (Figure 10).

Example 3: Subjecting mesophilic consortia to a temperature shock for increased salt tolerance

Materials and methods Aerobic batch tests were performed according to Vlaeminck et al. (2007). Biomass with AerAOB and NOB activity was used, originated from an oxygen-limited autotrophic nitrification/denitrification (OLAND) rotating biological contactor (Pynaert et al., 2003). The aerobic tests were performed in open Erlenmeyer flasks with a buffering solution with final concentrations of 1 g NaHC0₃ L^~\ 4.2 g KH₂P0₄ L^" and 5.8 g K₂HP0₄ L^" .The sludge was shocked at 40 °C overnight with ammonium (50 mg N L ) and nitrite (25 mg N L^" ) as substrate. In parallel, the control OLAND sludge was incubated overnight with the same substrate concentrations at 30 °C, the temperature d the OLAND RBC. After the overnight incubation, all microcosms were put at 30 °C and 25% of the medium was replaced in order to provide different salt concentrations (0, 10, 20 and 35 g NaCI L^" ) and new substrate (ammonium, 50 mg N L^" ). Liquid samples were taken over time for ammonium, nitrite and nitrate analysis. All tests were performed in triplicate on a shaker.

Results

Batch activity tests were used to investigate whether a temperature shock can increase the resistance towards salt in a subsequent salt shock. In the control incubation at 30 °C, both AerAOB and NOB specific activities decreased with increasing salt concentrations, clearly reflecting the inhibition effect of salt on nitrification (Fig 1 1 ). The microcosms shocked at 40°C before the salt addition, did show a lower sat inhibition effect on AerAOB activity at the different salt concentrions (Fig 12). At 10 g NaCL/L, the most relevant tested concentration, the AerAOB activity was moreover stimulated compared with the control without salt addition. In contrast, for the NOB activity, the applied temperature shock could not improve the resistante towards high salt concentrations (Fig 12). These findings show that temperature shocks can be used to improve the resistance of AerAOB activity towards more saline wastewaters.

References

Abeynayaka, A., Visvanathan, C. 201 1 . Performance comparison of mesophilic and thermophilic aerobic sidestream membrane bioreactors treating high strength wastewater. Bioresource Technology, 102(9), 5345-5352.

Bouskova, A., Dohanyos, M., Schmidt, J.E., Angelidaki, I. 2005. Strategies for changing temperature from mesophilic to thermophilic conditions in anaerobic CSTR reactors treating sewage sludge. Water Research, 39(8), 1481 -1488.

Caldas, T., Demont-Caulet, N., Ghazi, A., Richarme, G. 1999. Thermoprotection by glycine betaine and choline. Microbiology -Uk, 145, 2543-2548.

De Clippeleir, H., Courtens, E., Mosquera, M., Vlaeminck, S.E., Smets, B.F., Boon, N.,

Verstraete, W. 2012. Efficient Total Nitrogen Removal in an Ammonia Gas Biofilter through High-Rate OLAND. Environmental Science & Technology, 46(16), 8826-

8833.

den Besten, H.M.W., van der Mark, E.J., Hensen, L, Abee, T., Zwietering, M.H. 2010.

Quantification of the Effect of Culturing Temperature on Salt-Induced Heat

Resistance of Bacillus Species. Applied and Environmental Microbiology, 76(13), 4286-4292.

Dincer, A.R., Kargi, F. 1999. Salt inhibition of nitrification and denitrification in saline wastewater. Environmental Technology, 20(1 1 ), 1 147-1 153.

Fletcher, S.A., Csonka, L.N. 1998. Characterization of the induction of increased thermotolerance by high osmolarity in Salmonella. Food Microbiology, 15(3), 307- 317.

Golovacheva, R.S. 1976. Thermophilic nitrifying bacteria from hot springs. Microbiology,

45(2), 329-331 .

Greenberg, A.E., Clesceri, L.S., Eaton, A.D. 1992. Standard Methods for the Examination of Water and Wastewater. American Public Health Association., Washington DC.

Guo, J.H., Peng, Y.Z., Huang, H.J., Wang, S.Y., Ge, S.J., Zhang, J.R., Wang, Z.W. 2010.

Short- and long-term effects of temperature on partial nitrification in a sequencing batch reactor treating domestic wastewater. Journal of Hazardous Materials, 179(1 - 3), 471 -479.

Holtmann, G., Bremer, E. 2004. Thermoprotection of Bacillus subtilis by exogenously provided glycine betaine and structurally related compatible solutes: Involvement of Opu transporters. Journal of Bacteriology, 186(6), 1683-1693. Hies, P., Mavinic, D.S. 2001 . The effect of decreased ambient temperature on the biological nitrification and denitrification of a high ammonia landfill leachate. Water Research, 35(8), 2065-2072.

Kurian, R., Acharya, C, Nakhla, G., Bassi, A. 2005. Conventional and thermophilic aerobic treatability of high strength oily pet food wastewater using membrane-coupled bioreactors. Water Research, 39(18), 4299-4308.

Lapara, T.M., Alleman, J.E. 1999. Thermophilic aerobic biological wastewater treatment.

Water Research, 33(4), 895-908.

Lebedeva, E.V., Off, S., Zumbragel, S., Kruse, M., Shagzhina, A., Lucker, S., Maixner, F.,

Lipski, A., Daims, H., Spieck, E. 201 1 . Isolation and characterization of a moderately thermophilic nitrite-oxidizing bacterium from a geothermal spring. Ferns Microbiology

Ecology, 75(2), 195-204.

Moussa, M.S., Sumanasekera, D.U., Ibrahim, S.H., Lubberding, H.J., Hooijmans, CM.,

Gijzen, H.J., van Loosdrecht, M.C.M. 2006. Long term effects of salt on activity, population structure and floe characteristics in enriched bacterial cultures of nitrifiers.

Water Research, 40(7), 1377-1388.

Mulder, A. 2003. The quest for sustainable nitrogen removal technologies. Water Science and Technology, 48(1 ), 67-75.

Ortega, L, Barrington, S., Gulot, S.R. 2008. Thermophilic adaptation of a mesophilic anaerobic sludge for food waste treatment. Journal of Environmental Management,

88(3), 517-525.

Pynaert, K., Smets, B.F., Wyffels, S., Beheydt, D., Siciliano, S.D., Verstraete, W. 2003.

Characterization of an autotrophic nitrogen-removing biofilm from a highly loaded lab- scale rotating biological contactor. Applied and Environmental Microbiology, 69(6), 3626-3635.

Sanchez, O., Aspe, E., Marti, M.C., Roeckel, M. 2004. The effect of sodium chloride on the two-step kinetics of the nitrifying process. Water Environment Research, 76(1 ), 73-80. Shore, J.L., M'Coy, W.S., Gunsch, C.K., Deshusses, M.A. 2012. Application of a moving bed biofilm reactor for tertiary ammonia treatment in high temperature industrial wastewater. Bioresource Technology, 112, 51 -60.

Suvilampi, J., Rintala, J. 2003. Thermophilic aerobic wastewater treatment, process performance, biomass characteristics, and effluent quality. Reviews in Environmental

Science and Biotechnology, 2(1 ), 35-51 .

Vlaeminck, S.E., De Clippeleir, H., Verstraete, W. 2012. Microbial resource management of one-stage partial nitritation/anammox. Microbial Biotechnology, 5(3), 433-448. Vlaeminck, S.E., Geets, J., Vervaeren, H., Boon, N., Verstraete, W. 2007. Reactivation of aerobic and anaerobic ammonium oxidizers in OLAND biomass after long-term storage. Applied Microbiology and Biotechnology, 74(6), 1376-1384.

Vlaeminck, S.E., Terada, A., Smets, B.F., De Clippeleir, H., Schaubroeck, T., Bolca, S., Demeestere, L, Mast, J., Boon, N., Carballa, M., Verstraete, W. 2010. Aggregate Size and Architecture Determine Microbial Activity Balance for One-Stage Partial Nitritation and Anammox. Applied and Environmental Microbiology, 76(3), 900-909.

Welsh, D.T. 2000. Ecological significance of compatible solute accumulation by microorganisms: from single cells to global climate. Ferns Microbiology Reviews, 24(3), 263-290.

Yamamoto, N., Otawa, K., Nakai, Y. 2010. Diversity and Abundance of Ammonia-Oxidizing Bacteria and Ammonia-Oxidizing Archaea During Cattle Manure Composting. Microbial Ecology, 60(4), 807-815.

Zeng, G.M., Zhang, J.C., Chen, Y.N., Yu, Z., Yu, M., Li, H., Liu, Z.F., Chen, M., Lu, L.H., Hu, C.X. 201 1 . Relative contributions of archaea and bacteria to microbial ammonia oxidation differ under different conditions during agricultural waste composting. Bioresource Technology, 102(19), 9026-9032.

Claims

Claims

1 . A process to remove at least 80% of ammonium from ammoniacal waste water having a temperature higher than 40°C in a reactor comprising: a) subjecting at least one mesophilic nitrifying consortium to an increased osmolarity of at least factor 10 compared to the osmolarity which is present when subsequently contacting said consortium with said ammoniacal waste water having a temperature higher than 40 °C, in order to obtain a consortium comprising bacteria belonging to the phylum Proteobacteria and the genus Nitrospira that oxidizes at least 80% of the total amount of removed ammonium to nitrite and/or nitrate at a temperature between 40 °C and 45°C, or b) incubating samples from a thermophilic environment at a temperature of at least 50 °C at a pH below 7 for a period of at least 30 days during which ammonia and nitrite are given as only substrate at a concentration of maximum 20 mg N/L in order to obtain a thermophilic consortium comprising archaea belonging to the phylum Thaumarchaeota and bacteria belonging to the genus Nitrospira that oxidizes at least 80% of the total amount of removed ammonium to nitrite and/or nitrate at a temperature above 50 °C.

2. A process according to claim 1 wherein said removal of at least 80% of ammonium from ammoniacal waste water is 85, 90, or 95%.

3. A process according to claims 1 and 2 wherein said consortium obtained by increased osmolarity comprises bacteria belonging to the taxa Proteobacteria, Deinococcus-Thermus, Nitrospira, NC10, Chloroflexi, Firmicutes and Acidobacteria.

4. A process according to claims 1 and 2 wherein said consortium obtained from a thermophilic environment comprises archaea belonging to the taxa Thaumarchaeota and Crenarchaeota and bacteria to the taxa Proteobacteria, Deinococcus-Thermus, Nitrospira, NC10, Chloroflexi, Firmicutes and Acidobacteria.

5. A biological nitrification process according to any of claims 1 -4 wherein said increased osmolarity corresponds to an increased osmolarity of at least factor 15 compared to the osmolarity which is present when subsequently contacting said consortium with said ammoniacal waste water having a temperature higher than 40° .

6. A consortium comprising bacteria belonging to the phylum Proteobacteria and the genus Nitrospira that oxidizes at least 80% of the total amount of removed ammonium that is present in waste water to nitrite and/or nitrate at a temperature between 40 °C and 45 °C and that is obtainable by a process according to claim 1 .

7. A thermophilic consortium comprising archaea belonging to the phylum Thaumarchaeota and bacteria belonging to the genus Nitrospira that oxidizes at least 80% of the total amount of removed ammonium that iss present in waste water to nitrite and/or nitrate at a temperature above 50 °C and that is obtainable by aprocess according to claim 1 .

8. Use of a consortium according to claim 6 and/or a thermophilic consortium according to claim 7 to nitrify ammoniacal wastewater in a reactor at a temperature higher than 40 °C wherein the concentration of free ammonia in said wastewater is controlled through controlling the pH of said wastewater and/or through controlling the ammoniacal nitrogen concentration in the reactor so that the concentration of said free ammonia is below 0.5 mg N/L.

9. A process to increase the resistance of a mesophilic ammoniacal nitrogen consortium towards higher salinity comprising subjecting said consortium originating from a lower salinity environment to a transient temperature increase of at least 2.5 °C and maximum 30 °C above the waste water treatment temperature for a subsequent exposure of said consortium to a salinity increase of at least 3 g/L NaCI equivalents and maximum 19 g/L NaCI equivalents.

10. A process according to claim 9 wherein said transient temperature increase is at least 7°C and maximum 30°C and wherein the salinity incrase of the environment is higher than 7 g/L NaCI equivalents and lower than 20 g/L NaCI equivalents.

1 1 . A process according to claims 9-10 wherein said transient temperature increase is 10°C and wherein the said salinity increase of the environment is 10 g/L NaCI equivalents.