WO2018134391A1 - Production of biogas from algae - Google Patents

Abstract

This invention relates to a method for production of biogas. The present invention also relates to the use of algae for production of biogas and a system for production of biogas.

Description

PRODUCTION OF BIOGAS FROM ALGAE

FIELD OF THE INVENTION

[0001] The present invention lies in the field of biochemistry and relates to a method for production of biogas. The present invention also relates to the use of algae for production of biogas and a system for production of biogas.

BACKGROUND OF THE INVENTION

[0002] The steadily increasing global energy demand and limited fossil fuel sources have created tremendous efforts in developing renewable energy sources (Martinot et al., 2007; REN21, 2015; Verbruggen and Al Marchohi, 2010). Because of their high theoretical and practical areal productivities, microalgae are in focus of research for generation of third generation of biofuels (Bux and Chisti, 2016; Stephens et al., 2010). However, the generation of biofuels such as biodiesel or bioethanol is not economically relevant, nowadays, due to the actually high biomass generation and extensive energy or chemical downstream process costs. Methane generation via anaerobic fermentation represents an alternative way of generating gaseous fuel from biomass. Anaerobic digestion (AD) process is simple in application and highly efficient, since up to 88 % conversion efficiency can be reached with appropriate substrate (Raposo et al., 2011).

[0003] Nevertheless, today microalgae biomass is not regarded as suitable substrate for biogas generation in AD process mainly for two reasons: High recalcitrance towards microbial decomposition mediated by the rigid cell wall and unfavorable low C:N ratio of the biomass caused by high protein content (Klassen et al., 2016). The resistance of the cell wall can be overcome by application of physical and enzymatically pretreatments (Mahdy et al., 2014a; Mahdy et al., 2014b; Mahdy et al., 2015; Marsolek et al., 2014; Mendez et al., 2014; Schwede et al., 2013), thereby unfortunately increasing investment costs for biomass processing. Additionally, the continuous fermentation of this pretreated and thus completely accessible biomass as mono -substrate was shown to be not efficient (Klassen et al., 2016), mainly due to ammonia inhibition of methanogens, released from protein degradation (Mahdy et al., 2015; Mendez et al., 2015; Schwede et al., 2013; Yenigiin and Demirel, 2013).

[0004] Some research was performed in the past, for the minimization of the protein content in the biomass by applying limited amounts of nitrogen or phosphate to the culture media (Klassen et al., 2015; Markou et al., 2013). This strategy seems to be favoring not only lower protein content but also the accessibility of algae to microbial community, which was monitored by methane potential tests and counting intact cells before and after batch fermentation process (Klassen et al., 2015). Microalgae from three different genera Chlamydomonas, Chlorella and Scenedesmus lost subsequently the capability to resist the bacterial degradation with ongoing starvation status, leading consequently to higher C:N ratios (24-26) in the biomass and higher biomethane yields, with conversions rates near the theoretical maximum (Klassen et al., 2015; Klassen et al., 2016). However, these experiments were performed in batch fermentation trails, allowing conclusions only regarding accessibility of biomass towards anaerobic degradability and achievement of maximal possible methane yields. In regular case (industrial scale), fermentation of biomass is performed in continuous or semi-continuous mode since it is more efficient regarding volumetric productivity. In this mode other factors besides biodegradability can play a crucial role e. g. ammonia or ammonium inhibition (often caused by high protein content), long chain fatty acid inhibition (caused by high lipid content), enrichment of toxic compounds and unbalance of macro/micro nutrients (necessary for growth of microbial community) (Uggetti et al., 2016; Weiland, 2010). Additionally, variety of process parameters (HRT, OLR, temperature, pH) may need to be considered for optimal performance of the digester, otherwise it can lead to failure of the process (Speece, 1983; Klassen et al., 2016).

[0005] Hence, there is a need in the art for an efficient method that converts algae biomass into a state that allows its continuous or semi-continuous fermentation.

SUMMARY OF THE INVENTION

[0006] It is an object of the present invention to meet the above need by providing a method for production of biogas as described herein. Surprisingly, the present inventors found that algae cultured under limiting nutrient conditions can directly (without the requirement of additional pre-treatment steps, such as pre-treatments that degrade or destroy the cell wall of the algae) be used for further digestion by bacteria and/or archaea in a continuous or semi- continuous fermentation. The continuous or semi-continuous fermentation of algae biomass to biogas provides the advantage that this process allows the scale-up to volumes as big as required in industrial scale. However, the successful transformation of the batch fermentation to continuous or semi-continuous fermentation could not have been expected, as the continuous or semi-continuous fermentation is usually highly sensitive with regard to different parameters. These parameters include ammonia or ammonium inhibition, long chain fatty acid inhibition, enrichment of toxic compounds, HRT, OLR, temperature and pH, and transformation of the batch fermentation to continuous or semi-continuous fermentation constantly fail. Klassen et al., 2016, discloses in Table 3 several examples, wherein the batch mode approached complete biomass-to-biogas conversion and in contrast to this continuous or semi-continuous fermentation resulted only in 15% to 55% specific methane yield compared to the theoretical methane potential (SMY of TMP). Said examples also include experimental set ups using biomass with a C/N ratio of 12.2. Thus, in the prior art the difficulty to transform batch fermentation to continuous or semi-continuous fermentation without significantly losing fermentation efficiency is well-known.

[0007] In a first aspect, the present invention is thus directed to a method for production of biogas comprising: (a) culturing a plurality of algae in media, wherein said media contains limiting nutrient conditions; and (b) anaerobically digesting the plurality of algae of (a) by bacteria and/or archaea to produce biogas.

[0008] In preferred embodiments of the invention, the digestion step of (b) is a semi- continuous or continuous fermentation.

[0009] The scope of the present invention also encompasses preferred embodiments with the proviso that the method of the invention does not comprise chemical and/or physical treatment of the plurality of algae between steps (a) and (b). In more preferred embodiments, the above-described chemical and/or physical treatment degrades or destroys the cell wall of the plurality of algae. In still further preferred embodiments of the invention, the limiting nutrient conditions comprise limiting conditions for nitrogen, sulfur, phosphate, potassium, magnesium, calcium, iron, manganese, silicon, zinc, copper, molybdenum, boron and/or vanadium. In more preferred embodiments, the limiting nutrient conditions comprise limiting nitrogen conditions, wherein said limiting nitrogen conditions comprise a total amount of nitrogen in the media of less than 100 mg per liter, preferably less than 50 mg per liter. In other preferred embodiments, the algae biomass comprises (a) a ratio of total amounts of carbon to total amounts of nitrogen of more than 10, preferably more than 15; and/or (b) a peptide/protein of less than 50%, preferably less than 40%.

[00010] Also encompassed by the scope of the present invention is that in preferred embodiments the biogas is methane (CH₄), carbon dioxide (C0₂), nitrogen (N₂), oxygen (O2), hydrogen sulfide (H₂S), hydrogen (H₂) or ammonia (NH₃), preferably methane (CH₄).

[00011] In preferred embodiments, the algae is a chlorophyte, preferably selected from the group consisting of Chlamydomonas, Chlorella and Scenedesmus. In more preferred embodiments, the algae is Chlamydomonas reinhardtii.

[00012] In further preferred embodiments of the invention, (a) the bacteria are selected from the group consisting of Armatimonadetes, Bacteroidetes, Chloroflexi, Firmicutes, Planctomycetes, Proteobacteria, Spirochaetes and Synergistetes; and/or (b) the archaea are selected from the group consisting of Methanosaeta, Methanospirillum, Methanolinea and Methanoculles.

[00013] In a further aspect, the present invention relates to the use of algae for production of biogas, wherein the algae is (a) cultured in media, wherein said media contains limiting nutrient conditions; and/or (b) anaerobically digested by bacteria and/or archaea.

[00014] In preferred embodiments of the use of the invention, the biogas is methane (CH₄), carbon dioxide (C0₂), nitrogen (N₂), oxygen (0₂), hydrogen sulfide (H₂S), hydrogen (H₂) or ammonia (NH₃), preferably methane (CH₄).

[00015] In a third aspect, the invention relates to a system for the production of biogas comprising: (a) a first container comprising at least one algae species and media, wherein said media contains limiting nutrient conditions; (b) a second container comprising (i) the same at least one algae species of (a) and (ii) bacteria and/or archaea.

[00016] In preferred embodiments of the system of the invention, the second container is formed to allow semi-continuous or continuous fermentation of the algae by anaerobic digestion of the bacteria and/or archaea.

[00017] In further preferred embodiments of the system of the invention, (a) the limiting nutrient conditions comprise limiting conditions for nitrogen, sulfur, phosphate, potassium, magnesium, calcium, iron, manganese, silicon, zinc, copper, molybdenum, boron and/or vanadium; (b) the biogas is methane (CH₄), carbon dioxide (C0₂), nitrogen (N₂), oxygen (O2), hydrogen sulfide (H₂S), hydrogen (H₂) or ammonia (NH₃), preferably methane (CH₄); (c) the at least one algae species is a chlorophyte, preferably selected from the group consisting of Chlamydomonas, Chlorella and Scenedesmus; (d) the bacteria are selected from the group consisting of Armatimonadetes, Bacteroidetes, Chloroflexi, Firmicutes, Planctomycetes, Pwteobacteria, Spirochaetes and Synergistetes; and/or (e) the archaea are selected from the group consisting of Methanosaeta, Methanospirillum, Methanolinea and Methanoculles .

BRIEF DESCRIPTION OF THE DRAWINGS

[00018] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings.

[00019] Figure 1 shows a graphical abstract of the method of the invention.

[00020] Figure 2 shows photoautotrophic accumulation of algal biomass under replete N and low N culture conditions. Cultivation was performed at 300 μιηοΐ photons nT² s^_1 (white light) and by aeration with carbon dioxide-enriched air (3% v/v) for 10 days. Biomass accumulation was monitored by determination of volatile solids (VS). Harvesting for fermentation trials was performed at day 6 for both media conditions (indicated by arrow).

[00021] Figure 3 shows the average methane content in biogas produced by different methods. [00022] Figure 4 shows microalgae biomass characteristics. After harvesting, important parameters of the C. reinhardtii biomass for fermentation were periodically determined and presented as mean values. Error bars represent standard error (SE, n = 8). DW=dry weight, N=nitrogen, C=carbon, VS=volatile solids, COD=chemical oxygen demand TMP=theoretical methane potential, BM=biomass.

[00023] Figure 5 shows biogas and methane productivity via anaerobic fermentation of algal biomass in semi-continuous mode. The biogas productivity was monitored online and methane content was measured weekly (left = replete N BM, right = low N BM). Organic loading rate (OLR) is indicated by shades of gray in the background, thereby following biomass concentrations were applied: OLR1 = lg VS L ¹ d ¹, OLR2 = 2g VS L ¹ d ¹, OLR4 = 4g VS L ¹ d^{" 1}. Error bars represent mean productivity of previous 7 days (SE, n = 7). N=nitrogen, BM=biomass, VS=volatile solides.

[00024] Figure 6 shows bacterial diversity dynamics as assessed by high-throughput 16S rRNA amplicon sequencing and represented at the OTU level. The reactors fed with biomass cultivated with replete and low nitrogen content (replete-N BM and low-N BM) were exposed to increasing organic loading rates of 2g and 4g (OLR2 and OLR4). The inoculum and the sampling periods at the end of each OLR were chosen for microbial community monitoring.

[00025] Figure 7 shows an overview of the mean biogas and methane productivities for low-N and replete-N reactors as well as maize silage as predominant used renewable substrate for industrial scale fermentation. The values were summarized by distinct OLR-phases (OLR2 = 2g VS L^"1 d^"1, OLR4 = 4g VS L^"1 d^"1). Error bars represent standard error (SE, n = 8); a) literature values for maize silage from (Mahnert and Linke, 2009).

[00026] Figure 8 shows the concentration of total carbon and nitrogen during the experimental time course in replete-N BM digester (A) and low-N BM digester (B). Concentration of total organic and inorganic carbon (TOC and TIC) is shown for replete-N BM digester (C) and low-N BM digester (D). Measurements were performed in 3 technical replicates. Error bars represent standard deviation (sd).

[00027] Figure 9 shows the concentration of volatile solids and total solids during the experimental time course in replete-N BM digester (A) and low-N BM digester (B). Measurements were performed in at least 3 technical replicates. Error bars represent standard deviation (sd).

[00028] Figure 10 shows the concentration of chemical oxygen demand during the experimental time course in replete-N BM digester (A) and low-N BM digester (B). Measurements were performed in 3 technical replicates. Error bars represent standard deviation (sd).

[00029] Figure 11 shows the removal efficiency, which was calculated on the basis of volatile solids of the substrate feed in OLR of 2 and 4 and VS of the digester outflow during the OLR of 2 and 4, for replete-N BM digester (A) and low-N BM digester (B). Removal efficiency was calculated on the basis of chemical oxygen demand of the substrate feed in and VS of the digester outflow during the OLR of 2 and 4, for replete-N BM digester (C) and low-N BM digester (D).

[00030] Figure 12 shows the analysis of essential fermentation parameters of anaerobic digestion of algal biomass in semi-continuous mode (left = replete N BM, right = low N BM). Organic loading rate (OLR) is indicated by shades of gray in the background, thereby following biomass concentrations were applied: OLR1 = lg VS L^"1 d^"1, OLR2 = 2g VS L^"1 d^"1, OLR4 = 4g VS L^"1 d^"1. Error bars represent standard deviation (SD, n = 3). N=nitrogen, BM=biomass, VS=volatile solids, TAN=total ammonium nitrogen, FAN=free ammonia nitrogen, VFA=volatile fatty acids.

[00031] Figure 13 shows bacterial diversity dynamics as assessed by high-throughput 16S rRNA amplicon sequencing and represented at the phylogenetic level for bacteria (A) and family level for Archaea (B). The reactors fed with biomass cultivated with replete and low nitrogen content (replete-N BM and low-N BM) were exposed to increasing organic loading rates OLR2 and OLR4 (OLR2 = 2g VS L ¹ d ¹, OLR4 = 4g VS L ¹ d ¹). The inoculum and the sampling periods at the end of each OLR were chosen for microbial community monitoring.

[00032] Figure 14 shows the filtered sequences during amplicon processing. OTU=operational taxonomic unit, N=nitrogen, sd=standard deviation, OLR=organic loading rate, rep.=replicate. DETAILED DESCRIPTION OF THE INVENTION

[00033] The present inventors found that algae biomass can be converted into biogas by using the advantageous process of continuous or semi-continuous fermentation. Surprisingly, continuous or semi-continuous fermentation can be applied on algae if the algae biomass has previously been cultivated under limiting nutrient conditions, such as limiting nitrogen conditions.

[00034] Therefore, in a first aspect, the present invention is thus directed to a method for production of biogas comprising: (a) culturing a plurality of algae in media, wherein said media contains limiting nutrient conditions; and (b) anaerobically digesting the plurality of algae of (a) by bacteria and/or archaea to produce biogas.

[00035] The term "biogas", as used herein, refers to a gas produced by the biological breakdown of organic material in the absence of oxygen. Biogas is produced by anaerobic digestion of biodegradable materials. The biogas produced by the method of the invention is digested or produced by bacteria and/or archaea. In preferred embodiments of the invention, the biogas is one or any combination of the following gases: methane (CH₄), carbon dioxide (C0₂), nitrogen (N₂), oxygen (O2), hydrogen sulfide (H₂S), hydrogen (H₂) or ammonia (NH₃). Typically, the biogas comprises primarily methane and carbon dioxide. In more preferred embodiments, the biogas comprises or consists of methane.

[00036] The terms "culturing" or "cell culturing", as interchangeably used herein, refers to maintenance or growth of a cell, preferably an algal cell, in a liquid culture medium under a controlled or uncontrolled set of physical conditions. Culture media and conditions are well- known to the skilled person and are, for instance, described in Algal Culturing Techniques, 1st Edition, Robert A. Andersen, Academic Press, 2005. In preferred embodiments, the algae are cultured under non-sterile conditions. This includes that the media does not contain any ingredients that inhibit growth of microorganism, such as antibiotics. In addition, non-sterile conditions include that the media containing the algae may be in direct contact with the air of the surrounding environment and may not be separated from the environment by membranes, filters etc. In other preferred embodiments, the algae are cultured under a light intensity that is lower than 400 μΕ, preferably lower than 390 μΕ, lower than 380 μΕ, lower than 370 μΕ, lower than 360 μΕ, lower than 350 μΕ, lower than 340 μΕ, lower than 330 μΕ, lower than 320 μΕ, lower than 310 μΕ or lower than 300 μΕ.

[00037] In preferred embodiments, the cultivation is conducted in vessels with a capacity of more than 100 L to less than 1 L, more preferably between 100 L and 1 L, more preferably between 50 L and 2 L, more preferably between 10 L and 3 L.

[00038] In preferred embodiments, harvesting of the cultivated algae biomass for fermentation is performed at day 6.

[00039] The term "media", as used herein, refers to a liquid or gelatinous substance containing nutrients in which microorganisms, cells, or tissues, preferably algae, are cultivated. In this context "cultivated" refers to the growth of the cells or their maintenance. Cell growth relates to the increase in size (for example the volume of one or more compartments of the cell or its membrane) or the division of cells. In preferred embodiments, the media for culturing the algae is free of any (organic) carbon source, more preferably free of TRIS buffer.

[00040] The terms "one or more" or "at least one", as interchangeably used herein, relate to at least one, but preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25 or a plurality of molecules. In this connection, the term "plurality" means more than one, preferably 2-1000, more preferably 2-100, even more preferably 2-50, still more preferably 2-25 and most preferably 2-15.

[00041] The terms "alga" and "algae", as used herein, refer to any organisms with chlorophyll and, in other than unicellular algae, a thallus not differentiated into roots, stems and leaves, and encompasses prokaryotic and eukaryotic organisms that are photoautotrophic or facultative heterotrophs. The term "algae" includes macroalgae (such as seaweed) and microalgae. The terms "microalgae" and "phytoplankton", used interchangeably herein, refer to any microscopic algae, photoautotrophic or facultative heterotroph protozoa, photoautotrophic or facultative heterotroph prokaryotes, and cyanobacteria (commonly referred to as blue-green algae and formerly classified as Cyanophyceae). The use of the term "algal" also relates to microalgae and thus encompasses the meaning of "microalgal". The term "algal composition" or "algal biomass" refers to any composition that comprises algae, and is not limited to the body of water or the culture in which the algae are cultivated. An algal composition can be an algal culture, a concentrated algal culture, or a dewatered mass of algae, and can be in a liquid, semisolid, or solid form. A non-liquid algal composition can be described in terms of moisture level or percentage weight of the solids. An "algal culture" is an algal composition that comprises live algae. The algae of the disclosure can be a naturally occurring species, a genetically selected strain, a genetically manipulated strain, a transgenic strain, or a synthetic algae. Algae from tropical, subtropical, temperate, polar or other climatic regions can be used in the disclosure. Endemic or indigenous algal species are generally preferred over introduced species, where an open culturing system is used. Algae, including microalgae, inhabit all types of aquatic environment, including but not limited to freshwater (less than about 0.5 parts per thousand (ppt) salts), brackish (about 0.5 to about 31 ppt salts), marine (about 31 to about 38 ppt salts), and briny (greater than about 38 ppt salts) environment. Any of such aquatic environments, freshwater species, marine species, and/or species that thrive in varying and/or intermediate salinities or nutrient levels, can be used in the embodiments of the disclosure. The algae in an algal composition of the disclosure may contain a mixture of prokaryotic and eukaryotic organisms, wherein some of the species may be unidentified. Fresh water from rivers or lakes, seawater from coastal areas, oceans, water in hot springs or thermal vents, and lake, marine, or estuarine sediments, can be used to source the algae. The algae may also be collected from local or remote bodies of water, including surface as well as subterranean water. Preferably, the algal species for use in the embodiments of the disclosure may be isolated from water, wastewater storage ponds, or soil. It is not required that all the algae in an algal composition of the disclosure are taxonomically classified or characterized for the composition to be used in the present disclosure. Algal compositions including algal cultures can be distinguished by the relative proportions of taxonomic groups that are present.

[00042] In preferred embodiments, the algae is a chlorophyte, preferably selected from the group consisting of Chlamydomonas, Chlorella and Scenedesmus. In more preferred embodiments, the algae is Chlamydomonas reinhardtii.

[00043] The term "chlorophyte", as used herein, refers to is a division of green algae. In more detail, it refers to one of the two clades making up the Viridiplantae, which are the chlorophytes and the streptophytes. The Chlorophyta include only about 4,300 species. [00044] The term "limiting nutrient", as used herein, refers to a chemical element that is required for cellular maintenance, cellular growth or cell division and that determines the species population size, if absent or not accessible. For example, adjustment of concentrations of limiting nutrients may have the effect of modifying, for example, a population's size, with greater levels allowing population sizes to increase whereas reduced amounts have the opposite effect.

[00045] In still further preferred embodiments of the invention, the limiting nutrient conditions comprise limiting conditions for nitrogen, sulfur, phosphate, potassium, magnesium, calcium, iron, manganese, silicon, zinc, copper, molybdenum, boron and/or vanadium. It will be understood that limiting conditions concerning a specific element or combination of elements (such as for nitrogen, sulfur, phosphate, potassium, magnesium, calcium, iron, manganese, silicon, zinc, copper, molybdenum, boron and/or vanadium) include all compounds comprising said specific element(s). In more preferred embodiments, the limiting nutrient conditions comprise limiting nitrogen conditions, wherein said limiting nitrogen conditions comprise a total amount of nitrogen in the media of less than 100 mg per liter, preferably less than 50 mg per liter, and/or preferably at least 1, or 2, or 3, or 5 mg per liter, for example limited to 5-40 mg per liter. In other preferred embodiments, the algae biomass comprises (a) a ratio of total amounts of carbon to total amounts of nitrogen of more than 10, preferably more than 15, and more preferably more than 16; and/or (b) a peptide/protein of less than 50%, preferably less than 40%. The peptide/protein is determined in relation to the total amount of biomass. In various embodiments, the relation of the peptide/protein content to the total amount of biomass is the weight by weight relation.

[00046] In preferred embodiments, the concentration of elemental nitrogen in the biomass, obtained after cultivation under limited nitrogen conditions, is below 5 % of dry weight, preferably below 4 %, and in particular below 3 % of dry weight.

[00047] In more preferred embodiments, the limiting nutrient conditions comprise (a) limiting nitrogen conditions, wherein said limiting nitrogen conditions comprise a total amount of nitrogen in the media of less than 100 mg per liter, preferably less than 50 mg per liter; (b) limiting phosphorus conditions, wherein said limiting phosphorus conditions comprise a total amount of phosphorus in the media of less than 10 mg per liter, preferably less than 6 mg per liter; (c) limiting sulfur conditions, wherein said limiting sulfur conditions comprise a total amount of sulfur in the media of less than 5 mg per liter, preferably less than 2.8 mg per liter; (d) limiting iron conditions, wherein said limiting iron conditions comprise a total amount of nitrogen in the media of less than 5 mg per liter, preferably less than 3.6 mg per liter; (e) limiting magnesium conditions, wherein said limiting magnesium conditions comprise a total amount of magnesium in the media of less than 10 mg per liter, preferably less than 6 mg per liter; (f) limiting copper conditions, wherein said limiting copper conditions comprise a total amount of copper in the media of less than 0.1 mg per liter, preferably less than 0.03 mg per liter; (g) limiting potassium conditions, wherein said limiting potassium conditions comprise a total amount of potassium in the media of less than 20 mg per liter, preferably less than 11 mg per liter; (h) limiting calcium conditions, wherein said limiting calcium conditions comprise a total amount of calcium in the media of less than 1 mg per liter, preferably less than 0.6 mg per liter; (i) limiting manganese conditions, wherein said limiting manganese conditions comprise a total amount of manganese in the media of less than 0.5 mg per liter, preferably less than 0.07 mg per liter; (j) limiting zinc conditions, wherein said limiting zinc conditions comprise a total amount of zinc in the media of less than 0.5 mg per liter, preferably less than 0.04 mg per liter; (k) limiting molybdenum conditions, wherein said limiting molybdenum conditions comprise a total amount of molybdenum in the media of less than 0.5 mg per liter, preferably less than 0.1 mg per liter; (1) limiting boron conditions, wherein said limiting boron conditions comprise a total amount of boron in the media of less than 5 mg per liter, preferably less than 1.75 mg per liter; (m) limiting silicon conditions, wherein said limiting silicon conditions comprise a total amount of silicon in the media of less than 300 mg per liter, preferably less than 150 mg per liter; and/or (n) limiting vanadium conditions, wherein said limiting vanadium conditions comprise a total amount of vanadium in the media of less than 3 mg per liter, preferably less than 1 mg per liter.

[00048] In preferred embodiments of the invention, the algae biomass comprises (a) a ratio of total amounts of carbon to total amounts of nitrogen of more than 10, preferably more than 15, and in particular more than 16; (b) a ratio of total amounts of carbon to total amounts of phosphorus of more than 83, preferably more than 150; (c) a ratio of total amounts of carbon to total amounts of sulfur of more than 180, preferably more than 300; (d) a ratio of total amounts of carbon to total amounts of iron of more than 140, preferably more than 300; (e) a ratio of total amounts of carbon to total amounts of magnesium of more than 84, preferably more than 160; (f) a ratio of total amounts of carbon to total amounts of copper of more than 17000, preferably more than 25000; (g) a ratio of total amounts of carbon to total amounts of potassium of more than 45, preferably more than 100; (h) a ratio of total amounts of carbon to total amounts of calcium of more than 833, preferably more than 1500; (i) a ratio of total amounts of carbon to total amounts of manganese of more than 7150, preferably more than 13000; (j) a ratio of total amounts of carbon to total amounts of zinc of more than 12500, preferably more than 25000; (k) a ratio of total amounts of carbon to total amounts of molybdenum of more than 5000, preferably more than 10000; (1) a ratio of total amounts of carbon to total amounts of boron of more than 286, preferably more than 500; (m) a ratio of total amounts of carbon to total amounts of silicon of more than 3.3, preferably more than 6; and/or (n) a ratio of total amounts of carbon to total amounts of vanadium of more than 500, preferably more than 1000. It is understood that said ratios are weight by weight ratios.

[00049] The term "anaerobic", as used herein, denotes a system, which is devoid of molecular oxygen. In contrast, an "aerobic" system is a system in which there is a free supply of molecular oxygen and an "anoxic" system is a system in which there is no molecular oxygen, but where there is a free external supply of nitrates and nitrites and where anaerobic fermentation does not occur. The term "anaerobic digestion", as used herein, refers to a series of processes in which microorganisms break down biodegradable material in the absence of oxygen resulting in the production of biogas.

[00050] The term "bacteria", as used herein, refers to unicellular, prokaryotic microorganisms. Bacteria constitute a domain of prokaryotic microorganisms. Typically a few micrometers in length, bacteria have a number of shapes, ranging from spheres to rods and spirals. The bacterial cell is surrounded by a cell membrane (also known as a lipid, cytoplasmic or plasma membrane). This membrane encloses the contents of the cell and acts as a barrier to hold nutrients, proteins and other essential components of the cytoplasm within the cell. As they are prokaryotes, bacteria do not usually have membrane-bound organelles in their cytoplasm, and thus contain few large intracellular structures. They lack a true nucleus, mitochondria, chloroplasts and the other organelles present in eukaryotic cells. Bacteria include organisms from the phyla of Acidobacteria, Actinobacteria, Aquificae, Armatimonadetes, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospirae, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermomicrobia, Thermotogae and Verrucomicrobia. In further preferred embodiments of the invention, the bacteria are one or more selected from the group consisting of Armatimonadetes, Bacteroidetes, Chlowflexi, Firmicutes, Planctomycetes, Proteobacteria, Spirochaetes and Synergistetes.

[00051] The term "archaea", as used herein, is directed to a domain and kingdom of single-celled microorganisms. The archaea are prokaryotes, meaning that they have no cell nucleus or any other membrane -bound organelles in their cells. Archaea include the taxonomic groups Euryarchaeota, Aenigmarchaeota, Diapherotrites, Nanoarchaeota, Nanohaloarchaeota, Micrarchaeota, Pacearchaeota, Parvarchaeota, Woesearchaeota, Proteoarchaeota, Aigarchaeota, Bathyarchaeota, Crenarchaeota, Geoarchaeota, Korarchaeota, Lokiarchaeota, Thorarchaeota and Thaumarchaeota. In more preferred embodiments of the invention, the archaea is one or more groups consisting of Methanosaeta, Methanospirillum, Methanolinea and Methanoculles.

[00052] In preferred embodiments of the invention, the digestion step of (b) is a semi- continuous or continuous fermentation.

[00053] In preferred embodiments, the fermentation temperature is between 30 and 40 °C, and in particular 38 °C.

[00054] In various embodiments, the organic loading rate (OLR) is greater than 1, preferably greater than 2, more preferably greater than 3, and in particular 4 or greater than 4. In certain embodiments, the OLR is increased during the fermentation process, for example from 1 g VS L^"1 d^"1 in the beginning to 4 g VS L^"1 d^"1 (VS: volatile solids). The increase may be stepwise or continuously, e.g. over the course of 1 or more days. Volatile solids as used herein, means the amount of organic biomass (dry weight minus the ash content).

[00055] The term "batch fermentation", as used herein, refers to a discontinuous process, wherein a fermenter tank is prepared with the raw materials (including the inoculum) and after fermentation the content of the tank is removed. A fermentation cycle requires an independent preparation with the raw materials and the removal of the products. Thus, the independent fermentation cycles represent different batches. Each cycle of the batch fermentation comprises a lag phase, followed by an exponential phase followed by a stationary phase. A typical batch reactor consists of a tank with an agitator and integral heating/cooling system. These vessels may vary in size from less than 1 liter to more than 15,000 liter. They are usually fabricated in steel, stainless steel, glass-lined steel, glass or exotic alloy. Liquids and solids are usually charged via connections in the top cover of the reactor. Vapors and gases also discharge through connections in the top. Liquids are usually discharged out of the bottom. In preferred embodiments of the method of the invention, the digestion step of (b) is not a batch fermentation.

[00056] The term "semi-continuous fermentation", as used herein, refers to biochemical processes, in which customarily use is made of a fermenter for culturing and at defined time points a part of the fermentation medium present is withdrawn and replaced by fresh medium (termed fed-batch culture). This maintains the organisms in the logarithmic growth phase. A semi-continuous fermenter may contain similar features as a continuous fermenter, such as regulation systems to control mixing of fermentation liquid, temperature, pH, add-on the raw materials and nutrients and/or removal of the fermentation product.

[00057] The term "continuous fermentation", as used herein, refers to a process of continuous sterile nutrient solution add-on to a bioreactor and the simultaneous removal of the equivalent amount of converted nutrient solution. This maintains the organisms in the logarithmic growth phase (also called steady state). A continuous fermenter may contain features, such as regulation systems to control mixing of fermentation liquid, temperature, pH, add-on the raw materials and nutrients and/or removal of the fermentation product. Two basic types of continuous fermentations using two different types of bioreactors can be distinguished:

[00058] A) Homogeneously mixed bioreactor: This type of bioreactor may run as either a chemostat or a turbidostat bioreactor. In the chemostat during the steady state, cell growth is controlled by adjusting the concentration of one substrate. Any required substrate (carbohydrates, nitrogen compounds, salts) can be used as a limiting factor. In the turbidostat, cell growth is kept constant by using turbidity to monitor the biomass concentration and the rate of feed of nutrient solution is appropriately adjusted. [00059] B) Plug flow reactor: In this type of continuous fermentation, the culture solution flows through a tubular reactor without back mixing. The composition of the nutrient solution, the number of cells, mass transfer, and productivity vary at different locations within the system. At the entrance to the reactor, cells must be continuously added along with the nutrient solution.

[00060] Different fermenters are well-known to the skilled person and are disclosed, for example, in Bioreactors: Design, Operation and Novel Applications, Carl-Fredrik Mandenius, WILEY- VCH, 2016.

[00061] The scope of the present invention also encompasses preferred embodiments with the proviso that the method of the invention does not comprise chemical and/or physical treatment of the plurality of algae between steps (a) and (b). In more preferred embodiments, the above described chemical and/or physical treatment degrades or destroys the cell wall of the plurality of algae.

[00062] In this context, the term "physical treatment" refers to processes that allow the degradation of the cell wall of the algae by a physical process, such as grinding processes (with microbeads), freeze and thaw cycles of the algae, high temperature exposure, sonication, homogenization (for example by using a French Press). The term "chemical treatment" refers to processes that allow the breakdown of a cell wall by using chemicals, such as EDTA, alcohols, ether or chloroform, and/or enzymes, such as cellulases, chitinase, bacteriolytic enzymes like lysozyme, mannase, glycanase, etc. The terms "degradation" and "destruction" of the cell wall, describe processes that partially or completely damage the cell wall and forcing open the cell wall and spilling the contents of the cell.

[00063] In a further aspect, the present invention relates to the use of algae for production of biogas, wherein the algae is (a) cultured in media, wherein said media contains limiting nutrient conditions; and/or (b) anaerobically digested by bacteria and/or archaea.

[00064] In preferred embodiments of the use of the invention, the biogas is methane (CH₄), carbon dioxide (C0₂), nitrogen (N₂), oxygen (O2), hydrogen sulfide (H₂S), hydrogen (H₂) or ammonia (NH₃), preferably methane (CH₄).

[00065] In a third aspect, the invention relates to a system for the production of biogas comprising: (a) a first container comprising at least one algae species and media, wherein said media contains limiting nutrient conditions; (b) a second container comprising (i) the same at least one algae species of (a) and (ii) bacteria and/or archaea.

[00066] In preferred embodiments of the system of the invention, the second container is formed to allow semi-continuous or continuous fermentation of the algae by anaerobic digestion of the bacteria and/or archaea.

[00067] The second container of the system of the invention may be a bioreactor or fermenter as described herein. In preferred embodiments, the first and the second container of the system of the invention are directly connected by a pipe. However, in alternative preferred embodiments, the first and the second container are not directly connected. These embodiments include systems, wherein the first and the second container are each connected with a third container that may be used as a storage unit for the cultivated algae. A typical bioreactor that may be used as the second container comprises a tank and optionally an agitator and integral heating/cooling system. These vessels may vary in size from less than 1 liter to more than 15,000 liter. In preferred embodiments, the fermenter has a capacity between 100 L and 1 L, more preferably between 50 L and 1 L, more preferably between 10 L and 1 L. They are usually fabricated in steel, stainless steel, glass-lined steel, glass or exotic alloy. The used tank may comprise additional systems to establish anaerobic conditions within said tank.

[00068] The bioreactor used for the system of the invention may be chosen from one of the following ones: (1) continuous stirred tank bioreactor; (2) bubble column bioreactor; (3) airlift bioreactor; (4) fluidized bed bioreactor; (5) packed bed bioreactor and (6) photo-bioreactor.

[00069] In further preferred embodiments of the system of the invention, (a) the limiting nutrient conditions comprise limiting conditions for nitrogen, sulfur, phosphate, potassium, magnesium, calcium, iron, manganese, silicon, zinc, copper, molybdenum, boron and/or vanadium; (b) the biogas is methane (CH₄), carbon dioxide (C0₂), nitrogen (N₂), oxygen (O2), hydrogen sulfide (H₂S), hydrogen (H₂) or ammonia (NH₃), preferably methane (CH₄); (c) the at least one algae species is a chlorophyte, preferably selected from the group consisting of Chlamydomonas, Chlorella and Scenedesmus; (d) the bacteria are selected from the group consisting of Armatimonadetes, Bacteroidetes, Chloroflexi, Firmicutes, Planctomycetes, Pwteobacteria, Spirochaetes and Synergistetes; and/or (e) the archaea are selected from the group consisting of Methanosaeta, Methanospirillum, Methanolinea and Methanoculles .

EXAMPLES

Experimental methods and materials

Strains and growth conditions

[00070] Chlamydomonas reinhardtii strain CC-1690 from the Chlamydomonas Center (Duke University, Durham NC, USA) was used for all experiments. Liquid algal cultures were grown photoautrophically under continuous white light (300 μιηοΐ photons m ² s^_1; Osram L 36W/865, Osram Germany). Cultivations were conducted in glass bottles (DURAN® max. capacity 3.5 L, outer diameter 110 mm and 450 mm height, Schott Germany) with 3 L of algae culture, under continuous agitation on a magnetic stirrer. Carbon supply was achieved by bubbling with carbon dioxide-enriched air (3% v/v) with a flow rate of 10 L * h^"1. Nutrients were provided by a modified Provasoli based minimal medium (Provasoli et al., 1957). For replete nitrogen culture conditions, the following components and concentrations were applied: K2HPO4 0.57 mM; H3BO3 0.16 mM; MgS0₄ 4.87 mM; KC1 21.46 mM; NaNOs 11.77 mM; CaCl₂ * 2H₂0 2.72 mM; FeCl₃ * 6 H₂0 12.2 μΜ; Na₂-EDTA 12.5 μΜ; EDTA 103 μΜ; ZnCl₂ 2.2 μΜ; MnCl₂ * 4H₂0 16.7 μΜ; CoCl₂ * 6 H₂0 50.4 nM; CuCl₂ * 2H₂0 17.6 nM; Na₂Mo0₄ ^" * 2H₂0 24.8 nM and NaCl 17.1 mM. Low nitrogen cultivation conditions were realized according to (Klassen et al., 2015) by applying a limited amount of nitrogen (3.56 mM NaN0₃ equals to 50 mg of nitrogen per liter culture).

Determination of algal biomass concentration

[00071] The biomass concentration was determined by centrifugation of 15 mL of cell culture (3000*g for 5 min, at least three technical replicates per sample) and drying of the cell pellet in a pre-weighted glass tube at 105 °C for 24 hours. To determine the organic biomass fraction, the sample tubes were subsequently incubated at 550 °C for 5 hours and the residual ash determined by weighing. The amount of organic biomass (dry weight minus the ash content) was calculated and expressed as volatile solids (VS, g L^"1). Measurement of elemental N and C content in the biomass (C/N ratio)

[00072] Total carbon (C) and nitrogen (N) content of the algal biomass was determined via an element analyzer (VARIO EL III, Elementar Analysesysteme, Hanau, Germany) as described by Plainer et al. (Plainer et al., 2012).

Anaerobic fermentation and quantitative biogas measurement

[00073] Semi-continuous fermentation of algae biomass was performed according to the VDI 4630 guideline (VDI-4630, 2006). Fermentation was performed in B-Braun glass fermenters, max. capacity 2 L. Fermentation temperature of 38 °C was provided by external tempered water bath via water circulation thought a build in water jacket in the fermenter. Reactor content was stirred at 100 rpm via slices stirring system and in a 15 min. ON and 15 min OFF mode. The digester was operated with 1 L working volume (inoculated with microbial community from waste water treatment plant Bielefeld-Heepen, Germany) and constant hydraulic retention time (HRT) of 20 days. Feeding/withdrawing was performed manually with a syringe (first 50 mL rector content out thereafter 50 ml algae substrate in) daily. Biogas (water free after condensations column) evolution was measured continuously by a MilliGascounter® (MGC-1 V 3.0, 3,2 mL, Ritter, Germany) and evaluated via RIGAMO Software (Ritter, Germany), followed by normalization of the gas volume to standard pressure (1.013 bar) and temperature (0 °C) conditions. Organic loading rate (OLR) was increased subsequently and simultaneously in both digesters (reactor 1 fed with replete-N biomass (BM) and reactor 2 fed with low-N BM), from day 0 to 40 OLR = 1 g VS L ¹ d^"1, from day 40 to 100 OLR = 2 g VS L ¹ d^"1 and from day 100 to 160 OLR = 4 g VS L^"1 d^"1. The fresh algal substrate was obtained by centrifugation of the cultures at 3000*g for 5 min and removal of the supernatant. In order to avoid freezing or drying artifacts, biomass was diluted by addition of H₂0 to required concentration and stored by 2 °C prior feed (max. 2 weeks long).

Methane content measurement via gas chromatography (GC)

[00074] The determination of the methane content within the biogas was performed by GC analysis weekly in 9 technical replicates. Biogas from the fermenter was sampled with a gas tight syringe (5 mL) via the rubber seals and injected into a gas chromatograph Micro-GC-Anlage Micro Box III (Elster GmbH, Germany) equipped with an Micro packed HayeSep A-Column (Length: 65 cm, inner diameter: 0.3 mm) and a thermal conductivity detector (TCD). Column temperature in the first 50 s was at 50 °C with following linear increase 4 °C s^"1 to 165 °C, which was hold constant till the end by 120 s. Argon was used as the carrier gas and the calibration of the GC was performed with defined gas (Linde, Germany) containing 0₂ (0.103%), H₂S (0.208%), H₂ (0.498%), CH₄ (59.4%), C0₂ (34.4%) and N₂ (5.391%), mixed according to DIN EN ISO 6141.3.

Determination of biomass composition (Lipid, Protein and Carbohydrates)

[00075] Determination of lipid fraction was performed in 2 technical and 4 biological replicates from 50 mg lyophilized biomass each. After homogenization (3 x 30 s at 6,500 rpm using a Precellys 24, Peqlab, Erlangen, Germany), the total lipid fraction was extracted according to a modified Folch protocol (Folch et al., 1957) using a total of 4 ml methanol and 8 ml chloroform. Contaminants were removed by washing the extract with 3 ml of deionized water. After evaporating of solvents under nitrogen atmosphere lipid fraction was determined via gravimetrical measurement. The total cellular protein amount was determined by Lowry assay (Bio-Rad, CA, USA). Amount of carbohydrates was estimated as remaining fraction of DW after determination of protein and lipid fraction.

TAN / FAN determination

[00076] Total ammonium nitrogen (TAN) was determined using colorimetric verification via cuvette tests LCK302 (Hach Lange GmbH, Germany). Free ammonia nitrogen (FAN) was calculated from TAN value in respect to temperature and pH according to the formula given by Astals and colleges (Astals et al., 2012).

Other parameters

[00077] Total-, organic- and inorganic-carbon was measured via LCK381, total nitrogen was determined via LCK 338, (Hach Lange GmbH, Germany). COD was measured via cuvette test LCI400 and LCK014 (Hach Lange GmbH, Germany) according manufacture instruction.

VFA concentration determination

[00078] The determination of volatile fatty acids (acetate, propionate, iso-butyrate, n- butyrate, iso-valerate, and n-valerate) concentrations were performed by GC analysis. Sample preparation was done according to the (Clescerl et al., 1999) 5560D procedure and analyzed using a Shimadzu GC-2010 plus Gas Chromatograph equipped with a Macherey-Nagel OPTIMA® FFAPplus (Length: 30 m, inner diameter: 0.25 mm) column (Macherey-Nagel, Germany) and coupled to an FID detector (supplied with H₂ and synthetic air). Analysis was performed under constant pressure of 231,9 kPa with He as carrier gas and N₂ as makeup gas with constant flow rate of 60 cm s^"1. Column temperature in the first 2 min was at 100 °C with following linear increase to 175 °C within 15 min. For the calibration a VFA-Mix standard (46975-U, Supelco Analytical, Sigma- Aldrich, USA) at concentrations of 0,1 mM, 1 mM and 10 mM was used.

Microbial monitoring by high-throughput 16S rRNA amplicon sequencing

[00079] Genomic DNA was extracted as previously described by (Klocke et al., 2007). For the determination of the taxonomic profile of the biogas community, high-throughput sequencing of the hypervariable V3-V4 regions of the 16S rRNA gene was performed on the Illumina MiSeq system by applying the paired-end protocol, according to the manufacturer's instructions and by using of the Illumina recommended gene specific primer sequences.

Sequence data processing and data analysis

[00080] For the data processing and analysis, an amplicon analysis pipeline was used as recently described (Liebe et al., 2016; Theuerl et al., 2015). Briefly, raw sequences were merged by FLASH (Magoc and Salzberg, 2011) and further processed and analyzed using the UP ARSE pipeline (Edgar, 2013) based on Usearch 8.0 (Edgar, 2010) with default settings. Processed operational taxonomic units (OTU) were taxonomically classified using the RDP classifier 2.7 (Wang et al., 2007).

Abbreviations

[00081] C:N = carbon to nitrogen ratio, SE = standard error, FAN = free ammonia nitrogen, TAN = total ammonium nitrogen, OLR = organic loading rate, HRT = hydraulic retention time, AD = anaerobic digestion, CSTR = Continuously stirred tank reactor, SMY = specific methane yield, TMP = theoretical methane potential, VS = volatile solids. Example 1: Cultivation of algal biomass under replete N and low N conditions

[00082] Figure 1 shows a graphical abstract of the method of the present invention.

[00083] In previous work, it was elucidated that the composition and the recalcitrance of microalgal biomass strongly depend on growth conditions, in particular on nutrient availability and harvesting time point of the biomass (Klassen et al., 2015). In order to highlight the importance of nutrient availability, microalgae (C. reinhardtii CC-1690) biomass was generated using media with two different nitrogen concentrations (replete-N with 11.77 mM nitrogen and low-N with 3.56 mM nitrogen, supplied as NaN0₃). According to the results from previous work (Klassen et al., 2015), biomass harvesting was always performed after 6 days of cultivation for both conditions (Fig. 1). In order to avoid changes of biomass characteristics due to storage artifacts e.g. freezing (Gruber-Brunhumer et al., 2015), or drying (Mussgnug et al., 2010), algae biomass was cultured parallel to the fermentation experiments, after harvesting, it was concentrated and stored by 2°C for maximal 14 days before feeding into digester. The growth of the microalgae biomass in photobioreactors was periodically monitored by measuring organic biomass accumulation (Fig. 2).

[00084] The phototrophic algae with low and replete nitrogen concentrations in culture media, showed no significant differences in biomass accumulation rates between the two media (Fig. 2), at least until the time point 6 days (harvesting).

[00085] After 7 days of cultivation, an obvious starvation of biomass accumulation could be monitored in low-N media, due to nitrogen depletion (Fig. 2). In accordance with the expectation, biomass accumulation was observed in replete-N conditions up to day 10, however with significantly lower biomass accumulation rate after day 7, most likely due to stronger self- shading effect of the dense algae culture. In conclusion under this culture conditions, no obvious disadvantages could be observed (until day 6, harvesting time point), regarding biomass productivity by using nitrogen limited media.

[00086] The biomass composition of C. reinhardtii cultivated under replete-N and low-N conditions revealed significant differences regarding the protein and almost no difference regarding lipid content (Fig. 4), which is consistent with earlier obtained results regarding lipid content of C. reinhardtii CC-1690 under nitrogen deprivation (Bogen et al., 2013). Consequently, carbohydrates represent the main carbon sink in nitrogen starved C. reinhardtii cells. Based on biomass composition, the theoretical methane potential was calculated (Heaven et al., 2011) and revealed no significant difference with 554 ITLLN d^"1 g^"1 VS and 552 ITLLN d^"1 g^"1 VS between replete-N and low-N biomass, respectively. Furthermore, correspondingly to the 2.2 fold lower protein content, the concentration of elemental nitrogen in the low-N biomass was decreased to only 2.9 % of dry weight, whereas the nitrogen amount in the replete-N conditions resulted in 7.2 %. These values have direct impact on the C:N ratio in the biomass, which is one of the most crucial factors for continuous or semi-continuous fermentation (C:N ratio: replete-N = 6.9, low-N = 16.3, Table 1) (Bohutskyi and Bouwer, 2013; Zubr, 1986).

Example 2: Anaerobic fermentation of algal biomass in semi-continuous mode

[00087] Biomass generated under replete-N and low-N culture conditions was subsequently used for semi-continuous fermentation under anaerobic conditions, conducted under mesophilic temperature (38°C) conditions, corresponding to temperature conditions for anaerobic microbial community inoculum (source local waste water treatment plant (WWTP) operating at 38°C). The semi-continuous fermentation of algae biomass was performed under constant hydraulic retention time (HRT) of 20 days, and the organic loading rate (OLR) was subsequently increased from 1 g VS L^"1 d^"1 in the beginning to 4 g VS L^"1 d^"1 at the end of the experiment (Fig. 5).

[00088] Differences in the fermentation performance of these two types of biomass were already obvious in the beginning at OLR 1 (adoption phase), where the gas productivity was not only lower in "replete-N reactor", but also the adaptation process (defined by stable biogas production rate) was slower. During whole period at OLR of 2 in "replete-N reactor" as well biogas as methane productivities were lower and less stable compared to "low-N reactor". Right in the beginning of OLR 4 gas productivity of "replete-N reactor" started to decrease and reached the minimum level of biogas productivity of 62 ITLLN d^"1 g^"1 VS, at the end of the experiment. In contrast to replete-N biogas, the biogas as well as methane productivity of the low-N BM reactor was constantly high (Fig. 5) over the whole experimental time (exclusive adaptation period, OLRl). Despite the significantly lower methane concentration in the biogas of low-N digester 61 % compared to 65 % of replete-N digester (Fig. 3), the overall methane productivity was higher from low-N BM (Fig. 5) during the whole experimental time course. The overview of the mean biogas and methane productivities, presented in Figure 7 underlines again that microalgae biomass from replete-N conditions can be used only at OLR2 (max. 2g VS L^"1 d^{" x}), which is already critical since the biogas productivity was not constantly stable, however the achieved methane yield is higher than of maize at the same loading rate. However, higher loading rates (OLR 4) have a strongly negative effect on the biogas productivity from replete-N biomass. On the other hand fermentation of low-N biomass was observed to be stable over both experimental loading rates (OLR 2 and 4) with constant methane productivities of 464 ITLLN d^"1 g^{" 1} VS and 462 ITLLN d^"1 g^"1 VS, respectively (Fig. 7). In comparison to maize the methane productivity of low-N algae biomass is 50 % more efficient on VS basis.

[00089] Despite of the fact that the theoretical potential of replete-N as well as low-N biomass are similar, the specific methane yield of low-N biomass is significantly higher compared to replete-N biomass (464 ITLLN d^"1 g^"1 VS and 416 ITLLN d^"1 g^"1 VS, respectively) at OLR2. The lower biogas and methane productivity of biomass grown under replete-N condition is surprising, since the TMP values are similar to that of low nitrogen biomass. In order to evaluate the possible reasons for this, some essential fermentation parameters were analyzed for both reactors (Fig. 8-12).

[00090] One of the most crucial parameters in the fermentation of protein-rich biomass is nitrogen, which is released during anaerobic decomposition of biomass in form of ammonium into the reactor supernatant. Monitoring of total ammonia nitrogen (TAN) release/concentration in reactor revealed indeed a huge difference between the protein-rich (replete-N BM) and low- protein (low-N BM) biomass (Fig. 12). The TAN-concentrations in low-N reactor were observed to be always constantly below 600 mg/L, however the TAN-concentration in replete-N reactor increased with the scale up of the OLR, reaching at OLR2 a value of 1500 mg/L, which is already close to inhibitory levels given by 1700-1800 mg/L (Albertson, 1961; Melbinger et al., 1971; Yenigiin and Demirel, 2013). These inhibitory levels were exceeded directly after loading rate of 4g VS L^"1 d^"1, reaching the maximum at day 140 with 3500 mg/L. However, free ammonia is known to be a more efficient inhibitor than ammonium and to have a strong effect primarily to the methanogens already at low concentration of 50 to 100 mg/L (Yenigiin and Demirel, 2013). Indeed, it was observed in replete-N reactor high free ammonia (FAN) concentration already at OLR 2, which could have an inhibitory effect on methanogens, indicated by simultaneous decline in methane productivity at days 45 to 60 (Fig. 12). Yet, despite further increase of FAN to 74 mg/L at day 77, methane productivity stabilized, which may be due to bacteria or archaea adaptation to these FAN-concentrations, whereupon the FAN- concentrations decreased again to 35 mg/L. During OLR4 the FAN concentration increased again and reached at day 112 comparable levels, which were observed at OLR2 (73 mg/L), however, this increase was accompanied by a simultaneous increase of VFA concentration (day 112). Despite the subsequent decrease in FAN (caused by the drop of pH, which was caused by constantly increasing VFA), the VFA concentration increased further and the methane productivity dropped further down until the end of the experiment (Fig. 5 and 12, replete-N BM). It can be assumed that adaptation of anaerobic microorganisms was not possible within the short time to changes in this important/crucial factors FAN, TAN and VFA, so that the inhibition could not be overcome, which consequently led to almost complete failure of the fermentation process. Similar observation were also made by other researchers in fermentation approaches of protein-rich algal biomass as mono-substrate in continuous manner (Jegede, 2012; Mahdy et al., 2015; Markou et al., 2013; Mendez et al., 2014; Samson and Leduyt, 1986; Schwede et al., 2013), where high TAN/FAN concentrations and consequently increasing VFAs led to decreased methane productivities.

[00091] On the other hand, the reactor fed with low-N biomass did not show any imbalances in fermentation parameters, which were constantly low through the whole experimental time (Fig. 12, low-N BM), especially FAN concentration were found to be lower than 5 mg/L far below inhibitory levels (Yenigiin and Demirel, 2013). This is also reflected in constant methane productivity at different loading rates (Fig. 5, low-N BM).

[00092] Since the fermentation of microalgae biomass, generated under nitrogen limited conditions was stable and produced constant methane amount, it was of interest to evaluate the conversion efficiency level of this process. For this purpose, the theoretical methane potential (TMP) of the biomass can be compared to the specific methane yields (SMY) reached in the experiment (VD 1-4630, 2006). According to the calculations, the conversion efficiency for low- N biomass to biomethane reached 84 % (calculation SMY (Fig. 7) of TMP (Fig. 4)) for both loading rates (OLR 2 and 4). At this point, however, it should be noted, that approximately 12- 15% of the organic matter is used for bacterial growth and maintenance requirements during the anaerobic digestion process (Raposo et al., 201 1 ). Based on this consideration, the conversion efficiency for low-N biomass represents nearly 99 % in this study, and is therefore one of most efficient processes so far described in literature for algal biomass as a mono -substrate (Klassen et al., 2016). For instance, Samson and colleges observed maximal methane productivity by digestion of Spirulina maxima of 350 ITLLN d^"1 g^"1 VS and a maximal conversion efficiency of 59 , however, these results were achieved only under OLR 1 and HRT of 30 days, whereas the productivities decreased significantly if higher loading rates were applied, due to pronounced ammonia inhibition (Samson and Leduyt, 1986). Even lower maximal productivities of only 267 mLN CH₄ d^"1 g^"1 VS (at OLR 4 and HRT of 20 days) could be observed in a recent study with Spirulina biomass (Nolla-Ardevol et al., 2015). Similar results could be achieved for green algae biomass in other studies, where only 160 mLN CH₄ d^"1 g^"1 VS could be reached for raw Chlorella vulgaris biomass, corresponding to 32 % conversion efficiency, however after thermal pretreatment of the biomass, the yield could be increased by 1.5 fold and still but reached only 233 mL_N CH₄ d ¹ g ¹ VS corresponding to only 49 % of TMP (OLR 0.8, HRT 15) (Mendez et al., 2015). Surprisingly low methane productivities of only 70 mLN d^"1 g^"1 VS were published by Mahdy and co-workers for C. vulgaris, corresponding to only 15 % conversion efficiency (OLR 1 , HRT 15), nevertheless parallel digestion of enzymatically pretreated algae biomass was 2.2 times more efficiently digested and resulted in 196 mLN CH₄ d^"1 g^"1 VS corresponding again to only 49 % of TMP (OLR 1 , HRT 20) (Mahdy et al., 2015).

[00093] The biomass-to-methane conversion efficiency demonstrated within this work by application of low-N algae biomass of 84 % is not only significantly higher compared to other long term fermentations trails with untreated biomass but also compared to results achieved after successful pretreatment of microalgae biomass. Furthermore this efficiency may represent the maximum practically achievable under this AD conditions, since bacterial and archaeal biomass evolution and their metabolic energy demand consume up to 12- 15 % of substrate energy content (Raposo et al., 201 1). Considering the energy consumption of microbial biomass the practical efficiency of the fermentation process presented here is at 96 - 99 %. Example 3: Determination of the microbiological population during the fermentation process

[00094] For the investigation of how this suboptimal and optimal performance of replete - N and low-N biomass digesters is reflected by microbial community, high-throughput 16S rDNA gene amplicon sequencing was accomplished. For the comparison of the dynamics of the bacterial community in the different conditions, samples of inoculum (WWTP) and the biogas fermenter, fed with replete-N and low-N biomass after OLR2 (after 100 days) and OLR4 (after 160 days) were chosen. In all investigated samples no evidence of eukaryotic plastid 16S rDNA could be found, suggesting that the DNA was completely disintegrated during the anaerobic fermentation. Based on the 16S rRNA gene amplicon database, the biogas producing microbial community was dominated by bacteria with 99 , whereby the archaea were only represented with approximately 1 % (Fig. 13). These findings have previously been reported (Carballa et al., 2011; Liu et al., 2009; Regueiro et al., 2012; Sundberg et al., 2013), and are in agreement with the fact that bacteria are involved in the first three steps of biomass transformation with a high variety of substrates whereas archaea are restricted to a very narrow substrate spectrum in terms of acetate, methyl-group containing compounds as well as C0₂ and H₂. However, many of bacterial 16SrRNA amplicon reads (27.26 % + 2.75 for inoculum, 28.94 % + 1.37 and 10.39 % + 0.43 for replete-N BM OLR2 and OLR4, as well as 48.01 % + 1.77 and 40.58 % + 1.59 for low- N BM OLR2 and OLR4, respectively, Fig. 13) could not be classified at the phylum levels, respectively, confirming that largely bacterial communities in AD reactors remain uncharacterized.

[00095] According to Bergey's Manual of Systematic Bacteriology (Vos et al., 2011) and The prokaryots: Other major lineages (Rosenberg et al., 2014), all identified bacterial community members are typically involved in the anaerobic degradation of the supplied feedstock as they are described to have cellulolytic, saccharolytic, glycolytic, lipolytic, proteolytic and/or acido- /aceto genie capacities.

[00096] The active sludge (inoculum) from WWTP revealed very high species diversity comprised of 603 + 52 OTUs (Fig. 14). Overall, 73% of the identified sequence reads could be assigned to 18 different phyla, with the most abundant among them the members of the phyla

Chloroflexi (26.78 %), Actinobacteria (17.96 %), Verrucomicrobia (7.80 %) and Firmicutes (7.01 %), whereas all other phyla were found only to a minor portion (Fig. 13). The bacterial diversity dropped significantly during the anaerobic fermentation of algal biomass, cultivated under replete-N and low-N culture conditions and revealed 178 + 34 and 111 + 7 OTUs, as well as 269 + 20 and 177 + 2 OTUs for OLR2 and OLR4, respectively (Fig. 14). This development indicates, that distinct bacteria species begun to dominate due to the selection pressure based on the certain substrate type and amount and other species were extinct. Similar results were also observed in other studies (Blume et al., 2010; Carballa et al., 2015). Furthermore, in the reactors with successful anaerobic digestion, the members of the phyla Bacteroidetes became dominant, followed by Chlorobi in the digester replete-N BM OLR2 or Spirochaetes in the low-N BM OLR2 and 4. Thereby, there are actually three main OTUs for the phyla Bacteroidetes (OTU_2, 3 and 26) (Fig. 6). OTU_26 is representing the genus Paludibacter of the family Porphyromonadaceae, which was described to ferment various sugars to acetate and propionate as the major fermentation products (Krieg et al., 2010), especially lowN-digester contained carbohydrate rich substrate, where Paludibacter was most abundant. The phyla Chlorobi is represented by only one member of the genus Ignavibacterium (OTU_36), which was described to utilize various carbohydrates (Rosenberg et al., 2014). The phyla Spirochaetes is mainly consisting of two OTUs of the order Spirochaetales (OTU_8 and 18), of which OTU_18 could be classified to the genus Treponema, which utilize carbohydrates and/or amino acids as carbon and energy source (Krieg et al., 2010). Interestingly, the overfed digester (replete-N BM, OLR4), which experienced acidosis because of the high FAN/TAN and VFA concentrations (Fig. 2 reple-N BM), showed a completely different bacterial population, with the members of phyla Firmicutes and Thermotogae being the most abundant in this samples. Thereby the Firmicutes were to 70 % represented by only OTU_108 of the genus Sporanaerobacter, and the Thermotogae 99.9 % by the species (OTU_125, Fig. 14) similar to Defluviitoga tunisiensis (Maus et al., 2015). Sporanaerobacter was described to be able to utilize some sugars, peptides and various single amino acids into acetate (Hernandez -Eugenio et al., 2002). Moreover, members of Thermotogae have been characterized for complex polysaccharide fermentation and hydrogen production (Conners et al., 2006), what might promote beneficial associations with hydrogenotrophic methanogens (Muralidharan et al., 1997). The phyla Bacteroidetes is also present in these samples, however it is in contrast to the well-performing digesters, mainly represented by other members of the family Porphywmonadaceae (OTU_78 and 111), which were described to be weakly saccharolytic in contrast to Paludibacter observed in well- performing digesters, since the bacterial growth is not significantly affected by carbohydrates, but is enhanced by protein hydrolysates (Vos et al., 2011), which is also in agreement to the fact that this digester was fed with protein-rich biomass.

[00097] In general, archaeal communities were much less diverse than bacterial ones (Fig. 13 A, B), with Methanomicrobiaceae, Methanobacteriaceae and Methanosaetaceae being the dominant families. The members of Euryarchaeota in the inoculum (active sludge of WWTP) are present to 1.18 % ± 0.13 and are consistent on the genus level of Methanobrevibacter, Methanolinea and Methanospirillum and Methanosaeta, with the last being the most abundant of the methanogenic community. Distribution, similar to the inoculum, could be observed in the well-performing digesters, with Methanosaeta sp. representing the most abundant archaea in the methanogenic community, followed by Methanoculleus sp. and Methanospirillum sp. and Methanolinea sp. (Fig. 13B). On the other hand, the archaeal community in replete-N BM digester OLR4 is dominated by Methanoculleus sp. and to lesser extent by Methanosaeta sp., suggesting an apparent redirection from the acetoclastic towards hydrogenotrophic methanogenesis. The increased abundance of Methanoculleus sp. could be probably attributable to the reducing source of acetate and a higher availability of hydrogen provided by certain bacterial species like the members of the phyla Thermotogae. Similar behavior could be also observed in other studies, whereby the authors described that the replacement of the dominant Methanosaeta sp. by Methanoculleus sp., might be a potential warning indicator of acidosis within the fermenter (Blume et al., 2010; Carballa et al., 2015; Goux et al., 2015).

[00098] The invention has been described broadly and generically herein. Each of the narrower species and sub generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject-matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. [00099] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The compositions, methods, procedures, treatments, molecules and specific compounds described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[000100] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. The word "comprise" or variations such as "comprises" or "comprising" will accordingly be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[000101] The content of all documents and patent documents cited herein is incorporated by reference in their entirety. References

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Claims

1. Method for production of biogas comprising:

(a) culturing a plurality of algae in media, wherein said media contains limiting nutrient conditions, wherein the limiting nutrient conditions comprise limiting conditions for nitrogen; and

(b) anaerobically digesting the plurality of algae of (a) by bacteria and/or archaea to produce biogas, wherein the digestion step of (b) is a semi-continuous or continuous fermentation.

2. The method according to claim 1, with the proviso that said method does not comprise chemical and/or physical treatment of the plurality of algae between steps (a) and (b).

3. The method according to claim 2, wherein said chemical and/or physical treatment degrades or destroys the cell wall of the plurality of algae.

4. The method according to any one of claims 1-3, wherein the limiting nutrient conditions comprise additionally limiting conditions for sulfur, phosphate, potassium, magnesium, calcium, iron, manganese, silicon, zinc, copper, molybdenum, boron and/or vanadium.

5. The method according to any one of claims 1-4, wherein the limiting nutrient conditions comprise limiting nitrogen conditions, wherein said limiting nitrogen conditions comprise a total amount of nitrogen in the media of less than 100 mg per liter, preferably less than 50 mg per liter.

6. The method according to any one of claims 1-5, wherein the algae biomass comprises

(a) a ratio of total amounts of carbon to total amounts of nitrogen of more than 10, preferably more than 15; and/or

(b) a peptide/protein content of less than 50%, preferably less than 40%.

7. The method according to any one of claims 1-6, wherein the biogas is methane (CH₄), carbon dioxide (C0₂), nitrogen (N₂), oxygen (O2), hydrogen sulfide (H₂S), hydrogen (¾) or ammonia (NH₃), preferably methane (CH₄).

8. The method according to any one of claims 1-7, wherein said algae is a chlorophyte, preferably selected from the group consisting of Chlamydomonas, Chlorella and Scenedesmus, more preferably said algae is Chlamydomonas reinhardtii.

9. The method according to any one of claims 1-8, wherein

(a) the bacteria are selected from the group consisting of Armatimonadetes, Bacteroidetes, Chloroflexi, Firmicutes, Planctomycetes, Pwteobacteria, Spirochaetes and Synergistetes; and/or

(b) the archaea are selected from the group consisting of Methanosaeta, Methanospirillum, Methanolinea and Methanoculles.

10. Use of algae for production of biogas, wherein the algae is

(a) cultured in media, wherein said media contains limiting nutrient conditions, wherein the limiting nutrient conditions comprise limiting conditions for nitrogen; and/or

(b) anaerobically digested by bacteria and/or archaea, wherein the digestion step of (b) is a semi-continuous or continuous fermentation.

11. The use according to claim 10, wherein the biogas is methane (CH₄), carbon dioxide (CO2), nitrogen (N₂), oxygen (O2), hydrogen sulfide (H₂S), hydrogen (¾) or ammonia (NH₃), preferably methane (CH₄).

12. A system for the production of biogas comprising:

(a) a first container comprising at least one algae species and media, wherein said media contains limiting nutrient conditions, wherein the limiting nutrient conditions comprise limiting conditions for nitrogen; (b) a second container comprising (i) the same at least one algae species of (a) and (ii) bacteria and/or archaea, wherein the second container is formed to allow semi-continuous or continuous fermentation of the algae by anaerobic digestion of the bacteria and/or archaea.

13. The system according to claim 12, wherein

(a) the limiting nutrient conditions comprise additionally limiting conditions for sulfur, phosphate, potassium, magnesium, calcium, iron, manganese, silicon, zinc, copper, molybdenum, boron and/or vanadium;

(b) the biogas is methane (CH₄), carbon dioxide (C0₂), nitrogen (N₂), oxygen (O2), hydrogen sulfide (H₂S), hydrogen (H₂) or ammonia (NH₃), preferably methane (CH₄);

(c) the at least one algae species is a chlorophyte, preferably selected from the group consisting of Chlamydomonas, Chlorella and Scenedesmus;

(d) the bacteria are selected from the group consisting of Armatimonadetes, Bacteroidetes, Chloroflexi, Firmicutes, Planctomycetes, Pwteobacteria, Spirochaetes and Synergistetes; and/or

(e) the archaea are selected from the group consisting of Methanosaeta, Methanospirillum, Methanolinea and Methanoculles.