How to select the inoculum for the biogas plant – Part II

The specific methanogenic activity (High school): realization and interpretation of the test results.

Re-publication of an article by Mario A. Rosé on Agronotizie

In Part I of this article we have illustrated how the biological activity of an inoculum is measured by introducing a certain amount of reference substrate into a test reactor and verifying that for each gram of COD (Chemical oxygen demand) are produced at least 350 Ncm3 of methane.

Let's analyze in this Part II of article le peculiarities of the test of Specific Methanogenic Activity (High school).

How to carry out the SMA test in practice

The scientific literature on the evidence High school it is very varied as regards the substrates to be used (Ref.[i]).
The first doubt that the beginner must resolve before starting a test of methanogenic activity is therefore: which substrate should be used? Generally speaking, any substrate 100% digestible will produce 350 Ncm3 CH4/gCOD, as the Table 1.

Table 1: Cod e Bmp(methane potential) of some pure substrates and 100% digestible, easy to find. Calculation of the COD carried out by the author based on the stoichiometry of total oxidation, and theoretical BMP obtained with the Buswel and Symons formula (UNI Norms 11723, appendix B.2)

From a formal point of view, But, a test carried out with a substrate that is not sodium acetate or acetic acid is certainly a test of biological activity, but a specific test of methanogenic activity cannot be considered. For example, a test carried out with sugar instead of acetate will involve two groups of microorganisms: fermentative bacteria, which will convert the sugar into acetic acid, and the Archaea, which will convert the acetic acid into methane and carbon dioxide. If the test gives a negative result: how can we say with certainty which of the two groups of microorganisms is the one missing or inhibited?

The second aspect to consider concerns the quantity of acetic acid or sodium acetate to be introduced into the test reactor. The ruleUNI / TS 11703:2018 recommends carrying out a positive control in parallel with each Bmp test to ascertain the quality of the inoculum. However, the norm does not establish what the inoculation ratio is: substrate (I/S) to be adopted, implying that it must be “greater than 2” as for any Bmp test. In the particular case of acetic acid, however, the I / S ratio should be much greater than 10, otherwise there is a risk of lowering the pH too much, and also the reaction of the acid with the carbonates and bicarbonates present in the inoculum will produce abundant foam, until the reactor overflows. Sodium acetate, instead, it does not alter the pH of the inoculum because it is a salt, but an I / S ratio too close to 2 It may cause “false negatives” because sodium is an inhibitor of Archaea, as we have already demonstrated in another article in this column (The electrical conductivity is unreliable for the management of the biogas plant).

The Photo 1 shows the example of a test carried out on the same inoculum sample, with different amounts of sodium acetate.

Photo 1: A test of anaerobic digestion of sodium acetate, made by the author on a single inoculum sample, but with different I / S ratios
Click on the image to enlarge

To practical effects, the evidence of methanogenic activity it must be made with a dosage of sodium acetate – or acetic acid – low enough not to trigger “false negatives” due to the inhibitory effect of these substrates. To avoid the “false negatives”, the sodium acetate dosage should never exceed the inhibition threshold. The scientific literature it is not unique on what this threshold value is. There are values ​​ranging from 7 g of acetate / l of inoculum (Ref.[i]), al range 4-8 g / l valid if – as often happens in agricultural biogas plants– the inoculum has pH> 7,5 (Ref.[ii]) up to a minimum of 2 g /l (Ref.[iii]). It should be noted that all the experiences indicated above refer to digestate from sewage treatment plants, the author has achieved good results with acetate concentrations up to 12 g / l of inoculum. For tests with digestates from agricultural plants, it therefore seems reasonable to assume a limit equal to 10 g of acetate / l of agricultural digestate. If you use acetic acid instead, the total concentration should not exceed i 2,4 g / l of inoculum, otherwise you risk lowering the pH too much (Ref. [iv]). If you don't have pure acetic acid, this concentration is reached with 40 ml of vinegar / l of inoculum(the vinegar contains about 6% of acetic acid by volume).

The unit of measurement of High school

The respiratory rate of any living being is expressed in Nm3 of gas per unit of live weight, per unit of time. In the case of a digestate, the mass of bacteria is represented by volatile solids (SV), usually expressed in g / liter. La curva di attività metanogenica si traccia riportando sull’asse Y la portata giornaliera di metano per unità di solidi volatili dell’inoculo, in funzione del tempo, sull’asse X. La SMA è il valore numerico che corrisponde al picco di tale curva, solitamente espresso in Ncm3 di CH4/ (g SVinoculation x giorno).

The Photo 2 mostra le curve di attività metanogenica corrispondenti alla stessa prova riportata nella Foto 1.
In generale, un valore di SMA > 10 Ncm3/g SV . d si considera buono.

Photo 2: Attività metanogenica di un digestato proveniente da un impianto agricolo, misurata con diversi rapporti I/S. In questo caso concreto, la massima Sma si è verificata con I/S = 4 corrispondente ad un dosaggio di 12 g acetato/l inoculo
Click on the image to enlarge

Un caso di studio reale

Un impianto di biogas richiede il riavvio in seguito a degli interventi di manutenzione straordinaria. Nella zona ci sono due impianti dai quali si potrebbe prelevare l’inoculo, a distanze diverse. Il costo di trasporto dell’inoculo dall’impianto A è 3,65 euro/tonnellata e quello dall’impianto B è 4 euro/tonnellata. Le Sma degli inoculi sono quelle indicate nella Photo 3. Analizziamo quale dei due inoculi conviene utilizzare.

Dal punto di vista puramente biologico, l’inoculo B è migliore dell’A, ma entrambi sono comunque validi, in quanto hanno Sma > 10 Ncm3/g SVinoc . d mentre i costi specifici sono differenti (inoculo A: 3,65 euro/14 = 0,26 euro per unità di SMA e inoculo B: 4 euro/18 = 0,22 euro per unità di SMA).

The criterio generale parte da un presupposto: se la quantità d’inoculo necessaria per avviare l’impianto è direttamente proporzionale alla attività biologica, allora dobbiamo verificare se conviene usare l’inoculo più economico in quantità maggiore o una quantità minore dell’inoculo più caro. In termini economici è più conveniente avviare l’impianto con l’inoculo B (curva blu, Photo 3), il cui trasporto è più caro ma, avendo una attività metanogenica maggiore, richiederà una quantità minore.

Photo 3: SMA di due inoculi provenienti da impianti diversi. Rosso = Impianto A, Blu = Impianto B
Click on the image to enlarge


La prova di Attività Metanogenica Specifica (High school) è un test abbastanza veloce ed economico: consente di selezionare l’inoculo biologicamente più attivo, in modo da velocizzare l’avviamento di un impianto di biogas con il minimo costo. La sua realizzazione è abbastanza semplice, ma non esiste ancora una norma che stabilisca una procedura univoca. Il segreto per una buona riuscita della prova è mantenere la concentrazione di acetato di sodio – or acetic acid– al di sotto della soglia d’inibizione delle Archaea, altrimenti si potrebbero generare dei “false negatives”. Generally speaking, un dosaggio di 10 g acetato/l inoculo, oppure 2,4 g acido acetico/l inoculo (equivalente 40 ml di aceto/l inoculo) si ritiene abbastanza ragionevole per testare inoculi anaerobici da impianti agricoli.

Bibliographic references and recommended insights

[i] Athar Hussain,  Shashi Kant Dubey; Specific methanogenic activity test for anaerobic degradation of influents; Appl Water Sci (2017) 7:535–542; DOI 10.1007/s13201-015-0305-z.
[ii] Arjen Rinzema, Jules van Lier, Gatze Lettinga, Sodium inhibition of acetoclastic methanogens in granular sludge from a UASB reactor, Enzyme and microbial technology, Volume 10, Issue 1, 1988, pages 24-32, ISSN 0141-0229.
[iii] Satoshi Fukuzaki, Naomichi Nishio and Shiro Nagai; Kinetics of the methanogenic fermentation of acetate; Applied and environmental microbiology, Oct. 1990, p. 3158-3163 Vol. 56, No. 10.
[iv] Souto, T.F., Aquino, S.F., Silva, S.Q. et al. Influence of incubation conditions on the specific methanogenic activity test. Biodegradation 21, 411–424 (2010).
[v] Franke-Whittle IH, Walter A, Ebner C, Insam H. Investigation into the effect of high concentrations of volatile fatty acids in anaerobic digestion on methanogenic communities. Waste Manag. 2014; 34(11):2080-2089.

Si veda inoltre:
M. A. Rosé, S. Stromberg; Quick restart of a biogas plant and micronutrients cost minimization with AMPTS II Light, Application note, Bioprocess control.

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