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Anaerobic digestion of sulphate- containing wastewaters Industrial wastewaters can, in general, be effectively treated using anaerobic digestion - produces large quantities of methane which can be burned to generate electricity or for heating - use of combined heat and power plants allows for generation of electricity and heat recovery Normally less than 10% of the biogas produced is required to operate the plant - also produces far less waste biomass than aerobic system = less disposal costs

Modern digester design makes the process more attractive - can operate at high rates and therefore smaller, cheaper digester can be used Usual procedure is to have first stage anaerobic and then small activated sludge plant to “polish” the effluent = achieve discharge standards. May need some nutrient removal or other tertiary steps depending on the fate of the effluent

Problematic industrial wastewaters Application to industrial wastewaters can occasionally be complicated by microbiological problems - typical example is treatment of sulphate containing wastewaters - examples of the type of negative microbial interactions which can occur in an engineered ecosystem Industrial wastewaters can contain high levels of recalcitrant organic chemicals (e.g. chloroform, carbon tetrachloride etc.), xenobiotic products and side products (insecticides, herbicides, detergents etc.)

Can also contain significant quantities of inorganics some of which may be highly toxic e.g. cadmium in tannery wastewater In anaerobic systems the presence of alternative external oxidising agents ( e.g. sulphate SO 4 2- ) can promote the development of a sulphate- reducing rather than a methanogenic population This will result in the channelling of electrons towards the formation of H 2 S not methane

SRB and anaerobic digestion Very complex systems - absolute need to fully understand the microbiology in order to control treatment plant operation - i.e on one hand a useable fuel is generated and on the other hand a malodourous atmospheric pollutant is produced under sulphidogenic conditions Presents a challenge to microbiologists because of the complexity of the systems and the technical difficulty in studying them

Examples of wastewaters that contain high- levels of sulphate include: 1.Molasses-based fermentation industries - e.g. citric acid production, rum distillery 2.Paper and board production 3.Edible oil refinery Many other industries use sulphuric acid in their processes - leads to sulphate in the ww

So what? In the absence of external oxidising agents (sulphate, nitrate, etc.) anaerobic ecosystems are methanogenic - flow of reducing equivalents is directed towards the reduction of CO 2 to CH 4 In the presence of sulphate - the flow may be redirected towards the reduction of sulphate to sulphide by sulphate reducing bacteria (SRB) In other words there is a competition between different microorganisms for substrate

What will determine the outcome of competition? Very important to know as on one hand a useable fuel is produced, while on the other hand a toxic, corrosive malodourus compound is produced

Bacteria that reduce sulphate to H 2 S are either assimilatory or dissimilatory: 1. Assimilatory Sulphate Reduction: Carried out by many different bacteria - purpose is to reduce sulphate to sulphide prior to uptake of S for assimilation into S-containing proteins etc. No major environmental effect only amount of sulphate needed for bacterial growth is reduced e.g Klebsiella sp. - only reduce 1 mg sulphate for every 200 mg (d.wt.) of cells produced

2. Dissimilatory Sulphate Reduction: Totally different process only carried out by a unique group of bacteria carrying out anaerobic respiration using sulphate as electron acceptor Consequently transform large amounts of sulphate to H 2 S during growth e.g. Desulphovibrio sp. - for every 1 mg of sulphate reduced, only mg (d.wt) of cells are produced

SRB exhibit enormous ecological, morphological and nutritional diversity - grouped together only on the basis of carrying out dissimilatory sulphate reduction - 1 common property Widely distributed in the natural environment - include both sporeformers (Desulfomaculum sp.) and non- sporeformers (Desulfovibrio sp.)

13 eubacterial and 1 archaeal genera Can be divided into two broad categories based on their metabolism: 1. Incomplete Oxidisers: carry out incomplete oxidation of organic compounds to acetate, CO 2 and H 2 S - can use a very wide range of starting organics e.g. aliphatic mono- and dicarboxylic acids, alcohols, amino acids, sugars, aromatic compounds etc.

Desulfomicrobium, Desulfobulbus, Desulfoboyulus, Thermodesulfobacterium, Desulfovibrio*, Desulfomaculatum* * Most common species

2. Complete oxidisers: Complete oxidation of starting organic substrates to CO 2 and H 2 S - same wide range of substrates, but can also grow on acetate, breaking it down completely to CO 2 Desulfobacter, Desulfococcus, Desulfosarcina, Desulfomonile, Desulfonema, Desulfoarculus, Archaeoglobus Chemolithotrophic species also common - grow on H 2 /CO 2 or on CO very common ability to grow on H 2 - very important in certain ecosystems

Basically use H 2 as energy source and fix CO2 (autotrophic as carbon source) 4H 2 + SO H > 4 H 2 O +HS -  G˚´ = -150KJ/mole Very favourable reaction energetically

SRB very versatile metabolically - in the absence of sulphate in their environment, they can switch from anaerobic respiration to chemoorganotrophic fermentation - energy gain by substrate level phosphorylation only V. important as allows maintainence of SRB in the absence of sulphate

2 types of fermentation possible: 1. Fermentation (independent of H2 conc.) Grow fermentatively on sugars, carboxylic acids, alcohols etc. Incomplete degradation to l.mwt acids and alcohols and CO 2

2. Growth as syntrophic OHPA species (H 2 dependent) Convert higher carbon-number alcohols, acids, ketones etc. to acetate + H 2 or acetate + CO 2 + H 2 Same restrictions as syntrophs

What happens during anaerobic treatment of sulphate containing wastewaters? Competition between SRB and other anaerobes for common organic and inorganic substrates Competition for energy and reducing equivalents Between SRB and Fermentative bacteria, between SRB and OHPA, between SRB and Acetoclastic and Hydrogenophilic methanogens

Carbon flow in anaerobic digesters: 1 = Hydrolytic/fermentative bacteria; 2 = Obligate hydrogen producing acetogens; 3 = Homoacetogenic bacteria; 4a = Acetoclastic methanogens; 4b = Hydrogenotrophic methanogens; 5 = Fatty acid synthesising bacteria (O'Flaherty, 1997).

Negative effects of competition 1. Reduction of potential methane yield - diversion of substrates/reducing equivalents to H 2 S 2. Sulphide Toxicity - H 2 S is toxic to all cells - toxicity is pH dependent Only the unionised form of H 2 S to membrane permeable

H 2 S H + + HS - HS - H + + S - At neutral pH approximately 20-50% of the H 2 S is present in the unionised form At pH 8-9 virtually all the H 2 S is undissociated - toxicity increases with increasing pH

With respect to AD, fermentatives are far less susceptible to H 2 S toxicity than syntrophs, methanogens or even SRB IC50 of mg/l H 2 S for methanogens 3. H 2 S in the biogas - H 2 S is very volatile, so will appear in the biogas causing problems of odour, corrosion, release of SO 2 during burning

May well have to be stripped from the biogas - costly 4. Dissolved sulphide in the effluent - odour, oxygen demand, post-treatment costs 5. Precipitation of Alkali metals - Fe, cobalt etc. 6. Sulphate toxicity - salt effects, not usually significant

HOW TO SOLVE THE PROBLEMS Eliminate sulphate from the process, occaisonally possible using chemical precipitation, often not Engineer the ww treatment process, need to understand the microbial ecology

Outcome of competition is determined by the following factors: COD/BOD concentration Chemical composition of the ww Sulphate conc. COD/BOD:sulphate ratio Bacterial population of the sludge pH of reactor operation Mass transfer limitations

Very complex microbiological problem: Theoretical predications can be made based on kinetic and thermodynamic considerations However, these do not correspond to what is measured in practical situations - especially for conversion of the key (70% of biogas) substrate acetate See: review for discussion