How To Measure (BOD) Biological Oxygen Demand in Water

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What is BOD5？

Biochemical oxygen demand (BOD) refers to the amount of dissolved oxygen consumed in the biochemical process of microbial decomposition of some oxidizable substances in water, especially organic substances under specified conditions.

The whole process of biological oxidation takes a long time—for example, more than 100 days when cultured at 20°C. We generally measure the dissolved oxygen before and after culture in 20 ± 1 ℃ for 5 days. The difference between the two is BOD5, expressed in mg / L of oxygen. The higher the BOD, the more organic substance in the water and the poorer water quality.

The whole process of biological oxidation takes a long time.For example, more than 100 days when cultured at 20°C.In general,we measure the dissolved oxygen before and after culture in 20 ± 1 ℃ for 5 days . The difference between the two is BOD5, expressed in mg / L of oxygen. The higher the BOD, the more organic substance in the water and the more poorer water quality.

How to pretreat the water sample for BOD5 determination

PH adjustment of water sample

Because most microorganisms in the water are suitable for survival in neutral or weakly alkaline (pH = 7.2 ~ 7.6) environment.

If the phosphate buffer solution in water is diluted, it does not meet the requirement. We can adjust it through the below step:

First, use a pH meter or a wide range of pH test paper to test the pH value, and use hydrochloric acid solution [C (HCl) = 0 5 mol / l] or sodium hydroxide solution [C (NaOH) = 0 5 mol / l] adjust the pH value of the water sample to neutral or weakly alkaline and then dilute it.

Note:

The addition amount of regulating solution should be no more than 0.5% of the volume of the water sample.

If the acidity or alkalinity of a water sample is very high, a high concentration alkaline solution or acid solution can be used for neutralization.

Regulation of initial water temperature

The water temperature is closely related to the growth of microorganisms and the speed of biochemical oxygen consumption in the water. Therefore, we must strictly control the water sample temperature at 20 ℃± 1 ℃ during the whole determination process to ensure the precision and accuracy of the determination.

1) Low-temperature water sample:

The temperature of collected water samples in winter, or refrigerated and cryopreserved, is far lower than 20 ℃. Open it in a 20 ℃ temperature water bath for rapid heating and balance by thermometer control. When the water temperature reaches (20 ± 1 ℃), measure it.

2) High-temperature water sample:

The temperature of collected water samples in summer or heated polluted water samples is often higher than 20 ℃. Open it into a 20 ℃ constant temperature biochemical incubator for rapid cooling and balance by thermometer control. When the water temperature reaches (20 ± 1 ℃), measure it.

Initial dissolved oxygen regulation

The biochemical reaction of microbial decomposition of organic substances must be done under aerobic conditions. The concentration of dissolved oxygen affects the reaction progress.

We must strictly control the water sample’s initial dissolved oxygen concentration before determining.

If the initial dissolved oxygen concentration of the water sample is lower than 2.0mg/l: we can improve the dissolved oxygen concentration of the water sample by dilution or inoculation dilution method. (reason: if it is not enough to provide sufficient dissolved oxygen for microbial physiological and biochemical activities, making the biochemical reaction difficult to do)

Supersaturation of dissolved oxygen: water samples collected from water areas with low water temperature (winter) or eutrophic lakes and reservoirs may contain supersaturated dissolved oxygen.

In case of this, adjust the initial water temperature of the water sample to 20 ℃, put all the water samples in a 1000 ml measuring cylinder, and shake the measuring cylinder 5 ~ 10 times to drive out supersaturated dissolved oxygen.

Regulation of salt content in the water sample

The biochemical reaction of microbial decomposition of organic substance must be done in an environment with sufficient nutrients.

Suppose the salt content of the sample is low. In that case, the conductivity is less than 125 μ S / cm, which can affect the microbial activity due to insufficient nutrients, thus affecting the determination of BOD5 in water samples.

Therefore, we must determine the conductivity of the water sample by conductivity meter before measurement, add four salt solutions of equal volume into the water sample if the low salt content (conductivity less than 125 μ S / cm), and adjust the conductivity of the water sample to 125 μ s /cm.

Negative value phenomenon:

When water samples are collected from water areas with high water temperature (summer) or heat pollution discharge outlets, in case of low dissolved oxygen concentration, adjust the water sample temperature before measurement to quickly cool to 20 ℃, then put all the water samples to a 1000 ml measuring cylinder, and shake the measuring cylinder for 5 ~ 10 times, making it close to equilibrium with the oxygen partial pressure in the air by atmospheric reoxygenation. Otherwise, it will cause the result to be low or even negative.

What are the determination methods of biochemical oxygen demand?

Common BOD determination methods include dilution inoculation method, biological electrode method, mercury differential pressure method, mercury-free differential pressure method, activated sludge method and coulometer method.

Dilution inoculation method: after diluting the water sample to a certain concentration, culture it at a constant temperature of 20 ℃ for 5 days, measure the dissolved oxygen in the water before and after culture, and then calculate the BOD (i.e., BOD5).

2. Mercury-free differential pressure sensing method: BOD is measured by breathing. Oxygen reduction in a confined space will produce a certain pressure difference, which can be measured by a pressure-sensing probe and converted into a BOD value.

3. Mercury differential pressure sensing method: the dissolved oxygen in the closed water sample is consumed by microorganisms to produce CO2 equivalent to the oxygen consumption. After being absorbed by the absorbent, the pressure decreases, making the mercury column show differential pressure. We can calculate the BOD of the water sample according to the pressure decrease measured by the differential pressure.

4. Microbial electrode method: use a specific method to contact the water sample with the microbial sensor. There is a quantitative relationship between the difference between the current and the biodegradable organic substance in the water sample and the reduction of oxygen. Based on this, we can convert the biochemical oxygen demand of the water sample.

5. Coulometer method: the oxygen consumed by microbial decomposition of organic substance is supplied with the oxygen produced by electrolysis in the closed system. The oxygen consumption is obtained from the oxygen required for electrolysis. The instrument automatically displays the measurement results and records the biochemical oxygen demand curve.

6. Activated sludge aeration degradation method: use the activated sludge to degrade the samples at the temperature of 30 ℃ ~ 35 ℃ for 2h.Digest the samples before and after biodegradation by potassium dichromate, and determine the difference in chemical oxygen demand before and after biodegradation, BOD.

Determination of BOD5 by dilution and inoculation method

For some surface water, most industrial wastewater and domestic sewage contain more organic substances; they need to be diluted and then cultured for determination to reduce their concentration and ensure that the degradation process is done under the condition of sufficient dissolved oxygen.

The specific dilution ratio of the water sample can be calculated by potassium permanganate index or chemical oxygen demand (CODcr). If BOD5 is not more than 6mg / L, it does not need to be diluted and can be determined directly.

For industrial wastewater without or little microorganisms, we should introduce microorganisms that can decompose the organic substance in the wastewater while determining BOD5. When organic substances in the wastewater are challenging to be degraded by microorganisms in ordinary domestic sewage at a regular rate or contain highly toxic substances, we should inoculate domesticated microorganisms.

The below method is suitable for determining BOD5 in surface water, industrial wastewater and domestic sewage. The method’s detection limit is 0.5 mg / L, the lower limit of the dilution inoculation method is 2 mg / L, and the upper limit of the determination is 6000 mg / L.

Steps:

1) Prepare the inoculation solution according to the instructions;

2)Inoculation dilution water: add a certain volume of inoculation solution to the prepared dilution water according to the source ;

3)Sample pretreatment:

a.adjust the pH of the sample to 6 ~ 8 with a hydrochloric acid solution of sodium hydroxide solution.

b.Remove residual and free chlorine.

c. Homogenize the sample, filter and remove green algae if necessary, and adjust the conductivity to more than 125 µ s / cm2;

4) If the dissolved oxygen of the sample is supersaturated, we should remove the excess oxygen by oscillation method. If the dissolved oxygen is insufficient, aeration is required;

5) Determine the dilution multiple according to the sample conditions, and dilute the sample with inoculated dilution water;

6) Use the electrochemical probe method to determine the mass concentration of dissolved oxygen in the sample before culture, strictly seal the dissolved oxygen bottle containing the sample and put it in a constant temperature incubator at 20 ± 1 ℃ to avoid light for 5 days ± 4 hours, and determine the mass concentration of dissolved oxygen in the sample after culture;

7) Calculate BOD5 according to the mass concentration and dilution multiple of dissolved oxygen in the sample and blank sample before and after culture.

Factors affecting the accuracy of BOD5 determination

1)Preservation and transportation of water samples: Different water samples will change due to various physical, chemical and biological effects from collection to analysis, which will affect the determination of BOD. Therefore, we must take a series of protective measures to send the water sample to the laboratory for testing as soon as possible.

For example, the water sample is preferably contained in a glass or polyethylene containers, which are filled and sealed during collection; During transportation, it is best to refrigerate (keep away from light at 2 ~ 5 ℃), and if necessary, equip special insulated containers and use refrigerants to ensure the original condition of water samples as much as possible; The water sample should be analyzed within 6h after collection, and the cold storage time should be no more than 24h.

2) Dilution water and inoculation dilution water: dilution water and inoculation dilution water are the main factors affecting the accuracy of determination results. The dissolved oxygen of dilution water should be controlled at 8 ~ 9 mg / L. too high or too low will cause poor results. Too high will produce bubble overflow and cause an error. It will not be enough to decompose organic substances in water if too low and no data can be measured.

2)Other influencing factors: after the sample is cultured at 20 ± 1 ℃ for 5 days, ensure the dissolved oxygen is consumed at least 2 mg / L, and the residual dissolved oxygen is at least 1 mg / L after culture, so that get the reliable BOD5 measurement. Therefore, the dilution of multiple samples is significant.

The nitrifying bacteria in the sample will produce additional oxygen demand, nitrifying oxygen demand. Therefore, some samples need to add nitrification inhibitors such as allyl thiourea to reduce the interference of nitrifying oxygen demand; Some water samples, such as those collected from water areas with low water temperature or eutrophic lakes, may contain supersaturated dissolved oxygen. At this time, the water sample should be rapidly heated to about 20 ℃ and fully vibrated and deflated without filling the container to remove the supersaturated dissolved oxygen;

In addition, it should clean the glassware, control the quality of water in the lab, and other factors.

How to determine BOD5 by mercury-free differential pressure method

Preparation:

1. Instruments: biochemical oxygen demand tester, analytical balance and constant temperature incubator

2. Reagent: ultrapure water, D – (+) – glucose (AR, dried at 130 ℃), glutamic acid (BR, dried at 130 ℃), granular sodium hydroxide, phosphate buffer, nutrient solution, strain solution, 100g / L sodium hydroxide (AR) solution

Step:

1. Sample handling

a) If the BOD concentration of the sample is more than 4000mg / L or contains heavy metal toxic substances, it needs to be diluted with diluted inoculum.

b) For industrial sewage or disinfected water and other samples containing fewer microorganisms, it needs to add 2% of inoculation liquid into the sample.

2. Adjust the pH of the sample

Use sodium hydroxide solution to adjust the pH of the sample to 6.7-7.5 (the best pH is 7.2. It is recommended to use a pH meter ).

3. Prepare test samples

Use a clean measuring cylinder or volumetric flask to measure the sample from the table and pour it into the culture bottle. Then put a stirrer in the culture bottle. If necessary, aerate in the incubator for 1h. Put on the reagent cup and put in 0.5g sodium hydroxide. Install the reagent cup and BOD tester on the culture bottle in turn and tighten them.

4. Culture test

Set the parameters on the BOD tester, put the culture bottle on the mixing tray in the incubator, turn on the power and start the test. After the instrument completes the test, take out the culture bottle and read the recorded results.

What reagents are used for biochemical oxygen demand detection?

Reagent preparation:

Nutrients: dissolve the whole bottle of nutrients in 300ml of experimental water, shake well, and add 3ml per liter of the water sample.

Buffer: dissolve the whole bottle of buffer in 100ml of experimental water and shake well. Add 1ml per liter of the water sample.

Propenyl thiourea: propenyl thiourea nitration inhibitor, dissolve 0.20g propenyl thiourea (c4h8n2s) in 200ml water, mix and store at 4 ℃. This solution can be kept for 14d. 2ml propylene thiourea nitration inhibitor should be added to each liter of the sample, and the process must be recorded in the test report.

Strain preparation: weigh 0.2g solid reagent, mash it, pour it into the beaker, add about 100ml distilled water, and continuously stir it in the incubator until the blocks are dispersed. After 24 ~ 48 hours, take the supernatant for use. The dosage of each 1L of the water sample is 5 ~ 10ml in summer and 10 ~ 15ml in winter.

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